Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone

Definitions

• the present invention relates to means for the treatment of HIV infection and the prevention of AIDS. It relates more particularly to the use of means for the treatment of HIV infection and / or the prevention of AIDS, as well as the means relating thereto, the main function of which is to restore the CD4 lymphocyte count, together with a maintenance of the number of CD8 lymphocytes in seropositive subjects or treated patients, whose CD4 / CD8 ratio is thus markedly increased.
• HIV1 Human immunodeficiency virus type 1
• HIV1 Human immunodeficiency virus type 1
• the manifestations of AIDS are of various types: opportunistic infections; Kaposi's sarcoma; lymphomas; neurological disorders and a drop in blood CD4 below 200 / mm ** - 1 .
• Contamination with this virus first shows up in the serum of the infected organism antibodies directed against this virus. The subject is then said to be HIV positive.
• the clinical manifestations accompanying this phase include lymphadenopathy in particular.
• ARC abbreviation for "AIDS Related Complex”
• the ARC demonstrations precede and announce the occurrence of AIDS, which follows them from a few months to two years. According to current knowledge, more than 90% of infected people will evolve, in the absence of treatment, towards AIDS, the final phase of HIV infection, within fifteen years of being infected.
• CD4 lymphocytes which are largely responsible for the body's defenses.
• the normal CD4 blood level is 900 ⁇ 300 per mm 3 .
• Activation of the CD4 lymphocyte initiates the process of virus multiplication within the infected CD4 cell. This first results in the appearance of viruses on the surface of the CD4 cell, then the death of the CD4 cell and the discharge into the blood stream of many newly manufactured infectious viruses. This leads to the fact that, in the last phase of infection, only 50 CD4 cells / mm 3 remain in the blood. Therefore the concentration of CD4 cells and the percentage of infected cells are such that these cells can no longer play their role in the body's defenses and AIDS appears. In the absence of treatment or even most often in spite of himself, the patient's death eventually occurs.
• HIV also infects blood monocytes and tissue macrophages. The consequences of infection of these cells on the development of AIDS are currently unknown.
• the course of the infection is slow and can last three to fifteen years or more after infection, depending on the individual.
• the mechanisms used by the affected organism to slow the progression from infection to AIDS are therefore complex.
• antibodies specific to the different constituents of the virus are produced by specialized cells (plasma cells) in the bone marrow. These antibodies are produced within three to twenty weeks of contamination, very exceptionally later. The contaminated subject is then declared HIV positive. Among these antibodies, some, by attaching themselves to various areas of the virus, are able to prevent the virus from entering new CD4 cells; this is why they are called neutralizing antibodies. Over time, the virus produced by lymphocytes is unfortunately progressively less and less neutralized. It is possible that the production capacity of neutralizing antibodies by plasma cells is ultimately exceeded by the importance of viral production. It is also possible that the rate of spontaneous mutations to which the virus genome is subject leads to even minor changes in the proteins of the envelope of the virus. The immune system may then no longer be able to use neutralizing antibodies adapted to these modified viruses.
• CD8 Other white blood cells, called CD8 (or T8) lymphocytes, also play an important role in the fight against viral production.
• CD8 cells whose normal blood level is around 500 / mm 3 , increase rapidly in the weeks following contamination
• CD8 is to specifically destroy any cell carrying foreign proteins (mainly of viral origin) on its surface. It is thus possible that infected CD4 cells are destroyed by CD8 cells as soon as viruses appear on their surface. There are most likely other mechanisms for destroying CD4 cells. During much of the course of HIV infection, destroyed CD4 cells (either by one of the two processes described above or by other mechanisms not yet clearly identified) are replaced by new CD4 cells from precursor cells. This replacement, whose physiology is poorly understood, is gradually becoming more and more incomplete as the infection progresses. Thus, the progressive disappearance of CD4 cells is the result of a complex process where the role of viral multiplication and the implementation of the body's defenses against the virus and against the cells which carry it are very entangled.
• the human immunodeficiency virus type 1 (HIV1) is a lentiretrovirus preferentially infecting CD4 lymphocytes, as well as macrophages, dendritic cells and other cells of the monocytic line. It causes a slowly progressing attack on the immune system, manifested mainly by a gradual and irreversible decrease in blood CD4 cells, an increase in CD8 lymphocytes and an increase in the concentration of serum in various classes of immunoglobulins. There is also an increase in biological markers of immune activation (microglobulin ⁇ 2 # neopterin). Acquired immunodeficiency syndrome (AIDS) manifests itself in a variable period (on average 8-10 years) after contamination.
• AIDS Acquired immunodeficiency syndrome
• cyclosporine a potent immunosuppressive drug substance
• CSA cyclosporine
• no increase in the number of CD4 cells has been noted.
• glucocorticoids or glucocorticosteroids, or abbreviated below "GCC”
• GCC glucocorticosteroids
• adrenocorticotropic hormone or corticostimulin
• corticostimulin is a clinical immunosuppressant and anti-inflammatory. It is also known that corticostimulin has the same sedative action on inflammatory and allergic phenomena as cortisone.
• HIV Long Terminal Repeat sees its action increased by glucocorticoids in tissue culture cells and by the state of pregnancy in placental and uterine tissues in transgenic mice. It is suggested by these authors that hormonal stimulation may influence proviral activation and that placental expression of HIV-LTR may contribute to the high perinatal transmission of HIV.
• cytokines constitute a complex network which regulates the expression of HIV, in the same way as the endogenous regulatory proteins thereof, and that a certain number of different physiological and pharmacological agents capable of interfering with Cytokine-mediated events, including glucocorticoids, antioxidants, and retinoic acid, have been shown to significantly increase HIV replication in vitro for glucocorticoids. It is noted that treatment with glucocorticoids alone of chronically infected Ul cells did not result in direct induction of HIV expression, and that in transient transfection systems glucocorticoids have been shown to both induce and suppress expression of HIV-LTR.
• dexamethasone does not increase the action of HIV-LTR.
• dexamethasone seems to be able to prevent the increase in HIV replication due to stimulation.
• corticosteroids do not seem to promote the expression of virus and that an acute in vivo administration of these compounds should not have the harmful effects hitherto reported by other authors.
• a primary object of the present invention is therefore the use of at least one glucocorticoid for obtaining a medicament intended for the treatment of HIV infection and the prevention of AIDS, in particular in a patient infected with the HIVl virus. .
• the invention also relates to means for obtaining a medicament for the treatment of HIV infection, and in particular the prevention and / or treatment of AIDS, in particular in a patient infected with the HIVl virus, characterized in that they include an effective amount in the application considered at least one glucocorticoid.
• These means are in particular a pharmaceutical composition comprising at least one glucocorticoid as active ingredient and presented and / or packaged for the treatment of HIV infection, in particular the prevention and / or treatment of AIDS.
• the present invention thus also relates to a pharmaceutical composition presented and / or packaged for the treatment of HIV infection, in particular for the prevention and / or treatment of AIDS, comprising as active ingredient at least one glucocorticoid and / or a salt or other pharmacologically acceptable derivative thereof.
• Said composition is advantageously in the form of tablets, tablets or lozenges, optionally effervescent, or alternatively solutions or suspensions for intramuscular or intravenous injections.
• the dosage forms of this composition comprise in practice per unit dose approximately 1 mg to 1 g of prednisolone (or ⁇ 1 - dehydro-cortisol) or a weight equivalent, on the basis of the anti-inflammatory pharmacological effect, of any other glucocorticoid natural or synthetic or their different salts.
• prednisolone or ⁇ 1 - dehydro-cortisol
• weight equivalent on the basis of the anti-inflammatory pharmacological effect, of any other glucocorticoid natural or synthetic or their different salts.
• Non-limiting examples of these compounds are in particular: fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, cortisone cypionate, sodium salt of phosphate d hydrocortisone, the sodium salt of hydrocortisone succinate, beclomethasone dipropionate, betamethasone, the sodium salt of betamethasone phosphate, betamethasone valerate, cortisone acetate, dexamethasone, dexamethasone acetate, sodium salt of dexamethasone phosphate, Flunisolide, methylprednisolone, methylprednisolone acetate, sodium salt of methylprednisolone succinate, paramethasone acetate , prednisolone, prednisolone acetate, sodium salt of prednisolone phosphate, prednisolone tebutate, triamcinolone, triamcinolone acetonide
• the present invention comprises the use of an effective amount of at least one compound chosen from the group of glucocorticoids.
• glucocorticoids also called glucocorticosteroids, adrenocortical steroids or 11-oxy-corticosteroids
• glucocorticoids are steroids secreted by the fasciculated zone of the adrenal corticosteroid and contain in their molecule a ketone or alcohol radical attached to the carbon atom in position 11. They constitute a chemical group comprising in particular: corticosterone, 11-dehydrocorticosterone, 17-hydroxycorticosterone or cortisol or also hydrocortisone, and 17-hydroxy-ll-dehydrocorticosterone or cortisone.
• Synthetic steroids such as prednisone and prednisolone, which belong to this same group, are also known.
• Prednisone (or ⁇ *** - -cortisone) is obtained by dehydrogenation in positions 1 and 2 of cortisone, while prednisolone (or delta-hydroxycortisone) is obtained by dehydrogenation in positions 1 and 2 of 1 • hydroxycort isone.
• the present invention comprises the use of an effective amount of at least one compound chosen from the group of glucocorticoids. More advantageously, said effective amount consists of daily doses of approximately 1 mg to 1 g of prednisolone or of a dose of pharmacologically equivalent anti-inflammatory effect of any other natural or synthetic glucocorticoid. These doses may, depending on the route used, the condition of the patient and the product used vary over time and be administered according to a dosage comprising administration in one or more times per day, per week or per month, preferably in the morning.
• a daily dose as mentioned above can itself be replaced by any other chronology and / or dose of administration of the active compound (weekly, monthly, quarterly administration) insofar as the dosage form of the active principle thus administered provides a pharmacological effect appreciably similar to that obtained with daily administration.
• the glucocorticoids can be administered by the oral, parenteral, for example intravenous routes, the preference going to the oral route.
• the glucocorticoids can be administered together with a physiologically acceptable carrier or carrier or diluent to thereby form a pharmaceutical composition.
• a physiologically acceptable carrier or carrier or diluent to thereby form a pharmaceutical composition.
• Such compositions advantageously contain more than 0.1% by weight of at least one glucocorticoid and can be prepared by conventional techniques allowing their formulation in conventional dosage forms, for example tablets, capsules, dragees, dispersible powders , syrups, elixirs, suspensions or solutions, drops, nebulizations, suppositories.
• Suitable diluents or pharmaceutical fillers include, for example, water, alcohols, natural or hardened oils or waxes, calcium or sodium carbonates, calcium phosphate, kaolin, talc and lactose, as well as appropriate preservatives, such as for example 1 • methyl hydroybenzoate, suspending agents such as methyl cellulose, tragacanth and sodium alginate, wetting agents such as lecithin, polyoxyethylene stearate and polyoxyethylene -sorbitan, granulating and / or disintegrating agents such as starch and alginic acid, cohesion agents such as starch, gelatin and gum arabic, and lubricating agents such as magnesium stearate , stearic acid and talc.
• compositions in tablet form can be coated by known techniques, so that the disintegration of the tablet and the absorption of the active ingredient in the gastrointestinal tract are postponed and so that a prolonged action is obtained over a long period.
• the intramuscular forms can be designed by the usual techniques, known to those skilled in the art, so as to release the active principle over an extended time.
• a preferred form is represented by the forms for oral administration which are the drops and the tablets.
• a person skilled in the art is able to determine in each individual case whether or not a compound is an equivalent of the glucocorticoids by resorting to routine tests and by proceeding if necessary iteratively, preferably on the basis of the indications and proposed methods provided below.
• Fig. 1 shows graphs showing the rescue of CD4 + T cells by glucocorticoids from activation-induced cell death caused by HIV1.
• Fig. 2 represents graphs showing the effects of activation markers of PBMC acutely infected by HIV and stimulated by PHA or the soluble antigen of the influenza A virus.
• Fig. 3 represents graphs showing the CD4 + T and CD8 + T cell counts of peripheral blood of patients seropositive for HIV1 before and under treatment with prednisolone (PDN), as well as the levels of immunological and virological markers under treatment with PDN.
• PDN prednisolone
• ZDV Zidovudine
• Prednisolone (hereinafter abbreviated as PDN) (marketed under the name Solupred by the company Laboratoire Houdé, Puteaux, France) was administered orally to each of the above patients at a daily dose of 0.5 mg / kg (that is to say more precisely in this case: 25 mg for individuals weighing from 46 to 55 kg, 30 mg for those weighing between 56 and 65 kg, 35 mg for those weighing 66 to 75 kg, and 40 mg for those weighing between 76 and 85 kg), taken once before breakfast.
• treatments have been associated with it, in the form of an additional potassium intake, at a rate of 1200 mg per day (product marketed under the name Diffu K, by the Delagrange Laboratory,
• PDN therapy was initially planned to last 3 months at the maximum dose. On the seventieth day of treatment according to the evolution of the CD4 cell count and in the absence of side effects, it was decided in agreement with the patients to continue the treatment for another period, of six months this time.
• the initial assessment was done in the two weeks before the start of PDN therapy (day 0). The evaluations were then scheduled to be carried out on days 15 and 70. In the case of the modification of the length of the test, the evaluation of the treatment was also carried out on days 125 and 170.
• the evaluation was carried out comprising the following points: complete clinical examination, including measurement of the lymph nodes (or nodes), measurements of weight and blood pressure, usual renal and hepatic chemistries, blood count with phenotypes of CD4 and CD8 lymphocytes (percentage of total lymphocytes and absolute values). For 8 patients, a more complete characterization of the peripheral blood lymphocyte phenotypes (CD19,
• CD4 + / DR +, CD4 + / CD45RO +, CD4 + / CD25 +, CD8 / DR +, CD8 + / CD57 + was performed with appropriate antibodies by simultaneous double immunofluorescence if necessary.
• Serum immunoglobulins (Ig) G, A and M, as well as the levels of microglobulin ⁇ 2 and neopterin were also measured.
• the TNF- ⁇ , IL4, IL6 and soluble IL2 receptor concentrations were determined when sufficient serum was available.
• the serum concentration of viral particles and the blood concentration of infectious cells were determined on days 0, 70 and 125.
• Table I are summarized the changes in hemoglobin, hematocrites, platelets, white blood cells (WBC), neutrophils and total lymphocytes.
• the hemoglobin level increased significantly from day 70, while the hematocrit remained constant; the platelet count remained stable.
• the WBC count increased significantly from day 15 and then remained stable.
• a significant increase in the neutrophil count has been observed since days 15 and 70; this then decreased to a level not significantly different (although higher) than that observed on day 0.
• a significant increase in the lymphocyte count appeared under administration of PDN from day 15.
• Table 2 below below below shows the individual evolution and the average evolution of the CD4 and CD8 lymphocyte subpopulations.
• CD4 cells absolute values, as well as percentages of total lymphocytes
• the absolute value of the CD8 cell count remained stable, while their percentage decreased significantly. This was due to a significant increase in the CD4 / CD8 ratio from day 15.
• Table 3 illustrates the evolution of the other lymphocyte phenotypes produced in 8 patients. The percentage of CD4 DR cells / CD4 cells decreased significantly, as did that of the CD8 DR subpopulation.
• Table 4 summarizes the evolution of the serological parameters tested. IgG decreased significantly from day 70, while IgA began to decrease from day 125. IgM remained stable.
• the p24 antigenemia content (pg / ml) of the 4 positive patients did not change throughout the treatment (day 0: 202, day 70: 187, day 170 (1 patient): 180; the eight patients without p24 antigenemia were remained negative).
• the evolution of the serum concentration in viral particles, as well as that of the blood concentration in infectious cells, are indicated in Table 4. The two parameters remained stable during the treatment with PDN.
• Glucocorticosteroids have been used for decades for a multitude of medical conditions, including viral infections. Their effects have also been tested in healthy subjects. In these studies, glucocorticosteroids have usually resulted in a very marked decrease in the lymphocyte count, mainly with regard to the CD4 lymphocyte subpopulation, which resulted in a very marked decrease in the CD4 / CD8 ratio (0.57 ⁇ 0.07 (P ⁇ 0.0001) at 80 days). It has been suggested that this lymphopenia results from a redistribution of the recirculating mass of extra-vascular lymphocytes. In two studies a temporary (less than four weeks) increase in CD4 cell count was observed, but it was associated with a parallel and similar increase in the CD8 cell subpopulation, which resulted in stable CD4 / CD8 ratio.
• peripheral lymphocytes may be due to the fact that the immunosuppressive action of glucocorticosteroids interrupts the autoimmune pathways which usually cause the death of CD4 cells.
• HIV using a drug with a broad immunosuppressive spectrum.
• HIV-induced immune activation causes CD4 cell depletion are not yet fully understood; they are probably multiple and could include overlapping: a) autoimmune processes involving all immune cells and triggered by HIV infection; b) the activity of follicular dendritic cells, probably necessary for the destruction of CD4 cells; c) apoptotic death of inappropriate CD4 cells.
• glucocorticoids which are preeminent anti-inflammatory agents, have been shown to be antagonists of activation-induced apoptotic cell death in mouse thymocytes and T cell hybridomas. It is thus demonstrated according to the present invention that Pharmacological doses of glucocorticoids (or GCC) are effective not only for saving CD4 + T cells from apoptosis caused by HIV1 in vitro, but also for preventing apoptotic CD4 depletion in patients infected with HIV1.
• FIG. 1 shows in diagram form the rescue of CD4 + T cells from cell death induced by activation caused by HIV1.
• Fig. 2 shows in graph form the effects of glucocorticoids on activation markers of PBMC acutely infected with HIV.
• Fig. 3 diagrammatically depicts the peripheral blood cell counts of HIV-positive patients.
• Apoptosis which is a form of cell death fundamentally different from acute pathological cell death (commonly called “necrosis”) has so far been recognized as representing an important physiological test to which most cells respond, both in developing and mature tissue.
• Activation-induced cell suicide due to apoptosis has been identified as a cell death mechanism to which human T lymphocytes infected with human immunodeficiency virus type 1 (HIV1) are subjected both in vitro and in vivo .
• HV1 human immunodeficiency virus type 1
• glucocorticoids which are the most widely used anti-inflammatory agents with life-saving potentials to protect the body from many types of harmful stimuli, have been shown to be effective in saving murine thymocytes and T cell hybridomas of activation-induced cell death, although GCC alone causes apoptosis of these cells in the absence of stimulators. Since circulating T cells in HIV-infected patients undergo apoptosis when mediated in vitro by mitogens or the CD3 / T cell receptor (TCR), it was necessary to examine whether GCCs were effective in inhibit apoptosis induced by activation in human T cells infected with HIV1.
• PBMCs peripheral blood mononuclear cells
• HPA phytohemagglutinin
• anti -CD3 a monoclonal antibody for CD3
• DNA is degraded to oligomers corresponding to fragments the size of an oligonucleosome, revealed by a pattern (plot) of DNA degradation in multiples of 200 base pairs (called fragmentation).
• DNA gel electrophoresis of PBMC highly infected with HIV1 following activation with anti-CD3 showed a corresponding characteristic pattern.
• the stimulator or the virus were removed, the cellular DNA no longer produced any discernible characteristic of such a degraded pattern of DNA, which suggested that apoptosis depended on both activation and infection. DNA fragmentation did not appear when cyclosporin A or hydrocortisone / prednisolone was added at the time of activation.
• GCCs effectively suppress apoptosis induced by activation in primary PBMCs acutely infected with HIV1
• a quantitative polymerase chain reaction (PCR) demonstrated that, although PBMC appeared to be infected with an equal number of copies of HIV RNA, total genomic HIV RNA (including extrachromosomal forms and integrated provirus ) was strongly lowered (10 to 100 times) by pretreatment of the cells with CSA, which indicated a blockage of viral reverse transcription. However, this did not happen when prednisolone was used to pretreat the cells.
• PBMCs infected in vitro the PBMCs of patients stimulated by anti-CD3 have given rise to apoptotic CD4 depletion in a manner dependent on the virus load.
• high concentrations of blood cells carrying infectious HIV1 correspond to a rapid depletion of CD4 + peripheral T cell counts.
• PBMCs of unstimulated patients a higher level of apoptosis was observed than in acutely infected PBMCs.
• Apoptotic cell death reached 20% of CD4 cells in 3 cases (patients 1-3) with the highest viral load, against only 7% and 5% in both cases (patients 4 and 5) with the lowest viral load.
• This spontaneous apoptosis of patients' T cells provides an explanation for the observation that the rescue of CD4 cells from apoptosis by GCC appeared to be less effective in naturally infected cells than in acutely infected PBMCs.
• GCC which usually cause CD4 + T lymphocytopenia in normal humans
• CD4 cell counts without modifying the CD8 subpopulation at 15 th day as well as at the 120th day (see Figs. 3a and 3b).
• An increased CD4 cell count (20-200%) was noted in 36 of 50 subjects (72%) after 15 days and in 29 out of 44 (68%) after 120 days of treatment, while a decrease in at least 20% of the CD4 count was seen in 5 cases (10%) iS 6111® day and in 4 cases (9%) 120® me day (Figs. 3a3 and 3a4).
• GCCs are agents that are capable of: 1) save CD4 + T cells from apoptotic cell suicide caused by HIV1 by cell activation mediated by lectin or CD3 / TCR in vitro;
• CD8 + T cells from HIV1 positive individuals have also been shown to undergo apoptosis upon TCR / CD3 binding in vitro, what has been demonstrated above emphasizes that the destruction of T cells induced by HIVl is essentially restricted to CD4 cells both in vitro and in vivo.
• the immense effects of GCCs on CD4 counts in infected patients are consistent with the rationale that apoptosis caused by HIV is an active process that takes place in the lymphoid organs and governs the gradual CD4 depletion observed throughout infection, leading to the new perspective that virus-induced pathogenic destruction of CD4 + T cells in vivo can be alleviated by treating infected patients with specific anti-apoptotic agents, such as GCC.
• Fig. la % of cell mortality (1 - ratio of viable cells to initial cells),
• Fig. lb the% of CD4 + T cells viable in PBMC acutely infected with HIV and activated by PHA or anti-CD3, in each case in the presence or absence of cyclosporin A, hydrocortisone and prednisolone.
• Fig. le % CD4 mortality (1 -% of viable control CD4)
• Fig. ld % of total cell mortality (1 -% of viable control PBMCs) in PBMCs acutely infected with HIV and activated by anti-CD3, in each case in the presence of the indicated concentrations of cyclosporin A, hydrocortisone and prednisolone .
• Data represents mean ⁇ SD
• PBMCs were freshly separated by Ficoll-Hypaque density gradient centrifugation from heparinized blood from normal HIV negative donors.
• the cells (2 x 10 ⁇ ) were incubated with primary HIVl isolates for 2 hours at a multiplicity of infection (MOI) of 2 x 10 " 4 units of infectious virus per cell (according to Poisson distribution, one unit infectious corresponds to 63% of positive cultures for a given dilution of viral stock), which corresponds to 1% of cells positive for HIV1 DNA (assay by polymerase amplification reaction or PCR) in PBMCs inoculated by quantitative analysis cells diluted in series (limiting dilution method).
• MOI multiplicity of infection
• the cells were cultured for a further 3 days in the presence or absence of the substances indicated above, and then harvested for counting viable cells by the blue dye test. trypan. The percentage of CD4 + cells in the final cell population was analyzed by flow cytometry (FACScan, Becton, Dickinson, Mountain View, CA) after reaction with a fluorescently labeled monoclonal antibody for CD4 (Leu-3a).
• Fig. 2a the effects of glucocorticoids on activation markers of PBMC acutely infected by HIV and stimulated by PHA or the antigen of the soluble influenza A virus.
• the results represent the mean ⁇ AND of measurements made in triplicate : 2al:% of transformed lymphoblasts (blast cells), 2a2:% of cells positive for the interleukin 2 receptor (IL2), 2a3: of [ 3 H] -thymidine binding to PHA, 2a4: of [ 3 H] -thymidine binding to
• Figs. 2b average% of fragmented DNA (ratio between
• PBMCs were infected with HIV1 and activated with PHA or influenza A virus antigen (10 ⁇ g / ml) in the presence of different concentrations of cyclosporin A and glucocorticoids such as indicated on the legend above for FIG. 1. After another 3 days of culture, a count of the blast cells was carried out in a hemocytometer and the cells positive for the IL2 receptor were evaluated by flow cytometry after their treatment with a monoclonal antibody directly labeled for fluorescence for the IL2 receptor (CD 25). Cell proliferation was determined by the technique of [ 3 H] -thymidine fixation.
• Low molecular weight DNA (including fragments of extrachromosomal DNA and degraded DNA) was separated from high molecular weight nucleosomal DNA by centrifugation (5000 xg for 15 min). DNA was isolated from both the supernatant and pellet fractions using a commercial DNA purification kit. The supernatant DNA was subjected to electrophoresis on a 1% agarose gel containing 0.5 ⁇ g / ml of ethidium bromide at 80 V for 2.5 h. To determine the ratio of DNA from the supernatant to DNA from the pellets (% fragmented DNA), the two fractions were quantified by spectrophotometry. PBMCs alone, infected with HIV or heat inactivated HIV (HI) (56 ° C, 40 min) served as controls.
• HI heat inactivated HIV
• Figs. 3a3 and 3a4 In the abovementioned counts, the number of individual patients was used for the analysis of the population and its representation in diagram.
• Fig. B Immunological and virological markers were used in 27 consecutive patients treated with PDN. Blood samples from 10 healthy donors negative for HIV were used as a control for each trial performed. The data were expressed by the mean ⁇ SD (Figs. 3bl to 3b4 and 3b6) or by the geometric mean ⁇ AND (Fig. 3b5) of the measurements.
• PBMC peripheral blood mononuclear cells isolated under gradient according to Ficoll- Hypaque and taken from 27 of the 28 consecutive subjects who reached the first 120 days of PDN treatment (the cells were not available for a patient). Briefly, the cell suspensions were divided into two aliquots and the pellets were either gently resuspended in 1.5 ml of a solution of propidium iodide (PI, 50 ⁇ g / ml in citrate 0.1% sodium plus Triton X-100 at
• the viral RNA was extracted, subjected to reverse transcription and amplified by PCR. Amplified HIV signal was quantified using the curve standard obtained with dilutions of RNA derived from virions. HIV genomic DNA was quantified in a PHA-stimulated PBMC overnight (reflecting total infection of blood cells by the virus) and viral RNA was quantified in a PHA-stimulated PBMC for 3 days (representing the number of viruses expressing / replicating in the patient's cells).
• the proliferative response of patients' PBMCs to stimulation by anti-CD3 and the booster antigen (influenza A) was obtained as indicated above (see section on Fig. 2a). Since these experiments were performed in an advanced phase of the present study, 14 of 27 patients did not have cell samples for a proliferative booster response.
• Platelets f / ⁇ D Average ⁇ SE 215 ⁇ 11,237 ⁇ 11,234 ⁇ 12,221 ⁇ 22,244 ⁇ 39 Probability * 0.002 0.02 0.722 0.753
• PBL peripheral blood lymphocytes

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