MXPA01011386A - Therapeutic uses for nitric oxide inhibitors - Google Patents

Abstract

Nitric oxide (NO) is an important growth regulator in an intact developing organism. The present invention relates to a method of increasing in a mammal a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, wherein the hematopoietic tissue is contacted with multiple doses of at least one inhibitor of NO, such as inhibitors of nitric oxide synthase (NOS). The present invention also relates to a method of increasing a population of cells in S phase in a tissue of a mammal, comprising contacting the tissue with multiple doses of at least one inhibitor of NO, such as an inhibitor of NOS. The invention also pertains to a method of regenerating tissue in an adult mammal comprising contacting a selected tissue (e.g., blood, skin, bone and digestive epithelium), or precursor cells, with multiple doses of at least one inhibitor of NO, thereby inhibiting differentiation and inducing proliferation of cells of the tissue.

Description

THERAPEUTIC USES FOR NITRIC OXIDE INHIBITORS RELATED APPLICATION (S) This application is a continuation in part of the US Application. No. 09 / 315,929, filed May 20, 1999 and entitled "Therapeutic Uses for Nitric Acid Inhibitors," which is a continuation in part of the US Application Ser. No. 08 / 969,475, filed on November 13, 1997 and entitled "Therapeutic Uses for Nitric Acid Inhibitors," which claims the benefit of the US Provisional Application. No. 60 / 030,690, filed on October 13, 1996, and the benefit of the US Provisional Application. No. 60 / 045,411, filed May 2, 1997. The teachings of the US Application. No. 09 / 315,929, of the US Application. ? 08 / 969,475, of the US Provisional Application ? 60 / 030,690 and the US Provisional Application ? 60 / 045,411 are here incorporated in their entirety. GOVERNMENT FINANCING The work described here was funded by Grant No. 5ROINS32764 from the National Institutes of Health. The Government of the United States has certain rights over the invention. BACKGROUND OF THE INVENTION The development of organs requires a tightly controlled program of cell proliferation followed by growth arrest and differentiation and, often, programmed cell death. The balance between the number of cell divisions and the degree of programmed cell death later determines the final size of an organ (reviewed by Bryant and Simpson, Quart, Rev. of Biol., 59: 387-415 (1984); Raft, Nature , 356: 397-400 (1992)). Although much of the cellular machinery that determines the timing of the appearance and cessation of division per se is well understood (reviewed by Hunter and Pines, Cell, 79: 573-582 (1994); Morgan, Nature, 374: 131 -134 (1995); Weinberg, Cell, 81: 323-330 (1995)), little is known about the signals that cause discrete groups of cells and organs to terminate growth by reaching the number of cells and the size appropriate. A greater understanding of the signals involved provides possible targets for the manipulation of the cellular machinery, resulting in therapeutic benefits for a number of conditions. SUMMARY OF THE INVENTION The present invention is based on the discovery that nitric oxide (NO) is an important growth regulator in an intact developing organism. Specifically, the present invention relates to a method for increasing in a mammal a population of hematopoietic cells (e.g., hematopoietic stem cells), including precursors of myeloid, lymphoid and erythroid cells, which are capable of hematopoiesis, differentiation and normal maturation in hematopoietic tissue, where the hematopoietic tissue contacts with multiple doses of at least one NO inhibitor, such as multiple doses of one or more inhibitors of nitric oxide synthase (NOS), thus producing a hematopoietic tissue that has a larger population of hematopoietic stem cells that are capable of experiencing hematopoiesis, differentiation and normal maturation. In one embodiment, the present invention relates to a method for increasing in a mammal a population of hematopoietic cells that are capable of experiencing hematopoiesis, differentiation and normal maturation in hematopoietic tissue, consisting of putting the hematopoietic tissue in contact with two inhibitors of nitric oxide synthase, thus producing a hematopoietic tissue that has a larger population of hematopoietic stem cells that are capable of experiencing hematopoiesis, differentiation and normal maturation. The method can be carried out in vivo or ex vivo. In addition, the method can be used to prevent the differentiation of erythroid cells and / or myeloid cells in the mammal. The method can also include contacting the hematopoietic tissue with at least one agent (e.g., a hematopoietic growth factor) that induces the differentiation of a selected hematopoietic maternal cell population. The present invention also relates to a method of treating a mammal for the purpose of increasing a population of hematopoietic stem cells in the hematopoietic tissue which are capable of experiencing hematopoiesis, differentiation and normal maturation in the hematopoietic tissue of the mammal. In the method, the hematopoietic tissue of the mammal contacts multiple doses of at least one NOS inhibitor, thus producing a hematopoietic tissue that has an increased population of hematopoietic stem cells that are capable of experiencing hematopoiesis, differentiation and normal maturation . In one embodiment, the present invention relates to a method for treating a mammal in order to increase a population of hematopoietic stem cells that are capable of experiencing hematopoie-sis, differentiation and normal maturation in the mammalian hematopoietic tissue, consisting of putting in contact the hematopoietic tissue of the mammal with two inhibitors of nitric oxide synthase, thus producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing hematopoiesis, differentiation and normal maturation. The method may further include contacting the hematopoietic tissue with at least one agent that induces differentiation of a selected hematopoietic stem cell population.

In an embodiment of the method for treating a mammal in order to increase a population of hematopoietic stem cells that are capable of experiencing normal hematopoiesis, differentiation and maturation in the mammalian hema-topoytic tissue, hematopoietic tissue is obtained that is to be transplanted, where the hematopoietic tissue to be transplanted can be obtained from the mammal being treated (autologous transplant) or from another mammal (heterologous transplant). The hematopoietic tissue to be transplanted contact with multiple doses of at least one NOS inhibitor. The hematopoietic tissue to be transplanted is transplanted into the mammal being treated, thus providing the mammal with hematopoietic tissue that has a larger population of hematopoietic stem cells that are capable of experiencing hematopoiesis, differentiation and normal maturation. In one embodiment, two NOS inhibitors are used. The method may further consist in treating the mammal with a NOS inhibitor (s) before or after haematopoietic tissue transplantation. Alternatively, the method may also include treating the mammal with an NOS (one or more) enhancer before or after haematopoietic tissue transplantation. The present invention also relates to a method of increasing a population of pro-genitous blood cells (e.g., red blood cells, white blood cells) that are capable of experiencing hematopoiesis, differentiation and normal maturation by putting in contact the progenitor cells (stem cells) of the blood with multiple doses of at least one NO inhibitor (eg, an NOS inhibitor). In one embodiment, the progenitor blood contacts two NOS inhibitors. The present invention also relates to a method of increasing a population of dividing cells in a tissue of a mammal, comprising contacting the cells with multiple doses of at least one nitric acid inhibitor. In one embodiment, the present invention also relates to a method of increasing a population of cells in S phase in a tissue of a mammal, which consists in contacting the tissue with multiple doses of at least one NO inhibitor. , such as a NOS inhibitor. In one embodiment, the method results in an increase in the size of an organ in which the tissue appears. Moreover, as described herein, S-phase cells can be used in gene therapy. The present invention also relates to a method of reducing a population of cells in S phase in a tissue of a mammal and inducing differentiation of cells, consisting in contacting the tissue with an NO enhancer (s). , such as a NOS increment. In one embodiment, the method results in a reduction in the size of an organ with which the tissue is associated. The present invention also relates to a method of coordinating the decisions of the development of a cell type in a mammal, consisting of introducing NO into the cell type or into a precursor of the cell type, thus inhibiting the proliferation of the cell type or of a cell type. precursor of the cell type and inducing differentiation of the cell type or of a precursor of the cell type. The present invention also includes a method of induction of differentiation in a mammalian cell population, which consists of contacting the cell population with NO or a NO-enhancer. The invention also relates to a method of regeneration, of tissue in an adult mammal, comprising contacting a selected tissue (eg, blood, skin, bone and digestive epithelium) or precursor cells of the selected tissue with multiple doses of less a NO inhibitor, thus inhibiting differentiation and inducing the proliferation of tissue cells, and then contacting the selected tissue with a compound (e.g., nitric oxide, a growth factor or a combination of both) that inhibits proliferation and induced differentiation. In one embodiment, the method involves repopulating an organ or tissue (e.g., muscle or nerve fiber) comprised of cells that do not normally divide by contacting a selected organ or tissue or precursor cells of the selected organ or tissue with multiple dose of at least one NO inhibitor, thereby inhibiting differentiation and inducing proliferation of the cells of the organ or tissue, then contacting the selected organ or tissue with a compound that inhibits proliferation and induced differentiation. The invention also includes a method of producing a subpopulation of hematopoietic cells. In the method, the hematopoietic tissue contacts multiple doses of at least one NOS inhibitor, thereby producing a hematopoietic tissue with a larger population of hematopoietic stem cells capable of undergoing hematopoiesis, differentiation and normal maturation, and at least one agent ( for example, a hematopoietic growth factor) selected to induce specific differentiation of the population of hematopoietic stem cells, thus producing a subpopulation of hematopoietic cells. In a particular embodiment, the hematopoietic tissue is contacted with two NOS inhibitors. The identification of NO as an important growth regulator in an organism provides several therapeutic applications in humans and other mammals. DETAILED DESCRIPTION OF THE INVENTION The results of the work described herein have shown that a transcellular messenger (nitric oxide (NO)) has a critical role in tissue differentiation and organ development. NO regulates the balance between cell proliferation and cell differentiation in the developing intact organism. A higher production of NO allows the cessation of division and the subsequent differentiation of the cells of a tissue, while the elimination of NO-mediated growth arrest promotes cell division. Accordingly, the present invention is related to a method for increasing in a mammal a population of hematopoietic cells (e.g., hematopoietic stem cells), including precursors of myeloid, lymphoid and erythroid cells, which are capable of hematopoiesis , differentiation and normal maturation in the dreaded hematopoietic, by contact of the hematopoietic tissue with multiple doses of at least one inhibitor (one or more) of NO, such as an inhibitor of NOS. As defined herein, "hematopoietic tissue" is a tissue involved in hematopoiesis, eg, bone marrow, peripheral blood, umbilical cord vein blood, fetal liver, and hematopoietic cell culture. long term. The present invention includes a method of treating a mammal to increase a population of hematopoietic stem cells, which are capable of undergoing hematopoiesis, differentiation and normal maturation in the mammalian hematopoietic tissue, where the mammalian hematopoietic tissue is brought into contact with multiple doses of at least one NOS inhibitor. The invention also relates to a method of producing a subpopulation of hematopoietic cells by contacting the hematopoietic tissue with multiple doses of at least one inhibitor of NOS, thus producing a hematopoietic tissue having an increased population of hematopoietic stem cells capable of suffering hematopoiesis, normal differentiation and maturation, and at least one agent selected to induce specific differentiation of the hematopoietic stem cell population, thus producing a subpopulation of hematopoietic cells. In a particular embodiment, two inhibitors of NO, such as two inhibitors of NOS, are used in the methods. For example, a combination of L-NAME and ETU can be contacted with the hemapoietic tissue to increase a population of hematopoietic stem cells in a mammal, to treat a mammal in order to increase a population of hematopoietic stem cells in the hematopoietic tissue in a mammal or to produce a subpopulation of hematopoietic cells. The present invention also relates to a method for increasing a population of pro-genitor blood cells, comprising contacting blood progenitor cells with multiple doses of at least one inhibitor (one or more) of NO (eg, inhibitor). of the NOS). In one embodiment, the present invention relates to a method for increasing a population of progenitor blood cells, which consists of contacting blood progenitor cells with two NOS inhibitors. Sources of blood progenitor cells include, for example, bone marrow, peripheral blood, umbilical cord vein blood, fetal liver and long-term haematopoietic cell culture. Using the method of the present invention, red blood cells and white blood cells can be increased (eg, granulocytes (neutrophils, basophils, eosinophils), monocytes, lymphocytes). The present invention also relates to a method for increasing a population of dividing cells in a tissue of a mammal, which consists in contacting the cells with multiple doses of at least one NO inhibitor. In one embodiment, the present invention can also be used to increase a population of cells (cells-blank) in S phase in a tissue of a mammal relative to a similar tissue in a mammal not treated by tissue contact with multiple doses of at least one NO inhibitor, such as a NOS inhibitor. In one embodiment, the method results in an increase in the size of an organ with which the tissue is associated. Conversely, the present invention can also be used to reduce a population of S-phase cells in a tissue of a mammal and induce differentiation of the cells, which consists in bringing the tissue into contact with at least one enhancer. of NO, such as an NOS increment. In one embodiment, the method results in a reduction in the size of an organ with which the tissue is associated. Furthermore, as described herein, S-phase cells can be used in gene therapy. The present invention also relates to a method of coordinating the decisions of the development of a cell type in a mammal, consisting in introducing NO into the cell type or into a precursor of the cell type, thus inhibiting the proliferation of the cell type or of the precursor of the cell type and inducing differentiation of the cell type or precursor of the cell type. The present invention also includes a method of induction of differentiation in a mammalian cell population, consisting of contacting the cell population with NO or with a cell enhancer.

DO NOT. The invention also relates to a method of tissue regeneration in an adult mammal. The method consists in contacting a selected tissue with multiple doses of at least one NO inhibitor, thus inhibiting differentiation and inducing the proliferation of tissue cells, then contacting the selected tissue with a compound that inhibits proliferation and induces the differentiation of proliferated cells into cells characteristic of the tissue. In one embodiment, the method involves repopulating an organ or tissue (e.g., muscle or nerve fiber) that has cells that do not normally divide, which method involves contacting a selected organelle or tissue with multiple doses of at least one inhibitor (s) of NO, thus inhibiting differentiation and inducing the proliferation of the cells of the organ or tissue, contacting the selected organ or tissue with a compound that inhibits proliferation and induces the differentiation of the proliferated cells to characteristic cells of the organ or tissue. Compounds that inhibit proliferation and induce differentiation include NO, an NO enhancer and a growth factor. One or more of these compounds can be used to inhibit proliferation and induce differentiation. Weaves that can be regenerated using the methods described herein include blood, skin, bone and digestive epithelium, nerve fiber, muscle, cartilage, fat or adipose tissue, bone marrow stroma and tendo-nes. The methods described herein may further include the step of contacting the target cells of the hematopoietic tissue (e.g., bone marrow) with at least one agent that induces differentiation of a population of selected hematopoietic stem cells to a particular cell type. (for example, erythrocytes, macrophages, lymphocytes, neutrophils and platelets). For example, in the embodiment in which a mammal is treated to increase a population of hematopoietic stem cells in the hematopoietic tissue of the mammal by contacting the hematopoietic tissue of the mammal with multiple doses of at least one inhibitor of NOS, the increased population of the Hematopoietic tissue can be contacted with an agent, such as a hematopoietic growth factor, which will cause or promote the differentiation of cells of a particular cell type. Among agents (e.g., such as hematopoietic growth factors) that can be used in the methods of the present invention to induce differentiation of the increased or expanded number of cells produced by the contact of cells with a NOS inhibitor, include, for example, erythropoietin, G-CSF, GM-CSF and interleukins, such as IL-1, IL-2, IL-3 and IL-6. Alternatively, the methods described herein may further include the step of contacting the hematopoietic tissue with at least one agent that further induces or maintains the proliferation of the selected hematopoietic stem cell population at a particular cell type (e.g., erythrocytes, macrophages, lymphocytes, neutrophils and platelets). NO inhibitors for use in the present invention include, for example, NO scavengers, such as 2-phenyl-4,4,5,5-tetraethylimidazolin-1-oxyl-3-oxide ("PTIO"), 2- ( 4-carboxyphenyl) -4,4,5,5-tetraethylimidazolin-1-oxyl-3-oxide ("Carboxy-PTIO") and N-methyl-D-glucamine dithiocarbamate ("MGD"), and inhibitors of NOS, such as methyl ester of N-nitro-L-arginine ("L-NAME"), N-monomethyl-L-arginine ("L-NMMA"), 2-ethyl-2-thiopseudourea (ETU), 2-methylisothiourea (SMT), 7-nitroindazole, aminoguanidine hemisulfate and diflenylenenyonium ("DPI"). In the methods of the present invention, multiple doses of at least one NO inhibitor (e.g., NOS inhibitors) may be used. When more than one inhibitor is used in the methods of the present invention, the inhibitors may be the same or different. In a particular embodiment, two NO inhibitors are used, such as two NOS inhibitors (eg, L-NAME and ETU) in the methods of the present invention. Moreover, in the methods of the present invention, the NO inhibitor (s) can be administered in a single dose or in multiple doses. As used herein, "multiple doses" refers to at least two doses of at least one NO inhibitor. Multiple doses can be administered in a day or over a period of days (for example, a period of about 2 days to a period of about 15 days or months). For example, the NO inhibitor (s) can be administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. In one embodiment, a mixture of two NO inhibitors (eg, L-NAME and ETU) is administered to the mammal or contacted with the cells twice a day for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. In a particular embodiment, L-NAME and ETU are administered to the mammal or contacted with the cells twice a day for 9 days. In the methods of the present invention, one or more NO enhancers can be used. Increases in NO include, for example, NOS enhancers and NO donors, such as sodium nitroprusside ("SNP"), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (SNOG, GSNO) , Diethylamine NONOate (DEA / NO), DETA / NO (NOC-18), 3-morpholinosidnonimine (SIN-1) and spermine NONOato (Sper / NO). NO is a second multifunctional di-fusible messenger that has been implicated in numerous physiological functions in mammals, from dilation of blood vessels to immune response and enhancement of synaptic transmission (Bredt and Snyder, Annu. Biochem., 63: 175-195 (1994); Nathan and Xie, Cell, 78: 915-918 (1994); Garth aite and Boulton, Annu. .Rev. Physiol. , 57: 683-706 (1995)). NO is produced from arginine by NOS in almost all cell types. A group of three chromosomal genes has been cloned, giving rise to numerous isoforms of the NOS, from mammalian cells (Knowles and Moneada, Biochem J., 298: 249-259 (1994); Wang and Marsden, Adv. Pharmacol., 34: 71-90 (1995)) and recently a Drosophila NOS gene has been isolated, whose coding structure resembles the gene for the mammalian neuronal isoform (Re-gulski and Tully, Proc. Nati. Acad. Sci. USA, 92: 9072-9076 (1995)). The cell division and subsequent programmed cell death in the imaginary discs of Drosophila larvae determine the final size of the organs and structures of the adult fly. The results described here show that NO is involved in the control of the size of body structures during the development of Drosophila. These results show that NOS is expressed at high levels in the imaginary discs under development. The inhibition of NOS in larvae causes hypertrophy of organs and their segments in adult flies, while the ectopic expression of NOS in larvae has the opposite effect. The blocking of apoptosis in the imaginary discs of the eyes unmasks the excess of cell proliferation and results in an increase in the number of ommatidia and component cells of the individual ommatidia. These results demonstrate the activity of NO as an antiproliferative agent during the development of Iro-sophila, controlling the balance between cell proliferation and cell differentiation. Moreover, the results shown here demonstrate that NO acts as a crucial regulator of hematopoiesis after bone marrow (BM) transplantation. The NO regulates the maturation of both erythroid and myeloid lineages. These data demonstrate that manipulations of NOS activity and NO levels during hematopoiesis can be used to alter (increase or decrease) the production of blood cells. This is useful for preventive and therapeutic intervention. During the development of Drosophila, the structure, size and shape of most of the organs of the adult fly are determined in the imaginary structures of the larvae (Cohen, Imaginal disc development, in The Development of Drosophila melanogaster, M. Bate and A. Martí-nez-Afias, eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), 747-841 (1993), Fristrom and Fristrom, The metamorphic development of the adult epidermis, in Development of Drosophila melanogaster, M. Bate and A. Martinez-Afias, eds (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), 843-897 (1993)). Imaginary discs, specialized groups of undifferentiated epithelial cells that are recruited during embryogenesis, are formed in the first instar larvae as integuments of the larval epidermis. The cells of the discs divide rapidly throughout the larval development and cease to proliferate at the end of the third instar period. In the discs of the legs, the wings and the halteres, the progression through the cell cycle stops in the G2 phase 3-4 hours before the formation of the puparium. It resumes 15-18 hours later (12-14 hours after pu-parisation) and then stops again in a defined spatial pattern after 12-14 hours (10-14 hours of pupal development) (Fain and Stevens, Dev. Biol., 92: 247-258 (1982); Graves and Schubiger, Dev. Biol., 93: 104-110 (1982); Schubiger and Palka, Dev. Biol., 123: 145- 153 (1987)). Although most dividing cells in late larvae and early pupae are already delivered to adulthood, they do not develop a fully differentiated phenotype until growth arrest is firmly established. Therefore, cell proliferation is temporarily separated from cell differentiation, which takes place later during metamorphosis. Experiments with transplanted imaginal discs suggest that the cessation of cell proliferation in these structures is controlled by mechanisms that, being intrinsic to the disc, are not completely independent of the cells (Bryant and Schmidt, "Cell Sci., Suppl. 13: 169-189 (1990); Cohen, Imaginal disc development, in The Development of Drosophila melanogaster, M. Bate and A. Martinez-Afias, eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), 747-841 ( 1993).) The signaling pathways that control the arrest of coordinated temporal growth in larvae and pupae and the subsequent arrest of terminal growth in pupae and adults are not known, but probably involve second intercellular and intracellular messenger molecules that have not yet been The transformation of imagined precursors in adult structures during the metamorphosis of flies involves the transition of cell proliferation to cell differentiation. The cessation of cell division is a necessary, although not sufficient, condition for cell differentiation to proceed. A temporary cytostasis occurs at the end of the larval period and there is a permanent arrest of the cell division during the development of the pupa. The? O, a diffusible messenger molecule, is capable of efficiently blocking cell division. The induction of OS initiates a change to growth arrest before the differentiation of cultured neuronal cells (Peuno-va and Enikolopov, Nature, 375: 68-73 (1995)). Therefore, the NOS can act as a permissive factor, making the subsequent development of the phenotype completely differentiated possible. The results described here show that NOS acts as an antiproliferative agent during the normal development of Drosophila, indicating that NO is an important regulator of growth in the intact developing organism. Through larval development, there is a gradual and spatially specific accumulation of NADPH-diaphorase activity in the developing imaginary discs, reflecting an increase in the overall NOS content. At the moment when the temporal cytostasis is being established in the imaginal discs, NADPH-diaphorase staining becomes particularly intense and gradually decreases during prepupal and pupal development. In addition to imaginal discs, other structures with intense NADPH-diaphorase staining include imaginal rings, histoblasts, and the brain. These structures undergo radical changes during the metamorphosis before giving rise to the organs of the adult. Its development includes periods of rapid cell division alternating with periods of cytostasis and, therefore, it must employ mechanisms for the coordinated cessation of DNA synthesis and cell division in a spatially defined pattern. Since NO can prevent cell division and can diffuse and act within a limited volume, the ability of NO to act by inducing coordinated growth arrest during the development of Drosophila was considered. Certainly, if it does NOT actively exert its antiproliferative activity during the development of the imaginal discs, then the inhibition of NOS before the temporary cytostasis is established at the end of the larval period could lead to the reversal of cell division arrest and induce additional divisions, which, in turn, could lead to a larger size of the body structures of the adult fly. Conversely, excessive or ectopic production of NO in the larvae could cause a premature cessation of cell division and lead to a reduction in the size of the structures in adults. Both predictions were confirmed in the experiments described here, in which the activity of NOS in the developing fly was manipulated. The inhibition of NOS in the larvae produced an increase in the number of cells in some parts of the body of the adult and an increase in its size, while the ectopic expression of the NOS transgene during development caused a reduction in the number of cells in some structures of the adult and a re-duction of its size, probably by fusion and partial reduction. In the developing leg, the segments that were most frequently affected when the NOS activity was inhibited and the segments that were most frequently affected when the activity was induced ectopically were non-overlapping and complementary. The most important thing is that its distribution corresponded to the distribution of the NOS in the imaginary discs, thus supporting the hypothesis that it does not play a causative role in stopping growth in normal development. The antiproliferative properties of NO suggest that NOS acts in development through its influence on DNA synthesis and cell division. The results described here with incorporation of BrdU in the discs of the legs with high and decreased production of NO corroborate this position and suggest a direct relationship between NO synthesis, the number of cells in S phase and the final size of the organ. In accordance with this idea, in many cases BrdU incorporation was not observed in regions highly enriched in NOS. The mechanisms for the arrest of the NO-mediated cell cycle (both temporal and terminal) are not clear, but probably involve the conventional cellular machinery for growth arrest, for example the cell cycle-dependent kinases and their inhibitors. Consistently with this, changes in the expression of these proteins were observed when cells cultured with NO were treated. An intriguing feature of the cells of the imaginal discs is that they stop dividing and accumulate in the G2 phase in the third instar late, preceding the period of temporal cytostasis (Fain and Stevens, Dev. Biol., 92: 247-258 (1982 ); Graves and Schubiger, Dev. Biol. 93: 104-110 (1982); Schubiger and Palka, Dev. Biol. , 123: 145-153 (1987)). This parallels a trend of PC12 cells treated with NO (Peunova and Enikolopov, Nature, 375: 68-73 (1995)) and treated with NGF (Buchkovich and Ziff, Mol. Biol. Cell, 5: 225-241). (1994)) to accumulate in phase G2. Interestingly, the imaginal disks are released from the G2 blockade and enter the S phase again 12-15 hours after puparization, at the moment when the diaphorase stain decreases at low levels in adult flies. These correlations between the cells of the imaginal discs and the cells treated with NO support the idea that NO can be a major inducer of cytostasis in the cells of the imaginary discs in the prepupal stage. The final number of cells in an organ or segment is determined both by cell multiplication and by cell death, which the structures in formation of the fly suffer as a normal stage in development (especially in the late stages of pupal development). ). The results described here indicate that changes in the size of the leg segments after manipulation of NOS activity correlated directly with changes in DNA synthesis and the number of dividing cells. Moreover, no significant changes in apoptosis were detected in the discs of the larval and prepupale legs after the inhibition or ectopic expression of NOS, in comparison with control discs, when cell death was followed by staining with acridine orange or by means of a TUNNEL assay. This suggests that it is cell multiplication, rather than changes in programmed cell death, which leads to changes in the size of the appendages. On the other hand, apoptotic death may involve excessive cell proliferation in other developing organs. The effect of the absence of programmed cell death on potential excessive cell proliferation was also assessed. Transgenic flies were used in which programmed cell death in the developing eye was suppressed by recombinant p35, an inhibitor of apoptosis, to reveal excessive proliferation upon inhibition of NOS. Under these circumstances, several cell types and structures are over-represented, the most no-table change being a global increase in eye size due to the greater number of ommatidia. In addition, other cell types (e.g., secondary and tertiary pigment cells, cones cells and sow cells) proliferated upon inhibition of NOS at levels greater than those achieved by blocking apoptosis with p35 (Hay et al., Dev. ., 120: 2121-2129 (1994)). These data demonstrate that the elimination of the suppressor influence of NO leads to an increase in the size of the adult organ, unless this effect is masked by programmed cell death, and indicate that the final cell number in the organ of the adult is under the double control of cell proliferation and programmed cell death. Moreover, these data provide independent support for the hypothesis that NO directly regulates the number of cells during development. Following the inhibition of NOS with either of the two structurally unrelated compounds, excessive growth was observed in most structures of the adult flies that derive from the imaginal discs and the histoblasts, in varying degrees for the different ones. organs. The most obvious changes were observed in the segments of the legs whose primordia showed the highest levels of NOS. There did not appear to be any substantial number of cases in which a duplication of a larger structure (eg, leg or wing segments) occurred. This indicates that there is an extra proliferation of the cells under the influence of the NOS inhibitors after the fate of the development is determined for the largest of the cells in the imaginal discs. This suggests that, in most cases, it CAN NOT be more important for the induction of the cessation of growth and the subsequent differentiation of already destined cells than for the destiny of the development and the establishment of the cellular identity in the embryo or in the larva . Only some of the axes of the developing structures were affected by manipulations of the NOS activity. For example, in the developing legs only the anteroposterior and dorso-ventral axes were affected, but not the proximodistal axis, due to the inhibition of NO production. Conversely, when the NOS was expressed ectopically, only the proximodistal axis was affected. These results suggest that a gradient of NO could be involved in the process of establishing the polarity of the axes in the developing organ. Therefore, these results demonstrate that the inhibition of NOS in larvae leads to an increase of organs in adults and, conversely, that the ectopic expression of NOS in larvae leads to a reduction in the size of the organs in Adults. In addition, the distribution of the affected segments in the adult paw corresponds to the distribution of the NOS in the larvae and the changes in the size of the segments can be directly correlated with the changes in the DNA synthesis in the imaginary discs. after the manipulations of the NOS activity. The greater cellular proliferation that occurs in response to the inhibition of NOS is masked in some structures by programmed cell death and can be revealed by suppression of apoptosis. Taken together, these results demonstrate that the activation of NOS is a crucial stage in the development of Drosophila. They confirm that NO acts as an antiproliferative agent during cell differentiation and development of the organism and controls the number of cells in an intact organism in development. The expression of NOS can be induced at high levels in a large number of tissues and cell types by appropriate stimulation (Bredt and Snyder, Annu, Rev. Biochem., 63: 175-195 (1994)).; Forstermann et al., Adv. Pharmacol. , 34: 171-186 (1995)). In most cases, the distribution pattern of NOS in a developing organism differs greatly from the distribution in the adult organism. Moreover, the transient elevation of the expression of NOS in a given tissue often coincides with the cessation of the division of the targeted precursor cells. The brain of developing mammals provides an especially apt demonstration of this (Bredt and Snyder, Neuron, 13: 301-313 (1994), Blottner et al., Histochem J., 27: 785-811 (1995)). A strong increase in the activity of NOS in the cerebral cortical plate and the developing hippocampus on days 15-19 of prenatal development corresponds to the temporal course of cessation of proliferation of precursor cells, close growth arrest and cell differentiation; notably, NOS activity declines after the proliferation of the targeted neuronal precursors has been completed. NOS levels are also transiently increased in the lungs, bones, blood vessels and developing nervous system (Blottner et al., Histochem J., 27: 785-811 (1995); Collin-Osdoby et al., J " Cell Biochem., 57: 399-408 (1995); Cramer et al., J. Comp. Neurol., 353: 306-316 (1995); Shaul, Adv. Pediatr., 42: 367-414 (1995); Wetts et al., Dev. Dyn., 202: 215-228 (1995).) Elsewhere, the activity of NOS is very high in weaves in regeneration when the cessation of cell division is crucial for the prevention of unregulated growth (Roscams et al., Neuron, 13: 289-2Y9 (1994), Blottner et al., Histochem J. r 27: 785-811 (1995), Decker and Obolenskaya, J. Gastroenterol. Hep tol., 10, Suppl 1: 2-7 (1995), Hortelano et al., Hepat 21: 116-186 (1995).) In all these cases, a transient rise in NOS activity could trigger a change from proliferation to growth arrest and differentiation n, thus contributing to the proper morphogenesis and organ tej gone. The results described here support the position that NO production is necessary during embryonic development and during the regeneration of tissues in the adult organism for the appropriate control of cell proliferation. The antiproliferative properties of NO are particularly important in situations in which the terminal differen- tiation of the cells destined is temporarily separated from cell proliferation and is strictly dependent on the cessation of cell division. Given the multiplicity of NOS isoforms and their overlapping tissue distribution, it is conceivable that any group of cells in the embryo and in the fetus may be exposed to the action of NO. Moreover, recent data showing that NO can be transferred into the organism by hemoglobin (Jia et al., Nature, 380: 221-226 (1996)) raise the possibility that a developing mammalian embryo may also be supplied with NO exogenously by the mother. NO is an easily diffusible molecule and can, therefore, exert its antiproliferative properties not only in the cell that produces it, but also in neighboring cells (Gally et al., Proc. Nati. Acad. Sci. USA, 87 : 3547-3551 (1990)). This property is important when considering the mechanisms for the coordinated development of a group of neighboring cells destined to form a particular structure. These cells have to generate an intrinsic signal that tells them to stop dividing in a coordinated way after having reached a certain number. This cooperation and coordination is achieved in many cases by a tightly controlled paracrine regulation, which involves signaling between adjacent cells through gap junctions or segregated proteins. The results described here show that yet another way of coordinating the decisions of the development in groups of cells is by means of second diffusive antiproliferative messenger molecules., which can spread without the need for surface receptors or specialized systems for secretion and exert their influence in a limited domain. An efficient source of easily diffusible molecules can induce synchronized changes in adjacent cells in a limited volume of a tissue. What is more, several adjacent cells producing easily diffusible antiproliferative messenger molecules can share the total pool of these molecules produced by the neighbors, as well as by themselves. If a particular threshold level of a signal is needed to initiate a signaling chain that eventually leads to growth arrest, then the cells of this group could stop dividing when a certain number of cells is reached and, therefore, a determined local concentration of messenger molecules. In this way, by organizing groups of cells into functional groups and coordinating their decisions on proliferation and differentiation, the NO instructs the developing structures to complete their growth when they reach the appropriate size and shape and, thus, participates in the morphogenesis of tissues and organs. Also as described here, the role of NO in hematopoiesis was examined. To demonstrate the presence of NOS in bone marrow (BM) cells, the MO of adult mice was studied for the NDPH-diaphorase activity of NOS (which reflects the distribution of total enzymatic activity in a tissue ). It was found that OM contains a substantial proportion of cells (up to 12%) with strong diaphorase staining. The morphology of NADPH-diaphorase cells suggests that they are largely of the granulo-cyto-macrophage lineage at different stages of differentiation. This is in agreement with numerous data that show that NOS is present in the cells of the myeloid lineage and can be induced at high levels by appropriate stimulation. A mouse model of syngeneic MO transfer was used to evaluate the role of NO in hematopoiesis. The mice were irradiated to inhibit hematopoiesis in the recipient animal, the OM of syngeneic animals was transplanted and the animals were treated with specific inhibitors of NOS. This procedure allows the proliferation, differentiation and survival of only the transplanted cells. To study changes in hematopoiesis introduced by NOS inhibitors, spleen colonies were monitored to study the differentiation of erythroid cells and colony formation was monitored on the membranes located in the peritoneal cavity of the receptors to study. the differentiation of cells of the granulocyte-macrophage lineage. The role of NO on hematopoiesis was studied by injecting the animals with the NOS inhibitors specific and structurally unrelated L-nitroarginine methyl ester (L-NAME) and 2-ethyl-2-thiopseudourea (ETU). The inactive enantiomer D-NAME was used as control. The animals were sacrificed and the number and composition of colonies in the spleen (reflecting the cells that have undergone erythroid differentiation) and the colonies on the membranes (reflecting the cells that have undergone myeloid differentiation) were studied. Taken together, the results of these studies indicate that NO modulates hematopoiesis after BM transplantation. This confirms the role of NO as a major regulatory factor in the organism that controls the balance between proliferation and differentiation. This also shows that the manipulation of NO levels can be used for therapeutic intervention in order to increase the number of undifferentiated hematopoietic cells after a transplant of MO, change the proportion of cells suffering erythroid or myeloid differentiation and interfere in graft versus host disease or suppress it, which is a major cause of mortality in patients undergoing MO transplantation. Most of the tissues and organs in the adult organism are constantly undergoing regeneration and renewal, going through phases of rapid proliferation, determination, cessation of growth, differentiation and, often, programmed cell death. Many human diseases are caused by inappropriate or incomplete stages of differentiation, which result in a loss of function of a particular tissue or organ. This suggests that these diseases can be treated and, furthermore, that the appropriate function of the affected tissues and organs can be restored by directing and manipulating cellular and tissue differentiation. This work described here, which demonstrates the role of NO in cell proliferation and differentiation in an organism, provides several therapeutic applications in humans and other mammals. Specifically, this approach based on NO can be focused on renewable and regenerative tissues, such as blood, bone, skin and digestive epithelium. Additionally, a similar strategy can be used to repopulate organs with cells that normally do not divide, such as muscle and nerve cells. The work described here can also be used to increase the methods of gene therapy. For example, NOS can be used to bring a population of cells to the S phase, where the cells replicate. As is known in the art, cells that are replicating respond more to gene therapy methods (eg, introduction of genes by means of live vectors) than cells that are not replicating. Thus, the present invention provides a method of converting the cells to a state that makes the cells more receptive to gene therapy methods, where the cells contact a NO inhibitor (e.g., a NOS inhibitor) . Conversely, the pre-sentate invention provides a method of converting the cells into a state that renders them resistant to gene therapy methods. That is, the present invention provides a method of converting the cells into a state that makes them more resistant to gene therapy methods, where the cells contact NO and / or a NO-enhancer (e.g. the NOS). The results of the work described here support the ability of NO to act as a crucial regulator of hematopoiesis after bone marrow transplantation (BMT). The NO regulates the maturation of both erythroid and myeloid lineages. By interfering with NO production in the recipient animal after BMT, the number of undifferentiated stem cells and blasts that are then capable of further differentiation along the erythroid or myeloid lineages can be dramatically increased. Blast enrichment reaches 80 times for the myeloid lineage and 20 times for the erythroid lineage. The data described here demonstrate that manipulations of NOS activity and NO levels during hematopoiesis can be used for therapeutic purposes to influence the self-renewal and differentiation of hematopoietic stem cells and to replace damaged or defective cells . Areas of application include increased blood cell and myeloid cell formation following high-dose chemotherapy in the treatment of cancer, improved grafting after bone marrow or stem cell transplants, and gene therapy. Stem cells amplifying the undifferentiated cells of the erythroid and myeloid lineages and applying appropriate factors to induce terminal differentiation, and the regulation of the formation of various cellular components of the blood for the treatment of hematological and autoimmune disorders. The data also show that the change in NO production levels interferes with the differentiation of osteoblasts and chondrocytes. These results show that the manipulation of NO production can regulate the growth and differentiation of osteoblasts, chondrocytes or mesenchymal stem cells. This can be used for the amplification and subsequent differentiation of cells in the injured tissue, or for cellular implants (in combination with biocompatible vehicles, if necessary). Therefore, an NO-based approach can be used for regenerative therapy of damaged tissue, for post-injury repair, for age-related diseases, such as osteoporosis and osteoarthritis, and to reconstitute the stroma of the marrow after cancer chemotherapy at high doses. In addition, the data show that the change in NO production levels interferes with the differentiation of keratinocytes. The results described here show that the regulation of NO production can be used when a greater proliferation and subsequent differentiation of the cutaneous tissue is required (for example, during the healing of burns and wounds). Furthermore, NO can be used to control disorders caused by hyperproliferation of keratinocytes during psoriasis. Yet another potential application is to use NO-based preparations as exfoliating agents in cosmetic therapy. NO has been shown to act as a regulator of cell differentiation in neuronal cells. It has been shown that NO regulates the development of the brain in animals and helps to control the size of the brain in intact animals.

It has also been shown that, in certain contexts, NO mediates the survival effects of growth factors by activating an antiapoptotic program and that it can protect neuronal cells from death. Combined, these studies of the role of NO in neurons suggest that NO can be used to control the proliferation and subsequent differentiation of nerve cells in substitution therapy after neurodegenerative disorders caused by aging (for example, Alzheimer's disease or Parkinson), stroke or trauma. NO is actively produced in the smooth muscle cells of blood vessels and is subject to complex physiological regulation. These cells are highly susceptible to the suppression of DNA synthesis by NO. The very strong antiproliferative activity of NO can be used for the inhibition of the proliferation of smooth muscle cells and the formation of neointima for the treatment of restenosis following angioplasty. In addition, NO-based therapy has application for the treatment of conditions characterized by destruction of specific groups of cells. This includes the regeneration of hepatocytes after a toxic liver injury, the treatment of disorders of the reproductive system and the administration of differentiated pancreatic tissue for the treatment of type I diabetes. The methods of the present invention can be carried out in vivo or ex vivo. Administration of the NO inhibitor, the NO enhancer and / or the agent that induces differentiation can be accomplished using various delivery systems known in the art. The routes of administration include the intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural and intranasal routes. Any other convenient route of administration can be used, such as, for example, bolus infusion or injection, or absorption through epithelial or mucocutaneous coatings. In addition, the NO inhibitor, the NO enhancer and / or the agent that induces differentiation can be administered with other components or biologically active agents, such as adjuvants, pharmaceutically acceptable surfactants, excipients, carriers, diluents and vehicles. The administration can be systemic or local, for example by direct injection at the site containing the target cells. In the embodiment in which the NO inhibitor, the NO enhancer and / or the agent that induces differentiation are proteins or peptides, these can be administered by in vivo expression of genes or polynucleotides encoding them in a mammalian subject. . Various expression systems, such as live vectors, are commercially available, or may be reproduced according to recombinant DNA techniques for use in the present invention. The amount of NO inhibitor, NO enhancer and / or agent for use in the present invention that will be effective in the treatment of the particular disorder or condition will depend on the nature of the disorder or condition and can be determined by specific clinical techniques. tandar The precise dose for use in the formulation will also depend on the route of administration and the severity of the disease or disorder and will be decided according to the judgment of the practitioner and the circumstances of each patient. For example, the amount of NO inhibitor (s) for use in the methods of the present invention may be from about 1 mg / kg of body weight to about 1,000 mg / kg of body weight, of about 5 mg / kg from body weight to about 500 mg / kg of body weight and from about 25 mg / kg of body weight to about 100 mg / kg of body weight. In one embodiment, from about 25 mg / kg of body weight to about 1,000 mg / kg of body weight of L-NAME and / or from about 1 mg / kg of body weight to about 100 mg / can be used. kg of body weight in the methods of the present invention. In a particular embodiment, approximately 300 mg / kg body weight of L-NAME in combination with about 30 mg / kg body weight of ETU are used in the methods of the present invention. The following examples are offered for the purpose of illustrating the present invention and are not to be construed as limiting the scope of this invention. The teachings of all references cited herein are incorporated herein by reference. EXAMPLES EXAMPLE 1 Nitric Oxide Regulates Cell Proliferation During the Development of Drosophila Stocks of Drosophila The Oregon R strain of Drosophila melanogaster was used for most of the described experiments. Transgenic GMR-P35 flies (alleles 3.5 and 2.1, Hay et al., Dev., 120: 2121-2129 (1994)) were a generous donation of B. Hay and G.M. Rubin. Transgenic flies carrying the NOS mouse macrophage gene (NOS2) were generated under the heat shock promoter (hs-mNOS20 (2) and hs-mNOS 15 (2) alleles) by transformation of the germline mediated by the element P. A Notl fragment of 4,100 base pairs was cloned from the plasmid CL-BS-mac-NOS, which contained the entire NOS gene of mouse macrophage (Lowenstein et al., Proc. Nati. Acd. Sci. USA, 89: 6711- 6715 (1992)) at the Notl site in the P pP element vector (CaSpeR-hs) (Thummel and Pirrotta, Drosophila Information Service, 71: 150 (1992)), placing it under the control of the hsp70 promoter from Drosophila. The construct was coinjected into embryos (Spradling, P element-mediated transformation, in Drosophila: A practical approach, DB Roberts, ed. (Oxford: IRL Press), 60-73 (1986)) with the P element of help phs-II -? 2-3 (Misra and Rio, Cell, 62: 269-284 (1990)). A group of two independent homozygous transformants was established. The expression of the NOS2 transgene after thermal shock treatment of adult larvae and flies was confirmed by diaphorase staining and by protein and RNA analysis. In control experiments, identical heat treatment regimens of non-transformed flies did not induce any anatomical change per se. Histochemistry and electron microscopy "ADPH-diaphorase" staining was performed as described by Dawson et al., Proc. Nati Acad. Sci. USA, 88: 7797-7801 (1991), and Hope et al., Proc. Nati Acad. Sci. USA, 88: 2811-2814 (1991), with minor modifications. A staining of? ADPH-diaphorase insensitive to fixation reflects activity of several isoforms of NOS in mammals and Drosophila. Imaginary discs were mounted in 80% glycerol and photographed in a Zeiss Axiophot microscope with Nomarski optics. Cobalt sulphide staining of the pupal retinas was performed as described by Wolff and Ready, Dev., 113: 825-839 (1991). The labeling was performed with BrdU to identify the S-phase cells essentially as described by Schubiger and Palka, Dev. Biol. , 123: 145-153 (1987), and Baker and Rubin, Dev. Biol. , 150: 381-396 (1992), with minor modifications. The imaginal discs were removed, washed and incubated in Schneider's medium in a 50 μg / ml solution of BrdU for 30-40 minutes at room temperature. They were fixed in 4% formaldehyde, treated with a 1: 1 mixture of heptane and formaldehyde, washed, washed off by 1 M HCl, blocked by 1% sheep serum and incubated with anti-BrdU antibodies (Beck-ton Dickinson ). After extensive washing, the discs were incubated with secondary anti-mouse antibodies coupled to fluorescein (Boehringer-Mannheim). After washing, the individual imaginal discs were dissected, dehydrated in ethanol and mounted in Vectashield mounting medium (Vector Laboratories). Scanning electron microscopy was performed in the SUNY. Stony Brook Microscopy Center essentially as described by Kimmel et al., Genes Dev., 4: 712-727 (1990). The number of ommatidia was determined by analyzing series of scanning electron micrographs and analyzing heads of adults under blue fluorescent light in a Zeiss Axiophot microscope. Microinjection of larvae For the inhibition of NOS, larvae of the third instar were injected with L-nitroarginine methyl ester (L-NAME), its inactive enantiomer D-nitroarginine methyl ester (D-NAME) (both sigma) and 2-ethyl-2-thiopseudourea (ETU, Calbiochem). The chemical compounds were dissolved in Schneider's solution at concentrations of 0.1 M for L-NAME and D-NAME and 0.01 M for ETU and mixed with Freund's adjuvant (Sigma) in a 1: 3 ratio. . Amounts of 5-10 ni were microinjected at the third instar stage late using a glass needle. The injection times of the NOS inhibitors that gave the greatest efficacy (determined by changes in the phenotype of adults) in test experiments were determined and it was found to be more efficient when performed 5-12 hours before the puparization. This treatment did not affect the appearance of puparization or hatching. Ectopic expression of the NOS For the regulated ectopic expression of the NOS, carrier larvae of the mouse NOS2 cDNA were treated under the control of the heat shock promoter of Drosophila with thermal cho-at 36 ° C for 40 minutes in the first hour after of pupario formation. For the BrdU labeling experiments, third instar larvae were treated with thermal shock 5-8 hours after puparium formation. RESULTS The NOS is expressed in the imaginary discs during larval development. At the end of the third instar, the cells of the imaginal discs undergo a temporary arrest of the cell cycle. The cytostasis is released 12-14 hours after pu-parisation and is established again (permanently) in the late pupae and the faratous adult. The ability of NO to reversibly stop cell division and establish a temporary cessation of growth makes it a fac-tibie candidate to mediate cytostasis in imaginal discs. To investigate this possibility, imaginary discs of the third instar and early pupae were examined for the presence of NOS. The NOS gene of Drosophila (dNOS), which is expressed preferentially in the head of the adult, has been recently cloned and characterized (Regulski and Tully, Proc. Nati, Acad. Sci. USA, 92: 9072-9076 (1995) ). However, different species of AR? M related to NOS are present in the embryo, larvae and adult flies. These mRNAs can be produced by the cloned dNOS gene or by other potential Drosophila NOS genes, making it difficult to detect the relevant RNA species. Therefore, to visualize the expression of NOS in Drosophila during larval development, histochemical staining was used for NADPH-diaphorase (nicotinamide adenine dinucleotide phosphate reduced-diaphorase) activity of the NOS, which reflects the activity distribution total enzyme in a tissue (Dawson et al., Proc. Nati, Acad. Sci. USA, 88: 7797-7801 (1991); Hope et al., Proc. Nati Acad. Sci. USA, 88: 2811-2814 (1991); Muller, Eur. J. Neurosci. , 6: 1362-1370 (1994)). NADPH-diaphorase staining was observed in all the imaginary discs, imaginal rings, histoblasts and brain of the larvae, starting from the third instar. The staining became more intense as the development progressed, and in the larvae of the third instar late and in the early pupae, a highly specific and reproducible pattern of very intense staining was evidenced. In the imaginal disk of the legs, NADPH-diaphorase staining was initially observed very early in the third instar. The staining was confined to the center of the disc, corresponding to the presumed distal end of the leg. As the discs matured, the diaphorase staining intensified and, in the late third instar, practically obliterated the distinction between the individual concentric rings of the epithelial folds normally seen in axial view. At the end of the third instar stage, the staining of the disc center (distal end), which gave the darkest staining at the beginning of the third period, was weaker compared to the surrounding cells. Later in the development, when the disks began their eversion in the prepumps, the diaphorase staining of the paw in formation became less intense and a distinct characteristic pattern of staining of the individual segments was evidenced. At 2-4 hours after the formation of the puparium, an intense NADPH-diaphorase staining was observed in the pre-suntas tibiae, in the first and second tarsal segments and in the proximal part of the fifth tarsal segment of the paw in formation. . The staining was much weaker in the third and fourth segments and areas of intense staining were distributed unevenly across the regions of the suspected femur. There was also a weak staining of the coccyx and the body wall. The progression of staining patterns throughout larval development was highly specific and reproducible. The staining of the imaginal discs corresponding to the first, second and third pairs of legs was very similar. As with the imagined discs of the legs, other imaginal discs, imaginal rings and histoblasts exhibited an increasingly intense NADPH-diaphorase stain as the development of the larvae progressed. The discs of the wings, eyes, halters and genitals in the third instar had distinct and reproducible patterns of intense staining, which gradually decreased in a specific spatial pattern during early pupal development. These results show that there is a gradual and specific accumulation of NOS in the imaginary discs in development, which reaches the highest levels at the moment when the progression through the cell cycle becomes slower. DNA synthesis is affected by manipulations of NOS activity. If NO acts as an antiproliferative agent during the development of Drosophila in stages in which the cells of the imaginal discs enter temporary cytostasis, then their action could directly affect the DNA synthesis in the disks. It would then be expected that inhibition of NOS would release the blockade and increase the number of cells in S phase; on the contrary, high levels of NO would lead to a reduction in the number of dividing cells. To study this hypothesis and to map the degree and distribution of the antiproliferative effect of NO, DNA synthesis was monitored in the larval and pre-pupal discs while manipulating the levels of NOS activity. To inhibit the activity of NOS, specific inhibitors of NOS were injected into developing larvae. To increase NOS levels, the expression of the NOS transgene was induced in transformed larvae carrying the mouse NOS2 cDNA gene (Lowenstein et al., Proc. Nati, Acad. Sci. USA, 89: 6711-6715 (1992 )) under the control of the thermal shock promoter. NOS2 is an independent form of calcium from NOS that is capable of an efficient constitutive production of NO. The imaginal discs were marked with 5-bromodeoxyuridine (BrdU) and the degree and distribution of the labeling of the S-phase nuclei in the imaginary discs of the larval legs were compared after inhibiting the NOS, of N0S2 transformants after induction by thermal shock and untreated control larvae. The data show that there were significantly more BrdU-labeled cells in the imaginary discs of larvae in which the NOS activity was suppressed by the methyl ester of L-nitroarginine (L-NAME) than in untreated control larvae (or larvae treated with the inactive D-NAME isomer). The data also show that there were significantly more cells marked with BrdU in the imaginary discs of flies in which the NOS was uninhibited than in control flies. In contrast, there were markedly fewer BrdU-labeled cells in the imaginary discs of flies transformed by NOS induction than in non-induced controls. At the same time, these changes in the number of cells marked with BrdU after the inhibition or ectopic expression of the NOS appeared to be uniformly distributed throughout the disc. These data indicate that the modulation of NOS activity affects the number of S-phase cells in the imaginal discs, which is consistent with the observations that NO suppresses DNA synthesis and cell division. Inhibition of NOS results in hypertrophy of the leg segments. The highest levels of diaphorase staining occur during the period of development in which the synthesis of DNA and the rate of cell division in most of the cells of the imaginal discs slow down. The strong antiproliferative properties of NO and the specific pattern of diaphorase staining observed in mature imaginal discs implied that NO could act as a growth arrest agent in these structures, capable of inhibiting DNA synthesis and of supporting temporary cytostasis. during the change to metamorphosis. If NO certainly acts as an antiproliferative agent during late stages of larval development, then inhibition of NOS could lead to excessive organ and tissue growth, whereas ectopic overexpression of the NOS gene could have the opposite effect. To test this hypothesis, NOS activity was inhibited by injecting specific NOS inhibitors into the developing larvae at the end of the third instar, several hours before the metamorphosis. The majority of the larvae successfully completed the metamorphosis, giving rise to adult flies within the normal time frame. The resulting adults differed from normal flies in many ways, the most dramatic being increases in the appendages and other body structures of the fly. Changes included a) hypertrophy of the femur, tibia and tarsal segments; b) overgrowth of tissues that originate from the genital disc; in extreme cases, these cells contributed to more than a quarter of the body of the fly; c) an increase in the overall surface area of the wings; d) overgrowth of the tergite and sternite cells; e) hyperthermia of the humerus; f) occasional duplications of some areas of the eye; g) occasional actual formation of genital structures, legs and eyes, and h) occasional ectopic formation of poorly situated body structures. The changes were deeper in the adults and more often than not they affected their legs. The hypertrophy was particularly intense in the third pair of extremities, where the diameter of certain segments increased 3-4 times. The number of sows and the number of rows of sows also increased, confirming that hyperproliferation of the cells had occurred. The segments of the most affected limbs were those (first and second tarsal segments, tibia and femur) whose primordia had the highest levels of NOS in the larval and prepupal stages. The changes mainly affected the anteroposterior and dorsoventral axes, but not the proximodistal, in such a way that the length of the affected segments remained the same. Identical changes were observed when two non-structurally related NOS inhibitors, 2-ethyl-2-thiopseudourea (ETU) and L-NAME (but not D-NAME) were used, indicating that the observed effect occurred specifically as a result of blocking NOS activity. In summary, these data show that the inhibition of NOS in the later stages of larval-rio development leads to an excessive cellular proliferation and to a larger size of the body structures of the adult fly. The ectopic expression of a mouse NOS transgene results in a smaller size of the leg segments. The ability of NO to inhibit the synthesis of DNA and cell proliferation suggest that overexpression of NOS in developing larvae may result in decreased cell proliferation in the imaginal discs and in a reduction in the size of the organs of the adult fly. Transgenic flies expressing the mouse NOS2 transgene under the control of the heat shock promoter were studied. Transgenic larvae were subjected to thermal shock within one hour after puparization to induce ectopic expression of the NOS before the final cell divisions took place. This resulted in a reduction in the size of the fly members. The distal segments of the legs were the most frequently affected and to a greater extent. In extreme cases, the entire tarsus was shortened 1.5-2 times and the third, fourth and fifth segments were fused together with poorly defined boundaries. The number of sows in a row in the affected segments also decreased, although the number of rows did not change. Adult leg segments most frequently affected by NOS overexpression (third, fourth and fifth tarsal segments) were those that were not affected by NOS inhibitors and whose precursors exhibited particularly low levels of staining. of diaphorase in early prepubesional stages. The most terminal structures of the appendix, including the tarsal claw, remained intact in these defective legs. This suggests that the observed reduction in size was due to an incomplete growth of the distal area of the developing appendix, rather than to a complete loss of its distal structures. Contrary to the results on inhibition of NOS, the changes affected only the proximodistal axis, while the diameter of the affected segments remained the same. In addition to the reduction in the size of the paw segments, the changes included a reduction in the global surface of the wings, cuts in the wings and smaller size of termites and sternites. These results support the conclusion that the ectopic expression of NOS in the late stages of larval development leads to a decrease in cell proliferation and a reduction in the size of the structures of the body of the adult fly. Inhibition of apoptosis unmasks excessive proliferation. On imaginal discs, changes in the number of S-phase nuclei after manipulating NOS activity correlated directly with the size changes of adult members. However, in the imaginal disc of the eye, an increase in the number of S-phase cells was consistently detected after inhibition of NOS, but the resulting adult eye usually appeared normal. The possibility that the phenotype of the apparently normal eye was produced as a result of programmed cell death, which counteracts the excessive cell proliferation induced by the inhibition of NOS and restores the normal number of cells in the eye during metamorphosis, was studied. . To suppress programmed cell death, GMR-P35 flies were used (Hay et al., Dev., 120: 2121-2129 (1994), donated by Drs. B. Hay and G. Rubin), in which it is prevented largely apoptosis in the developing eye by expression of the recombinant baculovirus p35 protein. p35 is a potent inhibitor of apoptosis, which acts by inhibiting the converting enzyme-like proteases of inter-leukin IB and is capable of preventing apoptosis in multiple contexts. The GMR-P35 flies express p35 under the transcriptional control of the multimerized glass binding site of the Drosophila Rhl promoter. The promoter of the glass directs the expression of the transgene in all cells and in, and subsequently to, the morphogenetic groove in the eye disc (Ellis et al., Dev., 119: 855-865 (1993)). When NOS was inhibited in GMR-P35 larvae, the eyes of adult flies showed numerous changes, which reflected the excessive proliferation of various cell types in the developing eye. The most dramatic of these changes was the number of ommatidia in the adult eye, which increased from the nearly invariable complement of 750 in the wild-type flies (747 + 4) and in the untreated GMR-P35 flies (748 ± 6) to almost 820 (818 + 21) after inhibition of NOS in GMR-P35 flies. This, together with the high number of cells by omatidium, produced an increase in the overall size of the eye. Other changes in flies expressing p35 after inhibition of NOS, compared to control GMR-P35 flies, included a) more ommatidia with an irregular shape (perhaps due to the non-uniform increase in the number of various cell types) , b) more omati-god with an irregular arrangement of the rows and c) more ommatidia of a smaller size. Another manifestation of inhibition of NO production in GMR-P35 flies was an increase in the number of pigment cells, cones and sows. The wild-type ornaments contain, in addition to eight fo-tower cells, a group of four conic cells and two primary pigment cells, surrounded by an arrangement of six secondary pigment cells, three tertiary pigment cells and three sows (Wolff). and Ready, Pattern formation in the Drosophila retina, in The Development of Drosophila melanogaster, M. Bate and A. Martinez-Arias, eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), 1277-1326 (1993)) . The number of photoreceptor and ac-cesorias cells is normally constant and variations in this arrangement in the eyes of normal flies are very rare. In GMR-P35 flies, the number of secondary and tertiary pigment cells was increased from 12 to 25 (25 ± 4) cells per sample area (defined as described in Hay et al., Cell, 83: 1253-1262 (1995 )) as a result of programmed cell death suppressed. The inhibition of NOS in these flies resulted in a greater increase in the number of secondary and tertiary pigment cells to more than 35 (36 ± 8) per sample area. This number surpasses the maximum number of pigment cells saved from programmed cell death in untreated GMR-P35 flies and suggests that extra pigment cells arise as a result of excessive cell proliferation caused by the inhibition of NOS combined with suppression of cell death caused by p35. The number of ommatidia with extra primary pigment cells in GMR-P35 flies after inhibition of NOS was also increased compared to control flies, although it only slightly exceeded levels in untreated GMR-P35 flies. Moreover, the number of sows was increased in some areas of the eye in GMR-P35 flies after inhibition of NOS, up to 4-5 by omatidium instead of the three observed in normal flies and untreated GMR-P35 flies, and these were often poorly located.

Similarly, the number of cones was increased from four in normal and GMR-P35 untreated to five and six in many ommatidia of GMR-P35 flies after inhibition of NOS. There were also groups of ommatidia that contained one, two or three cones, which may correspond to improperly formed supernumerary ommatidia that did not achieve the appropriate group of cells. Therefore, the prevention of apoptosis by the baculovirus p35 protein in the developing eyes of transgenic flies revealed an excessive proliferation of various cell types after the inhibition of NOS in larvae, which would otherwise be masked by death programmed in larvae and pupae. EXAMPLE 2 Nitric oxide regulates hematopoiesis in erythroid differentiation of animals To study the formation of erythroid line cells in the spleen of irradiated recipient mice, the animals (female of CBAxC57Bl Fl hybrids weighing 22-24 g) were treated with 750 cGy of total body irradiation in 3-4 hours before transplant. It was found that this dosage was sufficient for the complete suppression of hematopoiesis in the irradiated recipient animals. The OM cells were removed by washing the femurs of syngeneic donors and injected intravenously (10 5 MO cells per mouse) into the receptors. The animals received twice daily injections of 100 mg / kg of L-NAME and D-NAME and 10 mg / kg of ETU for 7-10 days. The mice were analyzed 9-10 days after transplantation. The differentiation status of the co-lonias in the spleen was evaluated by morphogenetic criteria and by immunohistochemical tests in relation to the presence of receptors to several cytokines, which are present only in specific stages of erythroid cell maturation. The analysis of the colonies in the spleen of the control animals and of the animals treated with the specific enantiomer D-NAME (Table 1) showed that, according to numerous data, most of the colonies of the baso (> 60% ) contained erythroid colonies, minor fractions containing undifferentiated blasts (14%) or both erythroid cells and blasts (13%) and small colony fractions containing megakaryocytes (7.5%) and granulocytes (4%). In contrast, when animals were treated with NOS inhibitors after MO transplantation, most colonies of the spleen contained undifferentiated blasts (up to 85% of blasts colonies and mixed blasts and erythroid cells). The erythroid colonies constituted only 15% of the total number of colonies and the megakaryocyte and granulocyte colonies were not detectable. The results were similar with two structurally unrelated NOS inhibitors, confirming their specificity of action. Therefore, the prolonged treatment of the recipient mice after the transfer of the OM with a NOS inhibitor reversed the proportion of colonies containing blasts with respect to the colonies containing erythroid cells almost 16 times, effectively preventing differentiation erythroid. Table 1 Formation of hematopoietic colonies in spleens of irradiated mice after injection of NOS inhibitors Myeloid differentiation To study the formation of myeloid lymph node cells, cellulose acetate membranes were implanted in the peritoneal cavity of mice. After 7 days, when a layer of fibroblasts had covered the membranes, the mice were irradiated as described above. MO cells from syngeneic donors (105 MO cells per mouse) were injected into the peritoneal cavity of the receptors. The animals received injections of NOS inhibitors as described above. Membranes were isolated with growing colonies and analyzed 7-8 days later. The differentiation status of spleen colonies was evaluated by morphological criteria, reaction of myeloperoxidase and immunohistochemical tests with regard to the presence of receptors for several cytokines, which are present only in specific stages of the maturation of myeloid cells. The analysis of the colonies on the membranes in the control animals and in the animals treated with the inactive D-NAME enantiomer (Table 2) showed that, according to numerous data, most of the colonies (92%) contained granulocytic colonies. A much smaller fraction contained undifferentiated blasts (6%) and a very small fraction of colonies containing erythroid cells (1.3%). In contrast, when the animals were treated with NOS inhibitors after the MO transplant, most of the colonies on the membranes (up to 85%) contained undifferentiated blasts. Colonies with differentiated cells of the granulocytic lineage constituted only 15.6% of the total number of colonies and a negligible fraction of the colonies (<0.5%) contained erythroid cells. The results were similar with two structurally unrelated NOS inhibitors, confirming the specificity of their action. Therefore, the prolonged treatment of the recipient mice after transfer of OM with a NOS inhibitor reversed the proportion of colonies containing blasts with respect to the granulocytic colonies almost 80-fold, effectively preventing myeloid differentiation. Table 2 Formation of hematopoietic colonies on cellulose acetate in the peritoneal cavity of mice irradiated after injection of NOS inhibitors State of differentiation of transplanted OM cells To study the stage to which the transplanted cells have progressed, colonies in the spleen and on the membranes were studied with antibodies specific for receptors of various growth factors. This analysis allows us to visualize and evaluate the stage of the multistage differentiation process that eventually leads to erythroid or myeloid differentiation. We have used antibodies specific for the interleukin 3 (IL-3-R) receptors, the granulocyte-macrophage colony stimulating factor ("GM-CSF-R"), the granulocyte colony stimulating factor ( "G-CSF-R") and erythropoietin ("EpoR"). The appearance of each of these receptors marks a specific stage in hematopoiesis. The results of the analysis show that blasts from the colonies of the spleen (representing erythroid differentiation) have accumulated mostly in the phase of differentiation in which they have already acquired the receptor for IL-3, but not for erythropoietin , GM-CSF or G-CSF, while colonies with morphological signs of erythroid differentiation had accumulated EpoR. The blasts of the colonies on the membranes (representing myeloid differentiation) have accumulated mostly in the phase of differentiation in which they have already acquired the receptor for IL-3, but not for the erythropo-yetina, the GM -CSF or G-CSF, while positive myeloperoxidase colonies with morphological signs of myeloid differentiation had accumulated GM-CSF-R and G-CSF-R. Bone Marrow Stem Cells To study the maturation of hematopoietic cells in the bone marrow of the irradiated recipient mice, the animals were treated as described above and the OM cells of the syngeneic donor femurs were injected intravenously ( 105 MO cells per mouse) in the receptors. The animals received injections of NOS inhibitors (L-NAME, their inactive enantiomer D-NAME and ETU) as described above and the mice were analyzed at 7-10 days after transplantation. The cells of OM were studied for the presence of various growth factor receptors that serve as markers of the differentiation stage and indicate the presence of stem cells and pluripotent precursor cells. MO preparations were studied for cells that expressed receptors for HSF (c-kit ligand), GM-CSF, G-CSF and IL-3. The results of Table 3 show that the inhibition of NO synthesis in the recipient animals after MO transfer leads to a dramatic increase in the number of cells positive to c-kit and positive to IL-3-R, suggesting that the population of cells in MO is highly enriched in hematopoietic stem cells. At the same time, the number of cells expressing receptors for G-CSF, which marks the last stages of differentiation, decreases almost three times, while the number of cells positive for GM-CSF-R is slightly reduced. This suggests that the inhibition of NOS during hematopoiesis selectively enriches MO in undifferentiated stem cells that have already acquired the c-kit and IL-3 receptors, but have not proceeded to the subsequent phases in which the receiver for the G-CSF. Table 3 Presence of hematopoietic markers in MO cells of mice irradiated after the injection of NOS inhibitors Reversibility of the action of NOS inhibitors in MO cells The critical question is whether the undifferentiated stem cells that accumulate in the bone marrow as a result of treatment with NOS inhibitors have the ability to revert to the normal state and resume the normal process of hematopoiesis once the action of the NOS inhibitors has been suspended. Failing to do so could indicate that the cells are bound in their undifferentiated state, similar to several pathological conditions. To answer this question, the treatment of mice with NOS inhibitors was stopped 7-9 days after the transfer of OM and the cells of the MO were checked for the presence of markers of hematopoiesis 1-7 days after to finish the injections. The control mice continued to receive the daily injections. The results (Table 4) show that once the treatment with NOS inhibitors was suspended, the cells were able to resume their differentiation and proceed normally to the later phases. This indicates that enrichment in stem cells after treatment with NOS inhibitors is reversible and that it can be used to "reinforce" the number of stem cells before inducing them to proceed further along their differentiation pathways.

Table 4 Presence of hematopoietic markers in MO cells of mice irradiated after the injection of NOS inhibitors and subsequent suspension of treatment Inhibition of NOS and apoptosis In order to test whether prolonged treatment with NOS inhibitors affects the rate of programmed cell death in OM cells, the number of apoptotic cells in the preparation of MO cells was examined. The TUNNEL approach was used, thus revealing cells with intensely fragmented DNA, a hallmark of apoptosis, using DAPI staining at the same time to visualize the nuclei of all cells in the preparation. The results indicate that neither prolonged treatment with L-NAME nor with ETU affected the proportion of apoptotic cells (8 ± 3% in control animals versus 7 ± in those treated with L-NAME and 8% ± in those treated with ETU ). Similarly, suspension of inhibitor treatment did not affect programmed cell death in MO preparations (9 ± 4% of TUNNEL-positive cells). This suggests that the manipulation of NOS activity in animals following a BMT (bone marrow transplant), while having a profound effect on the differentiation and maturation of hematopoietic cells, does not affect the degree of programmed cell death in cells of MO, further supporting the viability of applications of NOS inhibitors for tera-pia. EXAMPLE 3 Nitric oxide regulates brain development in vertebrates It has recently been shown that nitric oxide (NO), a second multifunctional messenger, is involved in the differentiation of cells and tissues and in the development of the organism. NO synthase (NOS) controls the transition from cell proliferation to growth arrest and, as a result, regulates the balance between proliferation and cell differentiation in cultured neuronal cells, in developing Drosophila and during hematopoiesis in mammals ( Peunova et al., 1996; Kuzin et al., 1996; Michurina et al., 1977). Here, we studied whether NOS is involved in brain development in vertebrate animals. Xenopus laevis was chosen as the model organism for these studies, focusing research on brain formation. The Xenopus NOS gene was cloned and the distribution of NOS-positive neurons in the developing brain was studied. It was found that the inhibition of NOS dramatically increases the number of cells in the developing brain and that it increases the overall size of the brain. The results suggest that NOS is directly involved in the control of cell proliferation and neuronal differentiation in the brain of developing vertebrates. Cloning of the Xenopus NOS gene Using the information about the known NOS genes, the Xenopus NOS cDNA (XnNOS) was cloned. The analysis of its primary structure suggests that the cloned gene represents the homolog of the neuronal NOS isoform of mammalian Ca2 +. The analysis of the gene reveals a remarkable degree of evolutionary conservation with long stretches of amino acid sequences identical to those of humans, mice, rats and Drosophila. The cloned gene produces enzymatically active protein when transfected into cultured cells. The primary structure of the gene made it possible to obtain a specific antibody and the immunofluorescence analysis indicates that the diaphorase stain of the Xenopus in development correctly represents the distribution of the enzyme XnNOS. This notion is supported by in situ hybridization analysis of XnNOS transcripts in the brain of the tadpole. The cloned gene is now being used to isolate other possible NOS genes from Xenopus. The NOS is expressed in a spatio-temporal pattern consisting of the developing Xenopus brain. The brain of Xenopus suffers histogenesis beginning at stage 39-40; before that, the neural tube consists of rapidly dividing undifferentiated neuroepithelial cells. In the growing brain of the Xenopus tadpole, new cells arise in the narrow zone of the germinal layer in a defined pattern, which can be revealed by labeling with BrdU. The staining distribution of NADPH-diaphorase (which is indicative of NOS expression) in the brain of Xenopus from stage 40 to stage 50 was analyzed. The staining zones first appeared in state 43, the moment of the migration of young neurons from the neural tube and their differentiation. The staining appeared outside the germinal layer and became more intense as the development of the tadpoles progressed. The most intense staining was observed in large differentiated neurons alone in the roof and in the spinal cord and in the marginal zone of the ceiling composed of processes of differentiated neurons. The diaphorase staining gradient was lateromedial and reciprocal to the proliferation pattern, suggesting that the zones of active proliferation in the germ layer remained free of NOS activity through these stages. The inhibition of NOS in the developing brain gave rise to an excessive proliferation of young neurons. To verify whether NOS is involved in the growth of neuronal precursors in the developing Xenopus brain, NO production was blocked by inserting plastic pieces impregnated with inhibitors of NOS, L-NAME and ETU, in the ventricle of the brain of the tadpole in stage 43. After 3, 7 and 12 days, the animals were examined for changes in the patterns of cell division, differentiation, survival and morphology of the brain. Labeling with BrdU demonstrated a dramatic increase in the number of cells in the S phase of the cell cycle in brains treated with inhibitors, compared with the control brains. The number of BrdU positive cells in the ceiling increased consistently throughout the experiment. Staining of cell nuclei with DAPI revealed a higher number of cells in sections of the brain at each time interval of the experiment, indicating that excessive cells in the S phase successfully completed the cell cycle by mitosis. Inhibition of NOS and programmed cell death We studied whether the inhibition of NOS and the excessive proliferation of cells in the developing brain affects the programmed cell death of neurons in the ceiling. Using the TUNNEL technique to visualize the apoptotic cells in the brain, it was found that, on day 3, the number of cells positive to TUNNEL was the same in the control ceiling and in the ceiling treated with inhibitor. However, after 7 and 12 days, there were more apoptotic cells in the brains of animals that received inhibitors of NOS than in those of the control animals. The increase in the number of cells positive to TUNNEL is not due to the toxicity of the inhibitors, since the cells continued to incorporate BrdU very effectively. Identical changes were observed with two inhibitors not structurally related to NOS, indicating that the effects occurred specifically as a result of blocking NOS activity. These data suggest that the ex-cessation of cell proliferation in the roof leads to the activation of programmed cell death, which acts to eliminate surplus neurons. Alternatively, this may indicate that differentiated neurons become dependent on NO to survive, similar to the situation in totally differentiated PC12 cells (Peunova et al., 1996). Neuronal differentiation in the brain is affected by the inhibition of NOS To see if the excessive cell proliferation induced by NOS inhibitors affects the distribution and differentiation of neurons in the brain of Xenopus, antibodies were used for specific neuronal markers that they have a specific and highly reproducible pattern of expression during the development of Xenopus. It was seen that the distribution of neurons positive for Islet-1, N-tubulin and N-CAM changed after the inhibition of NOS. Specifically, the neurons moved to the marginal zone, the neurons of the intermediate layer were more heterogeneous and with shorter branches than in the control brains and the structure of different layers of the ceiling was altered. In addition, the number of motor neurons positive to Islet-1 was increased after the inhibition of NOS. The inhibition of NOS leads to an ectopic proliferation of neuronal precursors. The brain of Xenopus has a fi-na architecture. Groups of neighboring cells share the place and time of birth and are involved in common local circuits. The position of young and mature neurons in the brain is strictly dependent on the place of birth, migration and final differentiation and a characteristic pattern component. In the brains of animals treated with NOS inhibitors, it was seen, in addition to extra layers of young neuronal precursors in division, numerous ectopic sites of neuronal proliferation. Large groups of cells were observed in atypical location, occupying the marginal zone, various areas of the roof, the telencephalon and the posterior brain. Inhibition of NOS increases overall brain size Inhibition of NOS activity in the brains of developing tadpoles resulted in a greater number of cells in S phase, accompanied by a modest increase in programmed cell death in stadiums late Together, this increased the total number of cells in the brain and, consequently, increased the overall size of the brain. The most affected areas are the optical roof and the area immediately adjacent to the ventricle where the impregnated plastic part was inserted. In cases where the source of the NOS inhibitor was changed in the ventricle to regions of the telencephalon or posterior brain in the developing brain, an increase in the size of the anterior and posterior parts of the brain was observed, respectively. Taken together, these results show that NO controls the number of neurons in the developing brain and the inhibition of NOS directly affects the brain size of Xenopus. This confirms the role of NO as a general regulator of cellular and tissue differentiation in the body. This suggests that manipulations at NO levels can be used for therapeutic purposes to control the proliferation and subsequent differentiation of nerve cells in substitution therapy after neurodegenerative disorders caused by aging (eg, Alzheimer's disease, Parkinson's or Huntington), stroke or trauma. EXAMPLE 4 Nitric Oxide and Enrichment of Hematopoietic Stem Cells Materials and Methods Female mice 8-12 weeks of age were used and these were of the following strains: C57B1 / 6, B6 CBAF1 / J, CBAB6F1 / J and DBA (purchased from Jackson Laboratories or Taconic Farms). All mice were bred and maintained in the CSHL Animal Care facility, with standard feed diet and acidified water ad libitum. Irradiation and bone marrow transplantation The recipient mice were exposed to 8.2-9.5 Gy of total body gamma irradiation using an Marc I irradiator from a source of Cesium-137 (Atomic Energy of Cana-da, Ottawa) , at a dose rate of 1.06 Gy / min 3-20 hours before bone marrow transplantation. The dose of irradiation is sufficient to suppress hematopoiesis in recipient mice. The action of NO on hematopoiesis was studied by MO transfer after total body irradiation. The donor mice were sacrificed by asphyxia with C02 or cervical dislocation and the femurs and tibias were isolated. Bone marrow cells were removed from the femurs and tibias by repeatedly washing the bones with Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL). Suspensions of a single cell were prepared by passing the bone marrow through a 21 gauge needle, followed by a 26 gauge needle and through a 70 mkm nylon cell filter. The cells were counted using a hematocytometer. 3-5xl04 nuclear bone marrow cells were injected into the tail vein, or IxlO6 was injected intraperitoneally. Spleens or testicles were cut into pieces and then passed through a 21 gauge needle and a 70 mkm nylon cell filter to obtain a single cell suspension. Spleen colony assays The spleen colony assay of Till and McCulloch (Till, J.E. and McCulloch, E.A., Radiat.Res., 14: 213 (1961)) was applied. 3xl04 bone marrow cells were injected into lethally irradiated mice (8.5-9.5 Gy total body irradiation from a source of Cesium 137 at a dose of 1.06 Gy / min). Spleens were removed on days 8 or 12 after transplantation, fixed in Carnua's solution (96% ethanol: chloroform: acetic acid, 6: 3: 1) or Bouin's (Sigma) and the colonies of the spleen were macroscopically visible. . A secondary transfer of bone marrow or spleen cell suspensions was studied 12 days after the primary transplant. The number of spleen colonies of day 8 and day 12 in secondary animals was counted. Administration of NOS inhibitors N-omega-nitro-L-arginine (L-NAME) (Sig-ma), N-omega-nitro-D-arginine (D-NAME) (Sigma) and hydrobromide 2- were used ethyl-2-thiopseudourea (Calbiochem) (ETU). To suppress the NOS activity in the recipient animals, they were injected intravenously or intraperitoneally with 0.3 ml of a mixture of two NOS inhibitors, L-nitromethyl ester (L-AME) at 300 mg / kg body weight and 2-ethyl-2-thiopseudourea (ETU) at 30 mg / kg body weight, immediately after bone marrow transplantation. These injections were repeated twice a day for 3-17 days. In different groups of experimental animals, the treatment was suspended after 3, 5, 7 or 9 days. The animals of the control group received injections of 0.3 ml of saline. FACS To prepare the cells for FACS, the mice were killed by cervical dislocation and the cells were washed from the bone marrow of both femurs and tibias using a 2 ml syringe with a 21 gauge needle, followed by a 26 gauge needle. The spleens and testicles were cut into pieces and then passed through a 21 gauge needle and a 70 mkm nylon cell filter to obtain a single cell suspension. The cells of the bone marrow, the spleen or the testicle were counted using a haemocytoma. After washing in MEM (Minimum Essential Medium, Gibco BRL) supplemented with a solution of 3% fetal bovine serum, the hematopoietic cells (bone marrow or spleen cells) were resuspended in PBS (phosphate buffered saline) containing 3% fetal bovine serum. The erythrocytes were lysed with buffer of ammonium chloride-potassium bicarbonate (154 mM ammonium dichloride, 10 mM potassium bicarbonate, 0.082 mM EDTA) for 5 minutes at room temperature. After washing, the cell suspensions were filtered through a nylon cell filter of 70 mkm pore size and counted using a hematocytometer. 3-5xl06 hematopoietic nucleated cells were incubated in 50 μl of PBS supplemented with 3% fetal bovine serum with 50 μl of antibodies for 20-40 minutes at 4 ° C in the dark. The cells were washed twice with PBS and fixed with 300-500 μl of 2% formaldehyde in PBS. For the two-step procedure, the hematopoietic cells were incubated, after washing with PBS, with second antibodies 20-30 minutes in the dark and then washed twice with PBS and fixed with 300-500 μl of formaldehyde. 2% in PBS. The negative controls were unstained cells or cells stained with only second antibodies. All the cells were kept on ice throughout the entire procedure. The fixed cells were kept in the refrigerator at 4 ° C until the flow cytometry analysis. The control and stained samples were analyzed using an EPICS Elite cell sorter (Coulter, Hialeah, FL).

Antibodies The antibodies used in the immunofluorescent staining included E13-161.7 (anti-SCA-1 [Ly-6A / E]), conjugated with phycoerythrin ("PE") (PharMingen); 2B8 (anti-c-kit), played with FITC (PharMingen); V-18 (anti-IL-3R alpha) (Santa Cruz Biotechnology, Inc.); M-20 (anti-EpoR) (Santa Cruz Biotechnology, Inc.); M-20 (anti-G-CSFR) (Santa Cruz Biotechno-1 ° -? Y / Inc.); Fluorescein-conjugated anti-rabbit IgG (FITC). anti-nNOS mAb, anti-macNOS mAb, anti-eNOS mAb and polyclonal anti-nNOS polyclonal antibodies were purchased from Transduction Laboratories. Anti-nNOS polyclonal antibodies were also purchased from Zymed. Mapping with BrdU To identify cells in S phase, mice were injected intraperitoneally with 50 μg / ml 5-bromodeoxyuridine (BrdU) (Beckton-Dickinson) once a day for 5 days. Extensions of bone marrow cells were prepared and fixed with 4% formaldehyde. S-labeled nuclei were visualized with BrdU after denaturation of the DNA in 2 M HCl, triton 0.5%, for 2 hours and incubation with fluorescein conjugated antibodies for 5-BrdU (Beckton Dickinson) according to the suggestions manufacturer. The samples were analyzed in a Zeiss Axiphot fluorescent microscope. For the visualization of the nuclei, the extensions were stained with DAPI, a fluorescent stain for DNA (Molecular Probes), at 1 μM. TUNNEL The analysis of apoptosis was carried out in extensions of bone marrow cells fixed for 15 minutes with 4% formaldehyde in PBS by means of the TUNNEL test (Boehringer Mannheim) according to the manufacturer's suggestions. NADPH diaphorase Staining of? ADPH-diaphorase was carried out essentially as described (Dawson, TM et al., Proc. Nati, Acad. Sci. USA, 88: 7797 (1991), and Hope, BT et al. , Proc. Nati, Acad. Sci. USA, 88: 2811 (1991)) with minor modifications. The cells were fixed in 3.7% paraformaldehyde for 1 hour, washed in PBS and incubated for 60 min at 37 ° C in the staining solution, containing 1 mM NADPH, 0.025% nitroblue tetrazolium salt and 0, 3% Triton. Peripheral blood was analyzed using standard methods. The leukocytes were counted in the hematocytometer and in peripheral blood smears fixed with methanol and stained with Giemsa. RESULTS Inhibition of NOS activity in experimental animals In order to increase the number of stem cells and early progenitor cells in the bone marrow, the following protocol was used: a) a mixture of two inhibitors of NOS, L was introduced -NAME (concentration, 300 mg per kg of body weight) and ETU (concentration, 30 mg per kg of body weight) intraperitoneally twice a day; b) treatment was stopped after 3, 4, 5, 7 or 9 days and the presence of specific markers in the bone marrow was analyzed by FACS analysis 1, 2, 3, 5, 7, etc. days after cessation of treatment. This protocol dramatically increased the proportion of early progenitor cells in the bone marrow. At first, the increase is minimal or actually reversed compared to control animals. However, several days after stopping the treatment, the proportion of progenitor cells (positive to c-kit) became much greater than in the control animals that received the saline solution.

The content of c-kit positive cells in the bone marrow was increased from 5.1% to 23.9%. The content of cells positive for IL receptor was increased from 4.3% to 25%. The content of Sea-positive cells in the bone marrow was increased from 1.7% to 5.1%. The content of Sea-positive cells and c-kit (Sca + c-kit + cells) in the bone marrow was increased from 0.4% to 1.48%. The highest increase for c-kit in the bone marrow occurred on day 1 after the cessation of treatment with NOS inhibitors, which had been carried out for 9 days. The highest increase for IL3-R in the bone marrow occurred on days 2-3 after cessation of treatment with NOS inhibitors, which had been running for 9 days. The greatest increase for Sea in the bone marrow occurred on days 1-2 after cessation of treatment with NOS inhibitors, which also had been underway for 9 days. Similar changes were observed in the spleens of the treated animals. The largest increase for c-kit in the spleen occurred on days 3-4 after cessation of treatment with NOS inhibitors, which had been underway for 9 days. The greatest increase for IL3-R in the spleen occurred on days 3-5 after cessation of treatment with NOS inhibitors, which had been underway for 9 days. The greatest increase for Sea in the spleen occurred 1-3 days after cessation of treatment with NOS inhibitors, which had been underway for 9 days. Therefore, changes in the content of early progenitor markers in the spleen followed the kinetics of maturation of hematopoietic precursor cells in the bone marrow. Taken together, these results demonstrate that a new protocol for the inhibition of NOS is especially effective for the enrichment of bone marrow and spleen in early hematopoietic progenitors.

NOS inhibitors and changes in peripheral blood The composition of peripheral blood in animals was followed after treatment with NOS inhibitors. It was seen that the neutrophil content increased dramatically and, 5 days after finishing the injection of the NOS inhibitors, it increased 4.5 times in comparison with the control animals, while 7 days after finalizing the content. of neutrophils was increased 7.4 times compared to the control. This showed that the progenitor cells whose content in the bone marrow was increased by treatment with the mixture of two NOS inhibitors, proceed successfully through hematopoietic differentiation and enter the peripheral blood as mature granulocytes. Importantly, no immature precursors were observed in the peripheral blood, suggesting that treatment with NOS inhibitors does not induce neoplastic transformation in the bone marrow.

Inhibition of NOS and DNA synthesis To assess the effect of inhibition of NOS on DNA synthesis in the bone marrow, the incorporation of BrdU in the nuclei of bone marrow cells was studied. For this, mice were treated for 7 days with a mixture of two inhibitors of NOS, L-NAME and ETU, as described above, except for the fact that the animals did not receive gamma irradiation and bone marrow transplantation. During the last 5 days of treatment, the animals received daily injections of BrdU at 50 mg / kg intraperitoneally. The bone marrow was isolated and extensions prepared, fixed using 4% formaldehyde and processed using anti-BrdU antibody as described in Materials and Methods. The proportion of cells marked with BrdU was significantly higher (up to 10 times) compared to the control bone marrow. This indicates that treatment with NOS inhibitors has a direct effect on the synthesis of DNA in the hematopoietic cells of the bone marrow. Inhibition of NOS and apoptosis To study whether prolonged treatment with NOS inhibitors affects the rate of programmed cell death in bone marrow cells, the number of apoptotic cells in the preparation of bone marrow cells was studied. The TUNNEL approach was used, thereby revealing cells with intensely fragmented DNA, a characteristic feature of apoptosis, using DAPI staining at the same time to visualize the nuclei of all cells in the preparation. The results indicated that prolonged treatment with the mixture of two inhibitors, L-NAME and ETU, does not affect the proportion of apoptotic cells in the bone marrow of the experimental animals compared to the control group. Similarly, suspension of inhibitor treatment did not affect programmed cell death in bone marrow preparations. This indicates that the manipulation-of NOS activity in animals after BMT, even having profound effects on the differentiation and maturation of hematopoietic cells, does not affect the degree of programmed cell death in MO cells, further supporting the feasibility of the application of NOS inhibitors for therapy. Retransplant of hematopoietic cells from bone marrow and spleen to secondary receptors As described here, the treatment of animals with NOS inhibitors after bone marrow transfer dramatically increased the number of cells expressing markers of stem cells and progenitor cells. This indicates that, as a result of treatment with NOS inhibitors, the bone marrow is enriched in early stem and progenitor cells. However, it was possible that this procedure only affected the expression levels of the markers, or that it increased the proportion of the immediate progenitor cells, but not of the pluripotent hematopoietic stem cells. To show directly that the inhibition of NOS activity leads to an increase in the number of stem cells, the proportion of colony-forming units in the bone marrow and in the spleen of experimental animals was studied by transferring bone marrow or spleen cells to secondary receptors. To obtain experimental animals, mice were irradiated at a dose of 8.2-9.0 Gy and injected intravenously with 3 -5x1O4 or intraperitoneally with lxlO6 bone marrow cells from syngeneic donors. Immediately after the bone marrow transplant, the experimental mice received intravenous or intraperitoneal injections of a mixture of two NOS inhibitors: L-NAME and ETU. Injections of the NOS inhibitors were repeated twice a day for 9 days. The mice of the control group were injected with saline. After 9 days, the injections were stopped. The experimental mice and controls were sacrificed 1 or 3 days after finishing the injections of NOS inhibitors. To evaluate the content in pluripotent stem cells ("CFU") and in pre-CFU, 3xl04 bone marrow cells or IxlO6 spleen cells from experimental mice or controls were transferred to secondary irradiated recipient mice intravenously. In addition, aliquots of bone marrow or spleen cells from the experimental animals were studied by FACS for the presence of c-kit or Sca-1 molecules or both on the cell surface. After 8 and 12 days, the secondary receptors were sacrificed and the hematopoietic colonies were counted in their spleen. The number of spleen colonies on day 12 was 3.5 times higher in the mice that received cells from the bone marrow of the experimental animals (primary receptors, treated with a mixture of two NOS inhibitors for 9 days and left untreated during 1 day) (3.5 ± 0.22) than in those who received them from the primary control recipients who received saline (1.0 ± 0.15). In contrast, the number of spleen colonies on day 8 was 2.9 times lower in the secondary recipients that received cells from the bone marrow of the experimental mice (1.50 ± 0.23) than in the secondary recipients who received Bone marrow cells from the control mice (4.38 + 0.76). This indicates that, under these experimental conditions, the number of more primitive CFU-12 in the bone marrow of the primary receptors is increased, while the number of CFU-8 more intended is decreased. The increase after 12 days corresponded to the increase in the number of c-kit positive cells in the bone marrow of the primary receptors, as determined by FACS analysis. When similar experiments were carried out using only one of the NOS inhibitors, either ETU or L-NAME, injected for 8-12 days, the number of colonies on day 8 and day 12 in the spleen of the secondary receptors it was increased 1.7-2.5 times, which indicates that treatment with a mixture of two NOS inhibitors a) is more effective than the use of any of the inhibitors alone and b) results in a specific enrichment of the population of bone marrow with more primitive stem cells (CFU-12). When experiments were performed with spleen cells transplanted from the primary to the secondary receptors, the number of spleen colonies on day 12 was 1.5 times higher in the secondary recipients that received spleen cells from the experimental animals (primary recipients, treated with a mixture of two NOS inhibitors for 9 days and left untreated for 3 days) compared to secondary receptors that received spleen cells from control group mice injected with saline. Taken together, these experiments directly demonstrate that the inhibition of NOS in the bone marrow from primary receptors resulted in an increase in the proportion of pluripotent hematopoietic stem (CFU) cells. These results indicate that exposure to NOS inhibitors is used therapeutically to increase the proportion of stem cells in the bone marrow. Even more, these experiments indicate that different inhibitors of NOS activity and their combinations can be used for enrichment with hematopoietic and progenitor stem cells. What is more, they suggest that, in addition to bone marrow, a similar approach can be applied to early blood stem cells and progenitors, such as umbilical cord vein blood or peripheral blood and other blood loss. body to increase its content. EXAMPLE 5 Neurogenesis in Xenopus To investigate the effect of exogenous NO delivery to the brain of the developing tadpole, pieces of the Elvax slow-release matrix impregnated with S-nitroso-α-acetylpenicillamine (S? AP, a widely used ? Or releasing? Or after hydrolysis) or with saline as control in the brain ventricle of stage 45 tadpoles. At 1 or 3 days after implantation, the following parameters were analyzed in the optical roof: the total number of cells (measured by the number of nuclei stained with DAPI), the number of proliferating cells in S phase (measured by BrdU incorporation), the number of apoptotic cells (measured by the TUNNEL assay), the relative numbers of BrdU-positive cells and TUNNEL by 103 total cells, the size of the optical roof and the cell density in the ceiling (Table 5). The data show that the exogenously supplied? O drastically reduces the number of proliferating cells on day 1 to only 5% of the control value (Table 5). After 3 days, the effects of NO on cell proliferation are less pronounced, which probably reflects the complete hydrolysis of the NO donor and the cessation of NO release. Both the cell density and the total number of cells were markedly reduced at 1 and 3 days. An increase in programmed cell death in response to NO probably contributes to the decrease in the total number of cells in the brain. However, the TU-NEL data show that both absolute and relative numbers of apoptotic cells were reduced in the presence of S? AP, indicating that programmed cell death can not be invoked to explain the effects of S? AP about the change in the total cell number. The close proximity of the proliferating cells and cells expressing NOS in the optical roof and the data from previous NO donor experiments are consistent with the proposed antiproliferative role of NO during the development of the Xenopus brain. To study the causative role of NO in cell cycle arrest of neuronal precursors and to determine whether NO production is necessary for cell cycle arrest and subsequent differentiation of neuronal precursors in the developing Xenopus brain , NOS activity in the brain was inhibited using Elvax matrix impregnated with a NOS inhibitor, either L-NAME or ETU, or with saline as control. Pieces of impregnated matrix were inserted into the cerebral ventricle of stage 45 tadpoles. After 3 days of treatment, the brains were examined for changes in cell division and differentiation patterns, number of cells and overall size and morphology. Both NOS inhibitors significantly increased the numbers of S-phase cells compared to the untreated or saline-treated control animals at each of the time points studied (Table 6). The excess BrdU-positive cells were numerous in the proliferative zone of the optical roof. They were also present in more lateral and rostral regions of the roof, which are normally occupied by different post-mitotic neurons. To assess whether this excess of S-phase cells in the animals treated with NOS inhibitors increased the total number of cells in the brain, sections were stained with DAPI to reveal the cell nuclei. In control brains, the cells of the optical roof occupy a crescent that extends along the medial and caudal borders of the roof lobe. Only a few scattered cell bodies are located in the region of the neuropil, where incoming axons are mixed with the dendrites of the roof cells. The brains of animals treated with NOS inhibitors have significantly more cells stained with DAPI than control animals not treated or treated with saline (Table 6), indicating that excess S-phase cells successfully completed the cycle cellular by division. The extra cells distorted the lamination pattern in the optic roof, formed ectopic islands and filled the neuropil of the roof. A reduction in programmed cell death probably contributes to the increase in cell numbers after treatment with NOS inhibitors. Thus, the number of apoptotic cells was studied with the TUNNEL test to verify the effect of the inhibition of NOS on programmed cell death in the brain of the developing tadpole. While the number of cells positive for BrdU and the total number of nuclei stained with DAPI increased significantly after a 3-day exposure to L-NAME, there was no corresponding change in the absolute number or even in the relative or in the distribution of the apoptotic cells positive to TUNNEL (Table 6). This shows that the excessive proliferation of neuronal precursors due to the inhibition of NOS was not accompanied by an immediate and significant change in cell death, indicating that programmed cell death did not contribute significantly to the increase in total cell number , which was the most important consequence of inhibiting NOS. To determine if the greater number of cells affects the size of the brain of the tadpole, the changes in the volume of the dorsal mid-brain and in the tectal cell density after the inhibition of NOS were determined (Table 6). The cell density in the optical roof after 3 days of treatment showed that there was no significant difference between the treated animals and the controls; however, after 7 days of treatment, the cells were more densely packed. A significant increase in mean brain volume was seen in animals treated with the drug compared to controls treated with saline using both inhibitors of NOS, ETU and L-NAME (29% increase over control on day 3 and an 8% increase on day 7 for L-NAME and a 23% increase on day 7 for ETU, see Table 6). To determine if the cells generated in the experiments that used inhibitors of NOS were able to undergo neuronal differentiation and to express neuronal markers, the distribution of the tectal neurons expressing the panneuronal markers β-II tu-buline and N-CAM was determined . In both the control and L-NAME-treated brains, β-II tubulin and N-CAM immunoreactivity was concentrated in the tectal neuropil; however, both antibodies also mark differentiated cell bodies. Importantly, the staining pattern also suggests that the leftover cells in brains treated with L-ÑAME differentiate and express neuronal antigens. Moreover, antibody staining revealed a large distortion in the overall organization of the optical roof. The region of cell bodies of the treated brains was thicker, while the highly disorganized neuropil was looser and chaotically packed with the neurites and the boundary between the neuropil and the region of the cell bodies was markedly irregular. The antibodies for Islet-1 stain a subgroup of ventral motor neurons in chicken, which are destined very early during the development of the brain of vertebrates. The inhibition of NOS did not affect the number and pattern of distribution of the cells expressing Islet-1, although the increase in brain size due to exposure to NOS inhibitors was apparent. This is consistent with the early differentiation of these neurons, which occurred before applying the inhibitors. These data indicate that the inhibition of NOS can influence the development of cells in the brain within a specific window of their development, when they leave the cell cycle and begin to differentiate. Taken together, the results indicate that NO is an essential negative regulator of the proliferation of neuronal precursors during vertebrate brain development. Methods Animals, NOS inhibitors and NO donors. Albino tadpoles of Xenopus laevis were obtained by couplings induced by human chorionic gonadotropin and reared under standard conditions. In stage 455, the animals were anesthetized in 0.02% 3-aminobenzoic acid (MS-222, Sigma) and a small piece (100 μm2 x 30 μm) of slow-release plastic polymer Elvax (Dupont) was inserted. in the tectal ventricle through an incision made in the skin that covered it with a 30-gauge needle. Elvax was prepared with stock concentrations of the NOS inhibitors, 2-ethyl-2-thiopseudourea (ETU, Sigma) or methyl ester of L-nitroarginine (L-NAME, Sigma) or the donor of NO S-nitrosoacetylpenicillamine (SNAP, Sigma), prepared as a 1:10 ratio of chemical compound to polymer matrix. Low molecular weight molecules, including L-NAME, are released at a constant rate over a period of up to 30 days, as the lyophilized Elvax matrix slowly hydrates. Preliminary experiments with a ran-go of stock concentrations of inhibitor (10 mM, 100 mM and 1M for L-NAME and 1 mM, 10 mM and 100 mM for ETU) indicated that L-? AME 1 M and ETU 10 mM increased the incorporation of BrdU without any sign of toxicity for the animals. Similarly, S? AP was studied in a range of concentrations and was found to be effective at 300 mM. Control animals were treated with Elvax impregnated in saline (0.1 M phosphate buffer). Histochemistry and antibodies. Immunocytochemistry was performed with fully assembled preparations and sections of the tadpole as described. Monoclonal antibodies were obtained for β-II tubulin,? -CAM (developed by U. Ru-tishauser) and Islet-1 (developed by TM Jessell) of the Developmental Studies Hybridoma Bank, developed under the auspices of? ICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Fluorescein-conjugated anti-mouse antibody (Boehringer Mannheim) was used as a secondary antibody. The specimens were visualized and photographed under fluorescence or optic? Omarski in a Zeiss Axiophot. For the visualization of the nuclei, brain sections were stained with DAPI, a staining for AD? fluorescent (Molecular Probes), at 1 μM. To identify S-phase cells, 50 μl / ml of 5-bromodeoxyuridine (BrdU) (Beck-ton-Dickinson) was injected into the tadpoles. After two hours of survival, the animals were fixed with 3.7% paraformaldehyde for 2 hours and then in 70% ethanol overnight. S-labeled nuclei were visualized with BrdU after denaturing the DNA in 2 N HCl, 0.5% Triton, for 2 h and incubated with fluorescein-conjugated antibodies for BrdU (Beckton Dickinson) according to the manufacturer's suggestions. The analysis of apoptosis during tadpole brain development was performed on 20 μm brain sections by means of a TUNNEL assay (Boehringer Mannheim) according to the manufacturer's suggestions. The samples were analyzed in a Zeiss Axiophot fluorescent microscope and a Noran confocal microscope. Three sequential corresponding dorsal 20 μm sections, representing most of the optical roof, of each animal were collected and used to determine the volume, the number of cells (after DAPI staining), the number of BrdU-positive cells and the number of cells positive to TUNNEL of the sections. Each brain sample (3 sections) represented approximately 1/5 of the complete middle brain. At least 5 animals were analyzed for each experiment.

Table 5. The NO SNAP donor reduces the volume, cell density and total number of cells in the brain of the tadpole. 15 Elvax pieces impregnated with the NO SNAP donor or saline solution were implanted in the brains control. Each group contained at least 5 animals. Changes in cell counts and brain volume were determined from 3 sequential 20 μm sections through the midbrain (see methods for details). The differences in the means of cell number, brain volume and cell density were analyzed by ANOVA. After 3 days of treatment with SNAP, the differences between the means became significant for the number of cells (p = 0.003, F = 43.3), for the volume of the brain (p = 0.027, F = ll, 7) and for cell density (p = 0.009 and F = 21.9). * p 0.05, ** p < 0.01.

Table 6. NOS inhibitors increase the volume, cell density and total number of cells in the brain of the tadpole. 15 They were implanted in Elvax brains impregnated with the NOS L-NAME and ETU inhibitors or sa- Line as a control. Each group contained at least 5 animals. Changes in cell counts and brain volume were determined from 3 sequential 20 μm sections through the midbrain (see methods for details). The differences in the means of cell number, brain volume and cell density were analyzed by ANOVA. The differences in the number of cells were significant after 3 days of treatment with L-NAME (p = 0.016, F = 15.9) and 7 days of treatment with L-NAME (p = 0.007, F = 25.7 ) or ETU (p = 0.018, F = 15, l), differences in brain volume were significant after 7 days of treatment with ETU (p = 0.024, F = 12.4) and differences in cell density - after 7 days of treatment with L-NAME (p = 0.015 and F = 16.9) or ETU (p = 0.027, F = ll, 5). * p 0.05, ** p < 0.01.

EXAMPLE 6: Neurogenesis in the adult rat. Several NOS issformas are expressed in the developing nervous system of mammals (Bredt, D. and Snyder, S.H., Neuron, 13: 301-313 (1994)). Moreover, in the brain of adult mammals, NOS is present in cells that carry the characteristics of neural stem cells (for example, they can form neurospheres, a typical feature of the plutro-ripotent neural stem cell) (Wang et al. ., Cell Tissue Res. 296, 489-497 (1999)). Thus, the distribution of NOS in the developing and adult brain is consistent with the anti-proliferative role of NO during neurogenesis in mammals. To study the role of NO as a negative regulator of cell proliferation in the mammalian brain, NOS activity in the rat brain was inhibited. The solution of NOS inhibitors was introduced into the lateral ventricles of the adult rat brain using osmotic microbombs. A 50 mM solution of L-NAME, D-NAME or saline was injected as a control over a 7-day time course. After 4 days of treatment, the rats were injected with a solution of 60 μg / g 8-bromodeoxyuridine (BrdU) 7 times at 12 hour intervals. Animals were sacrificed and brain sections were analyzed for the number of BrdU-positive cells using anti-BrdU antibodies (Beckton-Dickinson). The treatment with inhibitors of NOS increased the number of positive BrdU cells in the subventricular zone, in the rostral migratory current and in the hippocampus by 52.8%, 39.3% and 12%, respectively. An increase in the number of BrdU positive cells was also observed in the cortex, in the corpus callosum and in other areas of the brain. Taken together, these data indicate that NO acts as a negative regulator of cell proliferation in the brain of the adult rat and that it is possible to increase the degree of neurogenesis in the adult brain by blocking the production of NO. EXAMPLE 7: Mouse hematopoiesis. Expression of NOS in the bone marrow. Various forms of NO synthase occur in most types of drugs in humans and rodents. To examine the presence of protein isoforms or NOS mRNA in the bone marrow of the mouse, preparations of bone marrow isolated by Western blot and RT-PCR (reverse transcriptase-polymerase chain reaction) were analyzed. The NOS protein isoforms were not detected by probing the total bone marrow lysates with antibodies to neuronal, endothelial or inducible forms of NOS, probably due to their low content in the total bone marrow preparations. However, mRNA of each form of NOS was detected when using RT-PCR. The RT-PCR signals were reliable and specific for each of the NOS genes studied, since they were reproducibly obtained with various combinations of primers for each gene, they were produced by DNA fragments of the expected nucleotide sequence and generated DNA fragments of the expected size after digestion with restriction endonucleases. These results indicate that the three genes coding for the NOS isoforms are all expressed in the bone marrow. In vitro culture of murine bone marrow cells. To study the role of NO signaling in the hematopoietic maturation of bone marrow cells in vitro, lxlO7 of bone marrow cells from C57B1 / 6 mice were added to flasks with a preliminarily formed estro-adhesive adhesive layer. The feeder layer was prepared from the bone marrow of syngeneic mice plated 3 weeks before the experiment (1 femur per 50 ml flask). The NOS inhibitors were added and the appearance of the hematopoietic markers was studied at weekly intervals. After 2 weeks of treatment with NOS inhibitors, the number of c-kit positive cells was 2.7 times higher in cultures treated with a mixture of 0.5 mM ETU and 5 mM L-NAME and 1, 6 times higher in cultures treated with 1 mM L-NAME compared to control cultures. Taken together, this indicates that the inhibition of NOS activity can be used to increase the number of hematopoietic stem cells and early progenitor cells in vitro. In vitro culture of human umbilical cord blood cells. To study the role of NO signaling in the maturation of human hematopoietic cells, human umbilical cord blood cells ("HUCB") were used. When HUCB cells were grown in the presence of NOS inhibitors, the content of CD34-positive cells increased 1.8 fold after growing for 1 or 2 weeks in the presence of 1 mM L-NAME and 2.6 fold in presence of 0.5 mM ETU and 5 mM L-NAME. When the HUCB cells were allowed to grow for 3 weeks before adding the NOS inhibitors, the increase in the content of CD34 positive cells was even higher, reaching 4.7 times for 1 mM L-NAME after 2 weeks of treatment. When similar experiments were performed with adhesive stromal feeder layer prepared from the bone marrow of NOD / SCID mice, the increase in the content of CD34-positive cells was 1.7 and 1.4 times after 1 and 2 weeks, respectively. Taken together, these experiments indicate that the inhibition of NOS activity can be used to increase the number of human early hematopoietic stem cells and progenitor cells in vitro. Transplant of human umbilical cord blood cells to NOD / SCID mice. To study the potential of NOS inhibitors to affect human hematopoietic cells in vivo, 10-25xl06 25xl0e HUCB mononuclear cells were transplanted into subletically irradiated NOD / SCID mice. The animals were treated with NOS inhibitors or saline as control. The number of CD34-positive cells increased 1.8, 5.5 and 7.4 times after treatment with 30 mg / kg of ETU and 300 mg / kg of L-NAME for 6, 14 and 20 days, respectively, in comparison with control. These experiments suggest that NO acts by controlling the proliferation and maturation of human hematopoietic cells in vivo. EQUIVALENTS While this invention has been particularly shown and described in relation to preferred embodiments thereof, those skilled in the art will understand that various changes in shape and detail may be made therein without departing from the spirit and scope of the invention, as defined by the appended claims. Those skilled in the art will recognize, or will be able to determine using only routine experimentation, many equivalents of the specific embodiments of the invention specifically disclosed herein. Said equivalents are intended to be covered by the scope of the claims.

Claims (40)

1. An augmentation method in a mammal of a population of hematopoietic stem cells that are capable of normal haematopoiesis, differentiation and maturation in the hematopoietic tissue, which consists in contacting the hematopoietic tissue with multiple doses of at least one inhibitor of nitric oxide synthase, thus producing hematopoietic tissue that has a larger population of hematopoietic stem cells that are capable of normal hematopoiesis, differentiation and maturation.

2. The method of claim 1, wherein the inhibitor contacts hematopoietic tissue for a period of days selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.

3. A method according to Claim 1, wherein the contact stage is carried out ex vivo. .

A method according to Claim 3, which further includes the transplantation of hematopoietic tissue having a larger population of hematopoietic stem cells to a mammal in need thereof.

5. A method according to Claim 1, wherein the differentiation of erythroid cells is prevented.

6. A method according to Claim 1, wherein the differentiation of myeloid cells is prevented.

7. A method according to Claim 1, further including contacting the hematopoietic tissue with at least one selected hematopoietic growth factor to induce differentiation of a selected population of hematopoietic stem cells.

8. A method according to claim 1, wherein the nitric oxide synthase inhibitor is selected from the group consisting of L-nitroarginine methyl ester, 2-ethyl-2-thiopseudourea, aminoguanidine hemisulfate and N-monomethyl-L-arginine. .

9. A method of treating a mammal to increase a population of hematopoietic stem cells that are capable of normal hematopoiesis, differentiation and maturation in the mammalian hematopoietic tissue, which consists of contacting the mammalian hematopoietic tissue with multiple dose of at least one nitric oxide synthase inhibitor, thereby producing hematopoietic tissue that has an increased population of hematopoietic stem cells that are capable of normal hematopoiesis, differentiation and maturation.

10. The method of Claim 9, wherein the inhibitor contacts hematopoietic tissue during a period of days selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.

11. A method according to Claim 9, further comprising contacting the hematopoietic tissue with at least one selected hematopoietic growth factor to induce differentiation of a selected population of hematopoietic stem cells.

12. A method of treating a mammal to increase a population of hematopoietic stem cells that are capable of normal hematopoiesis, differentiation and maturation in the hematopoietic tissue of the mammal, consisting of the following steps: a) obtaining the hematopoietic tissue which has to be transplanted into the mammal, b) contacting the hematopoietic tissue to be transplanted with multiple doses of at least one nitric oxide synthase inhibitor, c) transplanting the hematopoietic tissue of step b) to the mammal that has of being treated, thus endowing the mammal with hematopoietic tissue that has a greater population of hematopoietic stem cells that are capable of normal hematopoiesis, differentiation and maturation.

The method of Claim 12, wherein the inhibitor contacts the hematopoietic tissue during a period of days selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.

A method according to Claim 12, further comprising: d) treating the mammal with a nitric oxide synthase enhancer after transplanting the hematopoietic tissue.

15. A method according to Claim 12, which also includes: d) treating the mammal with a nitric oxide synthase inhibitor after transplanting the hematopoietic tissue.

16. A method for increasing a population of progenitor blood cells that are capable of normal hematopoiesis, differentiation and maturation, consisting of contacting blood progenitor cells with multiple doses of at least one nitric oxide synthase inhibitor, increasing thus the population of blood progenitor cells.

The method of Claim 16, wherein the inhibitor contacts the blood for a period of days selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.

18. A method according to Claim 16, wherein the blood progenitor cells are obtained from hematopoietic tissue selected from the group consisting of bone marrow, umbilical cord vein blood, peripheral blood, fetal liver and long-term hematopoietic cell cultures. term.

19. A method according to Claim 16, wherein the nitric oxide synthase inhibitor is selected from the group consisting of L-nitroarginine methyl ester, 2-ethyl-2-thiopseudourea, aminoguanidine hemisulfate and N-monomethyl-L-arginine. .

20. A method of increasing a population of dividing cells in a tissue of a mammal, comprising contacting the cells with multiple doses of at least one nitric oxide inhibitor.

21. A method according to Claim 20, wherein the inhibitor is an inhibitor of nitric oxide synthase.

22. A method according to claim 20, which results in an increase in the size of an organ with which the tissue is associated.

23. A method of reducing a population of cells in S phase in a tissue of a mammal and induction of cell differentiation, consisting of contacting the tissue with multiple doses of at least one nitric oxide enhancer.

24. A method according to Claim 23, wherein the enhancer is a nitric oxide synthase enhancer.

25. A method according to Claim 23, which results in a reduction in the size of an organ with which the tissue is associated.

26. A method of regenerating a tissue in an adult mammal, comprising contacting a selected tissue with multiple doses of at least one nitric oxide inhibitor, thereby inhibiting differentiation and inducing proliferation of tissue cells, then contacting the selected tissue with a compound that inhibits proliferation and induces differentiation.

27. The method of Claim 26, wherein the compound that inhibits proliferation and induces digestion is selected from the group consisting of nitric oxide, a growth factor or a combination of both.

28. The method of Claim 26, wherein the nitric oxide inhibitor is an inhibitor of nitric oxide synthase.

29. The method of Claim 26, which results in an increase in the size of the organ with which the tissue is associated.

30. A method according to Claim 26, wherein the tissue is selected from the group consisting of blood, skin, bone, digestive epithelium, fatty tissue, bone marrow stroma, cartilage and tendon.

31. A method of repopulating an organ or tissue having cells that normally do not divide, consisting of contacting a selected organ or tissue with multiple doses of at least one nitric oxide inhibitor, thus inhibiting differentiation and inducing proliferation. of the cells of the organ or tissue, then contacting the selected organ or tissue with a compound that inhibits proliferation and induces differentiation.

32. The method of Claim 31, wherein the compound that inhibits proliferation and induces digestion is selected from the group consisting of nitric oxide, a growth factor or a combination of both.

33. The method of Claim 31, where the nitric oxide inhibitor is an inhibitor of nitric oxide synthase.

34. The method of Claim 31, which results in an increase in the size of the organ.

35. A method of Claim 31, wherein the organ or tissue is selected from the group con- sisting of muscle and nerve fibers.

36. A method of producing a subpopulation of hematopoietic cells in the hematopoietic tissue consisting of the following steps: a) contacting the hematopoietic tissue with multiple doses of at least one nitric oxide synthase inhibitor, thereby producing hematopoietic tissue having a greater population of hematopoietic stem cells that are capable of normal hematopoiesis, differentiation and maturation; and b) contacting the hematopoietic tissue with at least one hematopoietic growth factor selected to induce specific differentiation of the population of hematopoietic stem cells. , thus producing a subpopulation of hematopoietic tissue.

37. The method of Claim 36, wherein the inhibitor contacts the bone marrow for a period of days selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days.

38. A method according to Claim 36, wherein the nitric oxide synthase inhibitor is selected from the group consisting of L-nitroarginine methyl ester, 2-ethyl-2-thiopseudourea, aminoguanidine hemisulfate and N-monomethyl-L-arginine. .

39. A method of increasing a population of S-phase cells in a tissue of a mammal, consisting in contacting the tissue with multiple doses of at least one nitric oxide inhibitor.

40. A method according to Claim 39, wherein the S-phase cells can be used in gene therapy.