EP1748793A2 - Estrogen modulation of ifn-gamma-mediated conditions and diseases - Google Patents

Abstract

Particular aspects provide methods for: modulating IFN-Ϝ-mediated conditions and diseases (e.g. inflammation, HIV/AIDS, etc). Additionally provided are methods for: reducing IFN-Ϝ expression or secretion in cultured mammalian cells, comprising culturing in the presence of at least one agent selected from the group consisting of estrogen, estriol, estrone and a SERM; for ex-vivo therapy for an IFN-Ϝ-mediated condition or disease, comprising culturing isolated lymphoid cells in the presence of at least one of the above agents; for treating an IFN-Ϝ-mediated condition or disease in a mammal, comprising administering to a subject at least one at least one of the above agents; for treating an IFN-Ϝ-mediated condition or disorder in a gender-dependent manner; for conferring estrogen-responsive expression using a recombinant SNP IFN-Ϝ-179G/T promoter; and for gene therapy using same. Preferably, mammalian cells are human, selected from T cells, PBL, LPMC, and combinations thereof. In particular aspects, the cells are female.

Description

ESTROGEN MODULATION OF IFN-GAMMA-MEDIATED CONDITIONS AND DISEASES

FIELD OF THE INVENTION The invention relates to the modulation by estrogen (and related molecules) of processes, diseases and conditions generally associated with interferon gamma ("IFN-γ") expression (e.g., inflammation, HIV/AIDS, etc).

GOVERNMENT RIGHTS The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Contract DK43211, awarded by the National Institutes of Health.

BACKGROUND Many diseases and other physiological conditions relate to inflammation; in particular, to inflammation associated with expression of interferon gamma ("IFN-γ"). IFN-γ is a prominent immunoregulatory protein, vital to immune development and function. IFN-γ plays .an important role in host immune and inflammatory responses. Secretion of IFN-γ by activated T cells and natural killer cells is tightly controlled (Young and Hardy, J. Leukoc. Biol. 58:373, 1995; Lewis and Wilson, Pediatr. Infect Dis. J. 9:642, 1990), yet the regulatory mechanisms controlling expression are not fully understood. The capacity of T cells to produce IFN-γ is determined primarily at the transcriptional level. Therefore, the gene structure and promoter response elements of IFNG have been intensely investigated. The structure of human IFNG consists of four exons and three introns (Sehgal et al., J. Interferon Res. 6:39, 1986), with a remarkable degree of conserved structure across human and rodent species (Id). A number of enhancer and repressor cis-regulatory regions have been identified within the 5' promoter region. It is believed that the cooperative interaction of multiple cis-acting elements ultimately determines the level of transcriptional activation. Recent published reports have suggested the existence of regulatory sequences at regions 50 kb upstream, and downstream beyond the region of IFNG itself — far more distal than what has been previously assumed (Zhou et al., Proc. Nail. Acad. Sci. U S A 101:2440, 2004). In addition, histone hyperacetylation patterns and alterations in chromatin structure have been demonstrated within 24 kb of IFNG, as cells progress from naive ThO to IFN-γ-producing Thl cells (Zhou et al., Proc. Natl. Acad. Sci. USA 101:2440, 2004; Eivazova and Aune, Proc. Natl. Acad. Sci. USA 101:251, 2004). Inappropriate overexpression of IFN-γ by activated mucosal T cells is an important contributory factor in the pathogenesis of IBD (Powrie, F., Immunity 3:171, 1995; Strober et al., Immunol. Today 18:61, 1997; Sartor, R. B., Gastroenterology 106:533, 1994). In fact, disease severity is correlated with the level of IFN-γ expression (Monteleone et al., Gastroenterology 117:1069, 1999). The pathways leading to activation of mucosal lamina propria T cells are different from those of peripheral T cells (Pirzer et al., Eur. J. Immunol. 20:2339, 1990; Targan et al., J Immunol. 154:664, 1995). Lamina propria T cells are particularly sensitive to activation through the CD2 pathway (Targan et al., J Immunol. 154:664, 1995; Boirivant et al., Proc. Assoc. Am. Physicians 108:55, 1996). The CD2 pathway dominance is believed to result from a T cell activation process inherent to the mucosal immune compartment. Despite much effort devoted to elucidating these differences, the molecular mechanisms involved remain poorly defined.

Unique regulatory mechanisms in the mucosa have been previously demonstrated that are distinct from those in PBL and T cell lines (Gonsky et al., J Immunol. 160:4914, 1998; Gonsky et al., J Immunol. 164:1399, 2000; Gonsky et al., Eur. J. Immunol. 33:1152, 2003). In PBL, CD2 activation elements have been mapped mainly to the -108 to +64 bp region, encompassing the proximal and distal AP-1 binding sites (Gonsky et al., J. Immunol. 164:1399, 2000). In contrast, in LPMC a significant CD2 response element resides between -204 and -108 bp region, a region previously reported in PBL and T cell lines to possess an essential AP-1 binding site, but CD2-mediated activation of IFNG expression is not altered by deletion of this AP-1 site (Id). CD2 stimulation has also been shown to activate the Janus protein tyrosine kinase /STAT pathway in PBMC differently than in LPMC. In LPMC, CD2 signaling results in enhanced phosphotyrosine STAT1 and STAT4 and phosphoserine STAT1. In contrast, in PBMC, phosphorylation is largely restricted to phosphotyrosine STAT1. Moreover, in LPMC (but not in PBMC), CD2 signaling results in enhanced promoter activity through STAT protein binding to an IFNG intronic enhancer element (Gonsky et al., Eur. J. Immunol. 33:1152, 2003). These results suggest that not only are there differences between the cis and trans regulatory elements used in PBMC and those used in LPMC, but additional differences probably exist leading to selective pathway accessibility in these cell populations. Activated mucosal T-cells play a central role in the initiation and perpetuation of inflammation in both animal and human disease. Indeed, IFN-γ produced by mucosal T-cells plays a central role in the vast majority of animal models and, for example, a major subset of Crohn's disease patients. Thus, the level of production of IFN-γ is believed to be related to the initiation of the disease process, as well as to the severity of inflammation in a variety of settings. At the same time, however, IFN-γ production is critical for protection from many invading pathogens. As such, complete or long-term inhibition of IFN-γ production is likely to be detrimental to the host. Therefore, there is a pronounced need in the art for identification of mucosa-specific targets that could selectively modulate IFN-γ production (e.g, in PBL or mucosal T cells) without eliminating it would be a significant therapeutic advance. It is hypothesized that specifically modulating, but not eliminating IFN-γ production, would preserve systematically and locally produced normal levels of IFN-γ. There is a pronounced need in the art for a therapeutic approach to the treatment of inflammation — particularly to inflammation associated with IFN-γ — and the diseases and physiological conditions of which inflammation is a component of pathology. There is a pronounced need in the art for therapeutic methods to attenuate sex-based immune differences between males and females in disease. SUMMARY OF THE INVENTION Particular aspects of the present invention provide a method for reducing IFN-gamma expression or secretion in cultured mammalian cells, comprising: obtaining mammalian lymphoid cells; and culturing the cells in the presence of an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in the cultured mammalian lymphoid cells. In particular embodiments, the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. In certain embodiments, cultured the cells are female cells. Preferably, the mammal is human. Preferably, the mammal is a human female. Additional aspects provide a method for ex-vivo therapy for an IFN-gamma-mediated condition or disease, comprising: isolating lymphoid cells from a mammalian subject; culturing the isolated lymphoid cells in the presence of an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in the cultured mammalian lymphoid cells; and transferring at least some of the cultured cells back into the mammalian subject to provide ex-vivo therapy for an IFN-gamma-mediated condition or disease. In particular embodiments, the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. In certain embodiments, the cultured cells are female cells. Preferably, the mammal is human. Preferably, the mammal is a human female. Further aspects provide a method for treating an IFN-gamma-mediated condition or disease in a mammal, comprising administering to a mammalian subject in need thereof an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in one or more lymphoid cells of the subject. In particular embodiments, the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. In certain embodiments, the cultured cells are female cells. Preferably, the mammal is human. Preferably, the mammal is a human female. In other embodiments, the condition or disease comprises inflammation, HIV/AIDS, or an HIV-related cellular condition or disease. Yet further aspects provide a method for treating an IFN-gamma-mediated condition or disorder in a gender-dependent manner, comprising administering to a mammalian subject in need thereof an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in one or more lymphoid cells of the subject, wherein the extent of reduction of IFN-gamma expression or secretion is greater for females than for males. Preferably, the mammal is human. Preferably, the mammal is a human female Additional aspects provide a method for conferring estrogen-responsive expression of a desired sequence within a mammalian cell, comprising: placement, in an expression vector, of a sequence downstream from a SNP IFN-γ- 179G/T promoter; and introduction of the expression vector into a cell; wherein transcription of the downstream sequence is driven by the estrogen responsive SNP IFN-γ- 179G/T promoter. In particular embodiments, the SNP IFN-γ- 179G/T promoter comprises SEQ ID NO: 15). In additional embodiments, the method further comprises contacting the cell with at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to modulate

SNP IFN-γ- 179G/T promoter-driven expression in the cell. Still further embodiment provide gene therapy methods for introduction of the expression vector into a subject in need thereof.

Preferably, the mammal is human. Preferably, the mammal is a human female.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows, according to particular aspects, enhanced IFN- γ promoter SNP (-179G/T) expression in PBMC but not in LPMC. Figure 2 shows, according to particular aspects, the ER-alpha and ER-beta proteins bind to the IFN- γ SNP (- 179G/T) region. Figure 3 shows, according to particular aspects, that estrogen inhibits CD2-mediated activation of consensus IFN- γ ERE in PBL but not in LPMC. Figure 4 shows, according to particular aspects, that estrogen inhibits PMA/ϊonomycin activation of consensus IFN- γ ERE In both PBL and LPMC. Figure 5 shows, according to particular aspects, that estrogen inhibits IFN- γ secretion in PBL. Figure 6 shows, according to particular aspects, that estrogen inhibits IFN- γ mRNA in PBL. Figure 7 shows, according to particular aspects, that estrogen inhibits IFN- γ secretion in

LPMC. Figure 8 shows, according to particular aspects, that estrogen inhibits CD2-mediated activation of -6.8 kb IFN- γ in PBL but not in LPMC. Figure 9 shows, according to particular aspects, that estrogen inhibits PMA/ionomycin activation of IFN- γ in both PBL and LPMC. Figure 10 shows, according to particular aspects, that comparable expression of ERa in CD3+ T cells from PBL and LPMC. Figure 11 shows, according to particular aspects, inhibition of IFN- γ expression by estradiol. A series of successively truncated IFN-γ promoter-constructs was utilized to map the estrogen response elements. Estrogen inhibited CD2-mediated IFN g promoter activation in PBL, however, not all promoter constructs were repressed to the same extent. Estrogen sensitivity mapped to the region up-stream of the -204 bp basal promoter region and was selective for PBL. In contrast, CD2-mediated IFN g promoter activation in LPMC was relatively unaffected following estrogen treatment. Selective sensitivity of PBL to estrogen treatment was activation pathway dependent since following PMA/ionomycin activation, IFN g promoter expression was inhibited to a similar extent in both PBL and LPMC.

DETAILED DESCRIPTION DEFINITIONS: The term "estrogen" refers to the steroids commonly known as 17.beta.-estradiol (E2), estrone (El) and estriol (E3). Also included within the term "estrogen" are metabolites and derivatives of El, E2 and E3. Such metabolites and derivatives act as agonists of the estrogen receptor (ER.alpha. or ER.beta.) and have a similar core steroid structure as El, E2 or E3, but can have one or more different groups (e.g., hydroxyl, ketone, halide, etc.) at one or more ring positions. Those skilled in the art can readily determine whether such metabolites and derivatives are agonists of estrogen by in vitro assays that measure signaling through the estrogen receptor. Alternatively, the effects of metabolites and derivatives of estrogen can be assessed, and compared to the effects of known estrogens, using any of the in vivo and in vitro assays that report estrogen's effects, as described in the Examples, below. In particular aspects, the term "estrogen" is used herein to refer to 17β-estradiol, and additionally encompasses estriol and estrone. In additional embodiments the term "estrogen" encompasses a selective estrogen receptor modulator (SERM). In particular embodiments, the term refers to at least one of 17β- estradiol, estriol and estrone. In particular embodiments, the term estrogen refers to 17β- estradiol. In particular aspects, the term "estrogen" also encompasses non-steroidal estrogen analog that acts as an agonist of the estrogen receptor. Methods of identifying receptor agonists from libraries of compounds are well known in the art, and include binding assays (e.g., competitive and non-competitive radioimmunoassays) and signaling assays (e.g., transcription- based assays using reporter genes driven by an estrogen response element). Libraries of naturally occurring and synthetic compounds, including inorganic compounds, peptides, lipids, saccharides, nucleic acids and small organic molecules, are commercially available, and can be screened in high-throughput assays to identify estrogen analogs. The term "transfection" is used herein to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al., Virolos 52:456 (1973); Sambrook et al, Molecular Clonins, a laboratory manual Cold Spring Harbor Laboratories, New York, (1989); Davis et al, Basic Methods in Molecular Biolosv, Elsevier (1986); and Chu et al, Gene 13:197 (1981). Such techniques can be used to introduce one or more exogenous DNA moieties, such as a plasmid vector and other nucleic acid molecules, into suitable host cells. The term refers to both stable and transient uptake of the genetic material. "DNA" is meant to refer to a polymeric form of deoxyribonucleotides (i.e., adenine, guanine, thymine and cytosine) in double-stranded or single-stranded form, either relaxed or supercoiled. The term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes single- and double- stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular DNA molecules, sequences may be described herein according to the nonnal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA). The term captures molecules that include the four bases adenine, guanine, thymine and cytosine, as well as molecules that include base analogues which are known in the art. A "gene" or coding sequence" or a sequence which "encodes" a particular protein is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences; although one of skill in the art will readily appreciate that various polynucleotides do not operate in this fashion (e.g., antisense RNA, siRNA, ribozymes, wherein the RNA transcript is the product). With respect to protein products (i.e., not RNA products), the boundaries of the coding sequence are determined by a start codon at the 5' (i.e., amino) terminus and a translation stop codon at the 3' (i.e., carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence. Moreover, a "gene" (i) starts with a promoter region containing multiple regulatory elements, possibly including enhancers, for directing transcription of the coding region sequences; (ii) includes coding sequences, which start at the transcriptional start site that is located upstream of the translational start site and ends at the transcriptional stop site, which may be quite a bit downstream of the stop codon (a polyadenylation signal is usually associated with the transcription stop site and is located upstream of the transcriptional stop); and (iii) may contain introns and other regulatory sequences to modulate expression and improve stability of the RNA transcript. The term "control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present, so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. The term "promoter region" is used herein in its ordinary sense to refer to a nucleotide region including a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence. For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated "upstream," "downstream," "5'," or "3"' relative to another sequence, it is to be understood that it is the position of the sequences in the non- transcribed strand of a DNA molecule that is being referred to as is conventional in the art. "Homology" as used herein refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions which fonn stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids, respectively, match over a defined length of the molecules, as determined using the methods above. "Isolated" as used herein when referring to a nucleotide sequence, refers to the fact that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, an "isolated nucleic acid molecule which encodes a particular polypeptide" refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide. However, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the compositions. "Mammal" as used herein refers to any member of the class Mammalian, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or females, are intended to be included within the scope of this term. "Treatment" or "treat" as used herein refers to any therapeutic methodology to ameliorate a disease, lessen the severity of its complications, prevent is from manifesting, prevent it from recurring, merely prevent it from worsening, or a therapeutic effort to effect any of the aforementioned, even if such therapeutic effort is ultimately unsuccessful.

OVERVIEW It is widely recognized that there are sex-based immune differences between males and females, predominantly after puberty. Although the etiology remains unclear, a role for estrogen has been suggested in differential immune modulation. IFN-γ is a key immunoregulatory cytokine. As described above, the capacity to produce IFN-γ, and the amount thereof, is controlled primarily on a transcriptional level. Particular aspects of the present invention are based, at least in part, on the inventors' investigation of the potential role of estrogen in the regulation of IFN-γ expression in peripheral (peripheral blood lymphocytes :PBL; PBMC) and mucosal T cells (lamina propria mononuclear cells:LPMC), and the surprising discovery of molecular-based gender disparities in the response to estrogen treatment. In particular aspects, ERE-like elements within the IFN- γ gene provide targets for selective regulation of IFN- γ expression. In additional aspects estrogen has substantial utility for downregulating IFN- γ protein secretion in a gender-dependent and dose-dependent fashion. In further aspects, estrogen has substantial utility for inhibiting IFN- γ protein secretion in PBL (peripheral blood lymphocytes) and LPMC (lamina propria mononuclear cells). In yet further aspects, estrogen has substantial utility for inhibiting IFN- γ promoter expression if PBL but not LPMC. In additional aspects, estrogen has substantial utility for inhibiting IFN- γ promoter expression in an activation pathway dependent manner, inhibiting CD2-mediated activation of IFN- γ promoter expression in PBL but not in LPMC, but inhibiting PMA ionomycin activation of IFN- γ promoter expression in PBL and in LPMC . In yet further aspects, the IFN- γ promoter SNP (-179G/T) (see Bream et al., Genes Immun. 3:165, 2002), has been demonstrated herein to provide selective allele-specific enhanced expression compared to the common allele in PBMC, but not in LPMC. According to additional aspects, ERE transactivation is selectively sensitive to estrogen downregulation in PBMC but not in LPMC.

Particular aspects of the present invention are based, at least in part, on the present investigation of the role of estrogen in the regulation of IFN-γ expression, and the surprising discovery that estrogen has substantial utility to down-regulate IFN-γ expression and secretion, and the discovery of molecular-based gender disparities; estrogen downregulates IFN- γ protein secretion in a gender-dependent and dose-dependent fashion. In one embodiment, treatment with as little as 10 nM estradiol resulted in a 40% decrease in IFN- γ protein secretion in PBMC isolated from female donors, whereas little, if any, inhibition was detected in PBMC from male donors. A pronounced transcription down- regulation was likewise detected in distal promoter regions spanning up to -6.8 kb upon transient transfection of reporter constructs in PBMC isolated from female donors. Example 2, herein below, further summarizes these results. Activation-specific IFN-γ promoter response elements have previously been identified within the -204 and -108 bp region. A single site nucleotide polymorphism ("SNP"), known as - 179G/T, was recently detected within that region, which imparts tumor necrosis factor alpha ("TNF-α") stimulation of IFN-γ production and is linked to acceleration of acquired immune deficiency syndrome ("AIDS") progression. In studies to define the molecular basis for functional significance of the -179G/T SN in humans, the present inventors discovered a putative non-classic estrogen response element ("ERE") introduced by the SNP variant allele. Example 1, herein below, further summarizes these results. When peripheral blood mononuclear cells ("PMBC") isolated from female donors were transfected with reporter constructs containing the common or polymorphic allele, they exhibited allele-specific enhanced expression of the ERE SNP allele construct compared to the common allele. PMBC transfected from male donors lacked allele-specific enhancement in expression of the ERE SNP allele. Nucleo-protein binding to the ERE SNP region was inhibited by both classic ERE as well as variant, but not the common allele oligonucleotides. Moreover, a pronounced transcriptional downregulation was detected following estradiol treatment only in PMBC isolated from female donors. These results suggest a potential molecular basis for gender-based immune differences between males and females contributed by a single SNP. The present invention thus provides a novel approach for utilizing estrogen, estrogen receptors (e.g., ERα, ERβ), other estrogen receptor ligands, selective estrogen receptor modulators ("SERMs") and analogs of the same to regulate the inflammatory processes resulting from IFN-γ expression. Furthermore, in various embodiments of the present invention, estrogen and its related compounds (e.g., estrogen receptors, estrogen receptor ligands, analogs, etc.) may be used in connection with therapeutic methodologies to treat inflammation, and particularly inflammation generally associated with IFN-γ. Thus, in still further embodiments of the present invention, estrogen and its related compounds may be used in connection with therapeutic methodologies to treat diseases and other physiological conditions of which inflammation is a component of the pathology. Diseases that may be treated in accordance with alternate embodiments of the present invention may include, but are no way limited to, breast cancer, prostate cancer, autoimmune diseases, HTV/AIDS and related diseases and conditions, and disease flares in women during the menstrual cycle. More specifically, the present invention provides for a molecular mechanism for underlying gender-based immune differences in transcriptional control of a central cytokine that is important in protective and disease-associated immune responses. This allows for the targeted use of estrogen and its aforementioned related compounds in modulating expression of IFN-γ and disease response.

Specific Embodiments Particular aspects provide a method for reducing IFN-gamma expression or secretion in cultured mammalian cells, comprising: obtaining mammalian lymphoid cells; and culturing the cells in the presence of an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in the cultured mammalian lymphoid cells. In particular embodiments, the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. Preferably, the cultured cells comprise peripheral blood leukocytes (PBL). In certain embodiments, the cultured cells are female cells. Preferably, the agent is 17β-estradiol. In particular embodiments, the amount of the agent is selected from the group consisting of 1 nM to about 100 nM, 1 nM to about lOnM, 1 nM to about 75 nM, 1 nM to about 50 nM, at least 1 mM, at least 10 nM, at least 50 nM, at least 75 nM, and at least 100 nM. In certain embodiments, the amount of the agent is at least 1 nM, at least 10 nM, or at least 100 nM. In some embodiments, the amount of the agent is at least 10 nM. Preferably, the mammal is human. Preferably, the mammal is a human female. Additional aspects provide a method for ex-vivo therapy for an IFN-gamma-mediated condition or disease, comprising: isolating lymphoid cells from a mammalian subject; culturing the isolated lymphoid cells in the presence of an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in the cultured mammalian lymphoid cells; and transferring at least some of the cultured cells back into the mammalian subject to provide ex-vivo therapy for an IFN-gamma-mediated condition or disease. In particular embodiments, the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. Preferably, the cultured cells comprise peripheral blood leukocytes (PBL). In certain embodiments, the subject is female. Preferably, the agent is 17β-estradiol. In particular embodiments, the amount of the agent is selected from the group consisting of 1 nM to about 100 nM, 1 nM to about lOnM, 1 nM to about 75 nM, 1 nM to about 50 nM, at least 1 mM, at least 10 nM, at least 50 nM, at least 75 nM, and at least 100 nM. In certain embodiments, the amount of the agent is at least 1 nM, at least 10 nM, or at least 100 nM. In some embodiments, the amount of the agent is at least 10 nM. Preferably, the mammal is human. Preferably, the mammal is a human female. Further aspects provide a method of treating an IFN-gamma-mediated condition or disease in a mammal, comprising administering to a mammalian subject in need thereof an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in one or more lymphoid cells of the subject. In particular embodiments, the lymphoid cells comprise one or more cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. In certain embodiments, the lymphoid cells comprise one or more peripheral blood leukocytes (PBL). In particular embodiments, the condition or disease comprises inflammation. Preferably, the mammal is human. Preferably, the mammal is a human female. In particular embodiments, the condition or disease comprises FHV/AIDS or an HIV-related cellular condition or disease. In some embodiments, the subject is female. In particular embodiments, the agent is 17β-estradiol. In certain embodiments, the amount of the agent is sufficient to raise the serum concentration of the agent to within a range selected from the group consisting of 30 pg/ml to 1000 pg ml, 50 pg/ml to 500 pg/ml, and 100 pg/ml to 250 pg/ml. In other embodiments, the amount of the agent is sufficient to raise the serum concentration of the agent to within 100 pg/ml to 250 pg/ml. In yet additional embodiments, the amount of the agent is sufficient to raise the serum concentration of the agent to a value greater than 250 pg/ml. In particular embodiments, the method further comprises administration of an anti-HTV agent. Preferably, the anti-HTV agent is selected from the group consisting of: nucleoside reverse transcriptase inhibitors (NRTIs); non-nucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors (Pis); fusion inhibitors (FI), and combinations thereof. Preferably, the nucleoside reverse transcriptase inhibitor (NRTI) is selected from the group consisting of: lamivudine and zidovudine; FTC, emtricitabine; lamivudine, 3TC; abacavir/ lamivudine; zalcitabine, ddC, dideoxycytidine; zidovudine, AZT, azidothymidine, ZDV; abacavir, zidovudine, and lamivudine; tenofovir disoproxil/emtricitabine; enteric coated didanosine; didanosine, ddl, dideoxyinosine; Didanosine (ddl) delayed release capsules; tenofovir disoproxil fumarate; stavudine, d4T; abacavir, and combinations thereof. Preferably, the non-nucleoside reverse transcriptase inhibitor (NNRTI) is selected from the group consisting of: delavirdine, DLV; efavirenz; nevirapine, BI-RG-587, and combinations thereof. Preferably, the protease inhibitor (PI) is selected from the group consisting of: Amprenavir; indinavir, IDV, MK-639; saquinavir mesylate, SQV; saquinavir; lopinavir and ritonavir; Fosamprenavir Calcium; ritonavir, ABT-538; atazanavir sulfate; nelfinavir mesylate, NFV, and combinations thereof. Preferably, the fusion inhibitor (FI) comprises enfuvirtide, T-20. Preferably, the HTV- related cellular effect, condition or disease is selected from the group consisting of: AIDS; Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5°C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HTV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1 -month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent term); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HIV, and combinations thereof. Yet additional embodiments provide a method for treating an IFN-gamma-mediated condition or disorder in a gender-dependent manner, comprising administering to a mammalian subject in need thereof an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in one or more lymphoid cells of the subject, wherein the sensitivity or extent of reduction of IFN-gamma expression or secretion is greater for females than for males. In particular embodiments, the lymphoid cells comprise one or more cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. In preferred embodiments, the lymphoid cells comprise one or more peripheral blood leukocytes (PBL). In particular embodiments, the condition or disease comprises inflammation. Preferably, the mammal is human. Preferably, the mammal is a human female. In particular embodiments, the condition or disease comprises HIV/AIDS or an HIV-related cellular condition or disease. Preferably, the agent is 17β-estradiol. In particular embodiments, the amount of the agent is sufficient to raise the serum concentration of the agent to within a range selected from the group consisting of 30 pg/ml to 1000 pg/ml, 50 pg/ml to 500 pg/ml, and 100 pg/ml to 250 pg/ml. In some embodiments, the amount of the agent is sufficient to raise the serum concentration of the agent to within 100 pg/ml to 250 pg/ml. In other embodiments, the amount of the agent is sufficient to raise the serum concentration of the agent to a value greater than 250 pg/ml. Further aspects provide a method for conferring estrogen responsive expression of a desired sequence within a mammalian cell, comprising: operably-linked placement, in an expression vector, of a sequence downstream from a SNP IFN-γ- 179G/T promoter; and introduction of the expression vector into a mammalian cell; wherein transcription of the downstream sequence is driven by the estrogen responsive SNP IFN-γ- 179G/T promoter. Preferably, the SNP IFN-γ-179G/T promoter comprises SEQ ID NO: 15. In particular embodiments, the method further comprises contacting the cell with at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to modulate SNP IFN-γ-179G/T promoter-driven expression in the cell. In particular gene therapy embodiments, introduction of the expression vector is by gene therapy, into a subject in need thereof. Preferably, the mammal is human. Preferably, the mammal is a human female.

While the description above refers to particular embodiments of the present invention, it will be understood that many modifications may be made without departing from the spirit thereof. The presently disclosed embodiments and ensuing Examples are therefore to be considered in all respects as illustrative and not restrictive.

Dosage and Formulation In the methods of the invention, estrogen is administered at a dose sufficient dose to reduce the severity of the particular immune pathology exhibited by the mammal. The dose will depend, among other considerations, on the type of estrogen, its fonnulation and route of administration, the duration of therapy, the type and severity of the pathology, and on the weight and gender of the mammal. A "low dose" refers to an amount sufficient to raise the serum concentration above basal levels, but below about pregnancy levels. Human female physiologic concentrations of El and E3 are roughly equivalent to those of E2, which circulates at 10 to 1,000 pg/ml during the normal menstrual cycle, and up to 35,000 pg/ml during pregnancy. Thus, a low dose of estrogen can raise serum El, E2 or E3 to at least 10 pg/ml, such as 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 75 pg/ml, 100 pg/ml, 150 pg/ml, 200 pg/ml, 300 pg/ml, 400 pg/ml, 500 pg ml, 750 pg/ml, 1000 pg/ml, 1500 pg/ml, and generally will not raise serum El, E2 or E3 beyond-2000 pg/ml. The amount of estrogen to administer to achieve desired hormone levels in the serum is known in the art, and will depend, for example, on the weight of the mammal, the half-life of the particular estrogen, and the route and form of administration. The . efficacy of a particular dose of estrogen can be monitored and adjusted during therapy by examining standard disease parameters. Those skilled in the art can determine an appropriate time and duration of therapy to achieve the desired preventative or ameliorative effects on the immune pathology. Thus, the methods of the invention can be practiced so as to maintain evels of estrogen in the blood for several days, weeks, months or years, or over the course of the lifetime of the individual. For example, the therapy can be administered continuously to an individual at risk of developing an immune pathology, such as an individual with a genetic predisposition to a pathology, or with preclinical indications of the pathology. Likewise, estrogen can be administered continuously to an individual early or late in the course of the disease, or only administered during exacerbations of the disease until symptoms are controlled. Doses of estrogen can be prepared in any convenient form and administered by any convenient route known in the art. Preferably, for human therapy, estrogen will be administered orally, transdennally, subcutaneously, intravenously, intramuscularly, by a respiratory route (e.g., inhalation), intranasal, enteral, topical, sublingual, or rectal means. Estrogen can also be administered directly to the site of the pathology, such as skin lesions, inflamed joints, into the central nervous system, and the like. For continuous release of defined concentrations of estrogen, administration via micropumps, biopolymers, liposomes and other slow-release vehicles is advantageous. Optionally, estrogen therapy can be combined with administration of an immunotherapeutic agent. As used herein, the term "immunotherapeutic agent" refers to any compound used prophylactically or therapeutically to inhibit an immune response or to ameliorate an immune pathology. Preferably, the immunotherapeutic agent will be administered at a lower dose than that required for complete efficacy on its own, such that when combined with administration of a dose of estrogen, there will be a pronounced effect on reduction of disease severity not achieved by the immunotherapy alone. Administering a lower dose of immunotherapeutic agent reduces the risk of adverse effects, as well as reduces the cost of therapy. The immunotherapeutic agent can be administered in combination with estrogen or separately; either before, at the same time, or after estrogen administration; either by the same route or by a different route (e.g., any of the routes described above); and either at the same site or at a different site. Those skilled in the art can determine appropriate conditions for administering both a dose of estrogen and an immunotherapeutic agent to a mammal. The choice of immunotherapeutic agent to use and route and site of administration will depend on the particular immune pathology. A variety of agents with at least partial efficacy in treating immune pathologies are known in the art, and their mechanisms of action are often well understood. Other immunotherapeutic agents with similar mechanisms of action are in development. The activation of a T cell immune response requires interaction between a T cell receptor on the surface of the pathogenic T cell, and an antigenic peptide bound to an HLA (MHC) molecule on the surface of an antigen presenting cell or target cell. Any agent which disrupts this trimolecular complex can be effective in combination with low dose estrogen therapy in reducing the T cell immune response. Agents which disrupt the trimolecular complex can be either immunomodulatory agents or immunoblocking agents, or act by both an immunomodulatory and a blocking mechanism. As used herein, the term "immunomodulatory agent" is intended to refer to an agent that induces a host immune response, such as a tolerogenic response or an active immune response in a mammal. Exemplary immunomodulatory agents that cause a tolerogenic response, which leads to immunological unresponsiveness, are autoantigens targeted by pathogenic T cells in an autoimmune response. Autoantigens and the adminstration of these autoantigens by a variety of routes (including oral and intravenous routes) so as to induce tolerance are known in the art and described, for example, in U.S. Pat. Nos. 6,039,947; 6,019,971; 5,869,093; 5,858,968 and 5,856,446 (incorporated herein by reference).

Gene Therapy/Delivery Methods. With respect to novel uses of the SNP IFN-γ- 179G/T promoter described herein below, therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 mg to about 2 mg, about 5 mg to about 500 mg, and about 20 mg to about 100 mg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations which will affect the dosage required for ultimate efficacy of the subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of subgenomic polynucleotides or the same amounts re-administered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to affect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect. The therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 7:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 7:185; and Kaplitt, Nature Genetics (1994) (5:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated. Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. PatentNo. 5, 219,740; WO 93/11230; WO 93/10218; U.S. Patent No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Tfier. (1992) 5:147 can also be employed. Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J Biol. Chem. 2(54:16985 (1989)); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Patent No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. 14:2411 (1994), and in Woffendin, Proc. Natl. Acad. Sci. (1994) °i:11581-11585. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA 91(24): 11581 (1994). Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Patent No. 5,206,152 and WO 92/11033). Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., U.S. Patent No. 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., U.S. Patent No. 5,206,152 and WO 92/11033).

EXAMPLE 1

(Non-classical Estrogen-Like-Response-Element (ERE) introduced by a variant SNP alleles was determined herein to display selective regulation in (e.g., alter expression in) PBMC but not LPMC, and also was shown to display gender-selective regulation) Recently, a single site nucleotide polymorphism (SNP) has been identified within the

IFN-γ region (Bream et al., Genes Immun. 3:165, 2002). The variant SNP allele involves a -

179G/T substitution. Enhanced TNF- stimulation of IFN-γ expression can be detected in highly activated T cells transfected with the SNP allele (Bream et al., Genes Immun. 3:165, 2002).

Interestingly, TNF-α and IFN-γ expression have been reported to synergistically enhance AIDS progression, and the presence of the variant SNP allele is associated with acceleration of AIDS progression, as measured by CD4⁺ count (An et al., J Infect. Dis. 188:228, 2003). The variant SNP allele creates a binding site with similarity to, but with differences from, an AP-1 binding element. Applicants, and disclosed herein, have discovered a putative non-classic ERE introduced by the variant SNP allele that displays selective regulation in PBMC but not in LPMC. The present Example shows that a SNP (-179G/T) recently detected within that region (Bream et al., Genes Immun. 3:165, 2002), exhibits selective allele-specific enhanced expression compared to the common allele in PBMC, but not in LPMC. In PBMC transfected with reporter constructs containing the polymorphic SNP allele, there is a CD2-mediated doubling of promoter activity compared to the common allele. In contrast, in LPMC, no allele specific differences in expression were detected. Signaling through the CD2 pathway in PBL, but not in LPMC, leads to upregulation of nucleo-protein binding to the SNP sequence. Sequence and binding analysis suggest that a putative non-classic estrogen response element (ERE) is introduced by the variant SNP allele. Following CD2 signaling, PBMC exhibit selective activation of ERE transactivation compared to LPMC, although LPMC express similar, if not greater, number of ER. Moreover, ERE transactivation is selectively sensitive to estrogen downregulation in PBMC but not in LPMC. Materials and Methods. Isolation of mononuclear cells. PBMC were isolated from normal healthy female volunteers by separation on Ficoll-Hypaque gradients. Intestinal specimens were obtained from female patients undergoing surgical resection of the colon at Cedars-Sinai Medical Center, Los Angeles. Approval for the use of human tissue was granted by the Institutional Review Board at Cedars-Sinai Medical Center. In this study, all tissue specimens were taken from an uninvolved area of resected colon from patients with colonic carcinoma (normal), involved areas from patients with ulcerative colitis, and uninvolved and involved areas from patients with Crohn's disease. LPMC were isolated from the resection samples using a technique modified from that described previously (Shanahan et al., Gastroenterology 92:1951, 1987). Briefly, the intestinal specimen was washed with HBSS, and the mucosa was dissected away from the underlying layers. The mucosal layer was incubated in a shaking water bath (100 rpm) in calcium- and magnesium-deficient HBSS, containing 1 mM EDTA, 50 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml fungizone, with the solution changed every 30 min until the supernatant was free of epithelial cells. The remaining LP was minced into 1- to 2-mm pieces and digested for 10 min in RPM 1640 containing 10% FCS, 0.5 mg/ml collagenase B (Roche Applied Science, Indianapolis, IN), 1 mg/ml hyaluronidase (Sigma, St. Louis, MO), 0.1 mg/ml deoxyribonuclease I (Sigma), 50 μg/ml gentamicin, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml fungizone in shaker water bath (100 rpm). The supernatant was collected, filtered through 110-μm nylon mesh (Spectrum Laboratory Products, Houston, TX), and centrifuged at 500 x g for 5 min. The cell pellet was resuspended in 15 ml and centrifuged at 30 x g for 5 min to remove epithelial and other large cells. The supernatant was removed and lymphocytes were isolated by separation on Ficoll-Hypaque gradients. The cells were then washed three times with HBSS and resuspended in RPM 1640 containing 10% FCS. Stimulation of mononuclear cells. PBMC or LPMC were stimulated with anti-CD2 antibodies (clones CB6 and GD10, a gift from Chris Benjamin, Biogen, Cambridge, MA) 0.1 μg/10 cells at 37°C for the times indicated for each experiment. Stimulation of LPMC and PBMC resulted in the secretion of an average of 2,100 and 1,400 ng/ml IFN-γ/10⁶ cells, respectively. Gel Mobility Electrophoretic Shift Assay (EMSA). Double stranded oligonucleotide was end-labeled with adenosine 5'-triphosphate, [γ-³²P] and T4 polynucleotide kinase. 3-6 μg of nuclear extract protein was incubated at 25°C with 0.25 mg/ml poly (dl-dC), in 20% glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris pH 7.5 for 10 min. The oligonucleotide was then added (20,000 cpm) and the binding reactions incubated for an additional 30 min. Specificity was detennined by the addition of 100-fold excess unlabelled oligonucleotide as competitor. The DNA-protein complexes were separated from unbound probe on a pre-run native 6% polyacrylamide gel in low ionic strength buffer (22.3 mM Tris pH 7.4, 22.3 mM Borate, 0.5 mM EDTA pH 8.0). After 2 h, the gel was dried under vacuum and exposed to X-ray film. The -204bp IFNG wt or SNP variant oligonucleotide used was as reported (Bream and Young, European Cytokine Network 11 (special issue):50, 2000): 5'- ATC GTC AAA GGA CCC AAG GA (wt) 5'- ATC GTC AAA TGA CCC AAG GA -3' (SNP). Consensus ERE, RAR, CREB, cEBP, AP-1, NFKB, Ets and OCT oligonucleotides were obtained from Santa Cruz Biotechnology were as follows: ERE, 5 '-GGA TCT AGG TCA CTG TGA CCC CGG ATC-3 ' (SEQ ID NO: 1); RAR, 5'-AGG GTA GGG TTC ACC GAAAGT TCA CTC-3' (SEQ IDNO:2); CREB, 5'-AGA GAT TGC CTGACG TCA GAGAGC TAG-3' (SEQ IDNO:3); c/EBP, 5'-TGC AGA TTG CGC AAT CTG CA-3' (SEQ ID NO:4); AP-1, 5'-CGC TTGATGACT CAG CCG GAA-3' (SEQ IDNO:5); NFKB, 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (SEQ IDNO:6); Ets, 5'-GGG CTG CTT GAG GAA GTA TAA GAA T-3' (SEQ IDNO:7); and OCT, 5'-TGT CGAATG CAAATC ACTAGAA-3' (SEQ ID NO:8).

Antibodies to estrogen receptor α (ERα) and ERβ were purchased from Affinity BioReagents (Golden, CO). Recombinant ERα and ERβ protein were purchased from

Invitrogen (Carlsbad, CA). The direct interaction between ERα and ERβ and the -204 bp IFNG

ERE introduced by the SNP was initiated by adding ERα and ERβ protein to nuclear extract from unstimulated cells and EMS A was performed per standard methods. Transfection. -179G common (wt) or variant SNP -179T allele IFNG reporter constructs have been previously described (Bream et al., Genes Immun. 3:165, 2002). Multimeric consensus ERE reporter construct was a gift from Peter Kushner (University of San Francisco). Freshly isolated PBMC or LPMC were transfected following overnight culture in RPM 1640 medium containing 10% FCS. Cells were then washed and resuspended in 250 μl fresh medium at 2 x 10⁷ cells/ml and electroporated in the presence of 50 μg of reporter construct (600 V, for 9 pulses of 500 μsec, with 100 μsec between pulses) using 4 mm (gap width) cuvettes in a BTX Electro Square Porator ECM 830 (Genetronics, Inc., San Diego, CA). A control plasmid containing the β-actin promoter driving a Renilla luciferase (provided by Dr. Christopher Wilson, University of Washington) was co-transfected as an internal standard and values were normalized to correct for transfection efficiency with normalized fold increase calculated from the empty pGL3 vector. After electroporation, the cells were diluted in fresh medium, allowed to rest for 1 h prior to plating, and then stimulated with anti-CD2 monoclonal antibodies for 4 h. Luminescence was measured using a Promega (Madison, WI) luciferase assay kit and counted on a 6-detector Perkin Elmer Life Sciences (Gaithersberg, MD) 1450 Microbeta liquid scintillation counter with coincidence counting deactivated. Flow Cytometric staining of ER . Freshly isolated PBMC or LPMC were rested overnight and then stained with mouse anti-human CD3 (Caltag Laboratories) for 15 min at room temperature. Cells were fixed and permeabilized using IntraPrep™ permeabilization reagents (Beckman Coulter, Inc., Fullerton, CA) then stained with rabbit anti-human ERα (HC- 20, Santa Cruz Biotechnology, Santa Cruz, CA), or control rabbit IgG (Caltag Laboratories, Burlingame, CA) and detected with donkey anti rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Following a final wash, the cells were analyzed using a BD FACScan flow cytometer (BD Biosciences, San Jose, CA). Statistical Analysis. Tests for statistical significance was determined using JMP Statistical Software (SAS Institute GmbH, Heidelberg, Germany).

RESULTS Allele specific enhanced expression of IFNG -179G/T SNP construct in PBMC but not in LPMC. To further define the functional significance of the variant SNP allele and its relationship to selected mechanisms of regulation of IFN-y expression in the mucosa, PBMC and LPMC were transfected with -204 bp constructs containing identical sequences, aside from for a one base pair substitution of the common -179G or variant SNP -179T regions. CD2- mediated signaling of PBMC resulted in transactivation of both the common and variant SNP allele constructs with a significant allele-specific enhancement in expression displayed for the variant SNP as compared to the common allele (p < 0.01, paired T test) (FIGURE 1). In contrast, when LPMC were transfected with the common and variant SNP allele constructs, no allele specific difference in the level of expression was detected (p > 0.05). CD2 stimulation resulted in allele specific upregulation of nuclear protein binding to the -179G/T SNP region in PBL, but not in LPMC. To evaluate the events involved in allele specific functional activation of IFN-γ expression, nuclear proteins were isolated from PBL and LPMC, and EMSA analysis was performed to measure DNA-binding activity to the common or variant SNP allele oligonucleotides. CD2 activation of PBL resulted in a rapid (within 30 min) and sustained upregulation of trans-acting factors binding to the variant SNP -179T allele, but no binding was detected utilizing the same extracts to the common -179G oligonucleotide probe. In contrast, LPMC demonstrated constitutive binding to both the common and variant SNP alleles, regardless of activation by CD2. A further difference was detected between PBMC compared to LPMC in the character of the binding to the variant SNP allele. Binding to the variant SNP oligonucleotide probe was specific, as excess unlabeled variant SNP, but not common probe oligonucleotide, competed for the binding. Binding to the variant SNP region in PBL was competed by as little as 5-fold excess oligonucleotide. In LPMC, however, a 500-fold excess unlabeled oligonucleotide was required to partially compete for binding suggesting non- specific binding interactions .

The nucleotides flanking the variant SNP -179T nucleotide participate in nucleoprotein complex formation. The data presented above suggest a selective role for CD2 in the regulation of nucleoprotein binding to the SNP polymorphic region in PBMC but not in LPMC. To further define the region involved in CD2-mediated upregulation of binding to the variant SNP allele in PBMC, mutational analysis was carried out utilizing a series of mutant oligonucleotides (see TABLE 1), in which substitutions were introduced outside of the polymorphic SNP base. These mutant oligonucleotides were used in cold competition experiments to compete for binding to the variant SNP allele. Mutation the region flanking the -179T results in oligonucleotides no longer capable of competing for nucleoprotein complex binding to the variant SNP region. These results suggest that in addition to the polymorphic G/T base, additional sequences in the flanking region contribute to the stability of nucleoprotein complex formation. The variant SNP allele sequence was determined herein to create a potential ERE binding site. Although a previous study had suggested that the variant SNP sequence created a new potential AP-1 binding site, competition and supershift analysis failed to support this hypothesis (Bream et al., Genes Immun. 3:165, 2002). Detailed sequence analysis was carried out to identify potential cis and trans-acting factors capable of binding to this region, which differ from that seen for the common sequence. Emphasis was placed on including an expanded region flanking the -179T nucleotide, which appears to actively participate in stabilizing nucleo-protein complex formation. TABLE 1 displays a number of putative binding sites in addition to AP-1 that are located within this region, including one for C EBP, which is believed to play a central role in chromatin restructuring to allow for transcriptional activation. Surprisingly, two putative hormone response elements, retinoic acid and estrogen receptor binding motifs, which belonged to the nuclear receptor super-family of hormone inducible transcription factors, had also been generated by the variant SNP allele. These were of particular interest in light of early studies, which had identified regulation of IFNG promoter expression by glucocorticoids, as well as vitamins A and D (Cippitelli and Santoni, Eur. J. Immunol. 28:3017, 1998 ; Cippitelli et al., J Biol. Chem. 271:26783, 1996). Competition experiments were carried out with consensus oligonucleotides to the various potential cis-acting elements. TABLE 1 summarizes the results of these experiments. Aside from the variant SNP oligonucleotide, only a consensus oligonucleotide to the estrogen-response element successfully competed for binding to the variant SNP allele oligonucleotide.

Table 1. Competition with consensus oligonucleotides to various potential cis-acting elements

Element Sequence _N( Competition wt ATCGTCA^ AMGGA CCCAAGGA 9 SNP ATC|GTCA| AA|TGA CC[CAAGGA 10 SNPml ATCGTTC AAWGA CCCAAGGA 11 SNPm2 ATCJGTCA| AA|TGA AC|CAAGGA 12 SEQ ID Element Sequence Competition NO: SNPml2 ATCGTTC AAffGA ACCAAGGA 13 SNPm4 ATC|GTCA| AAJTCA CC|CAAGGA 14 ER GGATCTAGGTCAETGTGA CCCCGGATC 1 RAR CGAGTG CTTTCGG| GAACC|CTACCCTAGA 2 CREB AGAGATTGCC|TGA CG[TCAGAGAGCTAG 3 C/EBP 4 TGCAGATTGCGCAATC|TG CA| AP-1 CGCTTGA|TGA CT|CAGCCGGAΆ 5 6 NFKB AGTTGA GGGGACTTTCCCAGGC ETS GGGCTGCT|TGA GGIAAGTATAAGAAT 7 OCT TGTCGAATGCAAA|TCA CT[AGAA

Representative results of 2-3 experiments with similar results are shown.

Next, the possibility that the SNP introduces an ERE element, which serves as a site for CD2-mediated upregulation of promoter activation was examined. Examination of the sequence flanking the -179T variant SNP allele reveals imperfect homology to the palindromic consensus ERE sequence 5'-GGTCAnnnTGACC-3'. The -179T variant SNP allele corresponds with complete homology to the right half site of the ERE binding region while the 5' flanking sequence displays partial homology. However, many genes that contain EREs have been found to vary from the consensus sequence by one or more nucleotides. To further explore whether CD2 mediated activation results in binding to an ERE element, nuclear proteins were isolated from PBL and EMSA analysis was performed. CD2 activation of PBL resulted in upregulation of trans-acting factors binding to a consensus estrogen-response element. To further define the nature of this interaction, nuclear extracts were prepared from PBL stimulated with CD2 for 60 min, and nucleo-protein binding was carried out to a consensus estrogen-response element in the presence of excess competing oligonucleotide. Binding to the consensus estrogen-response element was competed by both a consensus estrogen-response element, as well as the variant SNP -179T allele oligonucleotide. A series of mutant oligonucleotides were generated, which were targeted to disrupt within -179T region exhibiting complete homology to the ERE binding region or displaying partial homology to the 5' flanking region. Not all mutations that disrupt classic ERE binding were capable of abolishing binding to the variant SNP allele oligonucleotide. Mutation of the -179T variant SNP region abolished its capability to compete for the binding of the protein complex, whereas mutation of the region flanking the -179T, which exhibited only partial homology to the ERE binding sequence, did not affect its ability to successfully compete for binding. These results suggests that nucleo-protein complex generated following CD2 activation requires the homology to the -179T variant SNP region for functional binding to the ERE. Supershift analysis was performed by preincubating nuclear protein extracts with antibodies specific to ERE-α and ERE-β. The analysis revealed supershift of the nucleoprotein complex binding to the -179T variant SNP region by anti-ERE-α but not anti-ERE-β. Furthermore, a direct interaction between estrogen receptor alpha and estrogen receptor beta and the variant SNP -179T region was analyzed by EMSA analysis using recombinant estrogen receptor alpha and beta proteins and DNA probes corresponding to the -179T variant SNP region. As seen in FIGURE 2, no binding was detected in extract from unstimulated PBMC. However, binding was initiated by addition of recombinant estrogen receptor alpha and beta proteins, with enhanced binding revealed when comparing estrogen receptor alpha with beta proteins. These data demonstrate that while both ERα and ERβ can bind onto the variant SNP -179T ERE region there is enhanced preference of binding of ERα.

Enhanced expression of a consensus ERE reporter construct was observed in PBL compared to LPMC. In order to establish CD2-mediated functional activation of an estrogen response element, transfection experiments were carried out utilizing a multimeric consensus ERE reporter construct. CD2 ligation resulted in transactivation of a consensus ERE construct in both PBL and LPMC (FIGURE 3). The increased in promoter activity induced in PBL was 9- fold while only a modest 2-fold response was seen in LPMC. Moreover, as seen in FIGURE 3, CD2- mediated expression of the estrogen response element was effectively blocked in the presence of estradiol in PBL (p < 0.01, Wilcoxon Sign-Rank), while expression in LPMC remained virtually unchanged (p > 0.05, Wilcoxon Sign-Rank). However, estrogen inhibited PMA/ionomycin activation of consensus ERE in both PBL and LPMC (FIGURE 4). Significantly, these results indicate that ERE like elements within the IFNG promoter function as targets for estrogen-mediated regulation of IFN-γ expression. Comparative levels ofERa expressed on both CD3⁺ PB and LP T cells. To determine whether differences in response between PBL and LPMC were attributed to the level of ER expression, CD3 PB-T and LP-T cells were analyzed by flow cytometry for expression of ERα (FIGURE 10). A similar level of expression was detected in both PB T and LP T cell populations indicating that modulation of promoter expression by estrogen was not merely a function of receptor availability.

Treatments effecting a selective immune compartments The experiment described below with the IFN-γ -6.8kb to -204bp construct (FIGURE 8) indicates that, in addition to the SNP, there are other estrogen response elements upstream of the -204bp position. Additionally, because there are differences in the mechanisms of E2 inhibition of IFN-γ expression between PBL and LPMC (e.g., transcriptional vs. post-transcriptional), additional aspects of the present invention provide for treatments affecting a selective immune compartment (e.g., PBL or LPL), which would allow for lowering of IFN-γ but not totally wiping it out (an advantageous result as described herein above). In this regard, a series of successively truncated IFN-γ promoter-constructs was utilized to map the estrogen response elements. FIGURE 11 summarizes the results. Estrogen inhibited CD2-mediated IFN g promoter activation in PBL, however, not all promoter constructs were repressed to the same extent. Estrogen sensitivity mapped to the region up-stream of the -204 bp basal promoter region and was selective for PBL. In contrast, CD2-mediated IFN g promoter activation in LPMC was relatively unaffected following estrogen treatment. Selective sensitivity of PBL to Estrogen treatment was activation pathway dependent since following PMA ionomycin activation, IFN g promoter expression was inhibited to a similar extent in both PBL and LPMC. Conferring Estrogen-Responsive Expression Therefore, particular aspects of the present invention provide a method for conferring estrogen responsive expression of a desired sequence to a cell, comprising: placement, in an expression vector, of a sequence downstream from the SNP IFN-γ- 179G/T promoter; and introduction of the expression vector into a cell; wherein transcription of the downstream sequence is driven by the estrogen responsive SNP IFN-γ- 179G/T promoter. In particular aspects the method further comprises contacting the cell with at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to modulate SNP IFN-γ- 179G/T promoter- driven expression in the cell.

Treatment of HIV/AIDS and Related Diseases or Conditions As discussed herein above, the single site nucleotide polymorphism ("SNP"), known as - 179G/T, imparts tumor necrosis factor alpha ("TNF-α") stimulation of IFN-γ production, and is linked to acceleration of acquired immune deficiency syndrome ("AIDS") progression. As seen in FIGURE 3, CD2-mediated expression of the estrogen response element was effectively blocked in the presence of estradiol in PBL. These results indicate that ERE like elements within the IFNG promoter function as targets for estrogen-mediated regulation of IFN- γ expression. Moreover, these results are consistent with those shown in Example 2 below, which show that Estrogen inhibits IFN-γ secretion, IFN-γ expression and IFN-γ promoter- reporter gene constructs in PBL, and further inhibits IFN-γ secretion in LPMC and also PMA Ionomycin activation of IFN-γ in both PBL and LPMC. Therefore, additional aspects of the present invention provide a method for treating HJV/AIDS and related diseases and disorders, comprising administration of a an amount of estrogen sufficient to reduce IFN-γ expression, wherein acceleration of acquired immune deficiency syndrome ("AIDS") progression is impeded. According to particular aspects, estrogen (and estriol, estrone, and selective estrogen receptor modulators (SERMs)) has substantial utility for treatment and/or prevention of HTV- related cellular effects, conditions and diseases, including treatment and/or prevention of AIDS. The CD4+ T- lymphocyte coordinates a number of important immunologic functions, and a loss of these functions results in progressive impairment of the immune response. Studies of the natural histoiy of HTV infection have documented a wide spectrum of disease manifestations, ranging from asymptomatic infection to life-threatening conditions characterized by severe immunodeficiency, serious opportunistic infections, and cancers. Other studies have shown a strong association between the development of life-threatening opportunistic illnesses and the absolute number (per microliter of blood) or percentage of CD4+ T- lymphocytes. As the number of CD4+ T-lymphocytes decreases, the risk and severity of opportunistic illnesses increase. Therefore, according to aspects of the present invention, estrogen (and estriol, estrone, and selective estrogen receptor modulators (SERMs)) has substantial utility of treatment or prevention of HTV infection and/or replication or progression and HTV-related cellular effects, conditions and diseases. The phrase "HTV-related cellular effects, conditions and diseases" or "HTV-related (or mediated) conditions or diseases" refers to those illnesses and conditions included in, but not necessarily limited to the CDC 1993 AIDS surveillance case definition, as follows: Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5 C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HTV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1 month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent term); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M. kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HTV. Preferably, the method further comprises administration of an anti-HTV agent selected from the group consisting of: nucleoside reverse transcriptase inhibitors (NRTIs); non- nucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors (Pis); fusion inhibitors (FI), and combinations thereof. Preferably, the reverse transcriptase inhibitor (NRΗ) is selected from the group consisting of: lamivudine and zidovudine; FTC, emtricitabine; lamivudine, 3TC; abacavir/ lamivudine; zalcitabine, ddC, dideoxycytidine; zidovudine, AZT, azidothymidine, ZDV; abacavir, zidovudine, and lamivudine; tenofovir disoproxil emtricitabine; enteric coated didanosine; didanosine, ddl, dideoxyinosine; Didanosine (ddl) delayed release capsules; tenofovir disoproxil fumarate; stavudine, d4T; abacavir, and combinations thereof. Preferably, the non-nucleoside reverse transcriptase inhibitor (NNRTI) is selected from the group consisting of: delavirdine, DLV; efavirenz; nevirapine, BI-RG-587, and combinations thereof. Preferably, the protease inhibitor (PI) is selected from the group consisting of:

Amprenavir; indinavir, IDV, MK-639; saquinavir mesylate, SQV; saquinavir; lopinavir and ritonavir; Fosamprenavir Calcium; ritonavir, ABT-538; atazanavir sulfate; nelfinavir mesylate, NFV, and combinations thereof. Preferably, the fusion inhibitor (FI) comprises enfuvirtide, T-20.

Applicants were motivated to determine whether additional non SNP estrogen regulatory elements might be present in the wild-type IFN-γ promoter. As shown in Example 2 below, this is in fact the case, thus further supporting the therapeutic use of estrogen to treat IFN-γ-mediated diseases and disorders (e.g., inflammation, HIV/AIDS, etc). EXAMPLE 2 (Estrogen was shown herein to inhibit IFN-γ secretion, IFN-γ expression and IFN-γ promoter- reporter gene constructs in PBL, and was further shown to inhibit IFN-γ secretion in LPMC and also PMA/ionomycin activation of IFN-γ in both PBL and LPMC) Particular aspects of the present invention are based, at least in part, on the present investigation of the role of estrogen in the regulation of IFN-γ expression, and the surprising discovery that estrogen has substantial utility to down-regulate IFN-γ expression and secretion, and the discovery of molecular-based gender disparities; estrogen downregulates IFN- γ protein secretion in a gender-dependent and dose-dependent fashion. In accordance with one embodiment, treatment with as little as 10 nM estradiol resulted in a 40%) decrease in IFN- γ protein secretion in PBMC isolated from female donors, whereas little, if any, inhibition was detected in PBMC from male donors. A pronounced transcription downregulation was likewise detected in distal promoter regions spanning up to -6.8 kb upon transient transfection of reporter constructs in PBMC isolated from female donors. Figure 4 shows, according to particular aspects, that estrogen inhibits PMA/ionomycin activation of consensus IFN- γ ERE In both PBL and LPMC. Figure 5 shows, according to particular aspects, that estrogen inhibits IFN- γ secretion in PBL. Figure 6 shows, according to particular aspects, that estrogen inhibits IFN- γ mRNA in PBL. Figure 7 shows, according to particular aspects, that estrogen inhibits IFN- γ secretion in LPMC. Figure 8 shows, according to particular aspects, that estrogen inhibits CD2-mediated activation of -6.8 kb IFN- γ in PBL but not in LPMC. Figure 9 shows, according to particular aspects, that estrogen inhibits PMA/ionomycin activation of IFN- γ in both PBL and LPMC. Figure 10 shows, according to particular aspects, that comparable expression of ERa in CD3+ T cells from PBL and LPMC. Figure 11 shows, according to particular aspects, inhibition of IFN- γ expression by estradiol. A series of successively truncated IFN-γ promoter-constructs was utilized to map the estrogen response elements. Estrogen inhibited CD2-mediated IFN g promoter activation in PBL, however, not all promoter constructs were repressed to the same extent. Estrogen sensitivity mapped to the region up-stream of the -204 bp basal promoter region and was selective for PBL. In contrast, CD2-mediated IFN g promoter activation in LPMC was relatively unaffected following estrogen treatment. Selective sensitivity of PBL to estrogen treatment was activation pathway dependent since following PMA/ionomycin activation, IFN g promoter expression was inhibited to a similar extent in both PBL and LPMC.

Claims

CLAIMS 1. A method for reducing IFN-gamma expression or secretion in cultured mammalian cells, comprising: obtaining mammalian lymphoid cells; and culturing the cells in the presence of an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in the cultured mammalian lymphoid cells. 2. The method of claim 1, wherein the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. 3. The method of claim 2, wherein the cultured mammalian cells comprise peripheral blood leukocytes (PBL). 4. The method of claim 1 , wherein the cultured mammalian cells are female cells. 5. The method of claim 1 , wherein the mammal is human. 6. The method of claim 1, wherein the agent is 17β-estradiol. 7. The method of claim 1, wherein the amount of the agent is selected from the group consisting of 1 nM to about 100 nM, 1 nM to about lOnM, 1 nM to about 75 nM, 1 nM to about 50 nM, at least 1 mM, at least 10 nM, at least 50 nM, at least 75 nM, and at least 100 nM. 8. The method of claim 6, wherein the amount of the agent is at least 1 nM, at least

10 nM, or at least 100 nM. 9. The method of claim 7, wherein the amount of the agent is at least 10 nM. 10. A method for ex-vivo therapy for an IFN-gamma-mediated condition or disease, comprising: isolating lymphoid cells from a mammalian subject; culturing the isolated lymphoid cells in the presence of an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in the cultured mammalian lymphoid cells; and transferring at least some of the cultured cells back into the mammalian subject to provide ex-vivo therapy for an IFN-gamma-mediated condition or disease. 11. The method of claim 10, wherein the cultured mammalian lymphoid cells comprise cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. 12. The method of claim 11, wherein the cultured mammalian cells comprise peripheral blood leukocytes (PBL). 13. The method of claim 10, wherein the subject is female. 14. The method of claim 10, wherein the mammal is human. 15. The method of claim 10, wherein the agent is 17β-estradiol. 16. The method of claim 10, wherein the amount of the agent is selected from the group consisting of 1 nM to about 100 nM, 1 nM to about lOnM, 1 nM to about 75 nM, 1 nM to about 50 nM, at least 1 mM, at least 10 nM, at least 50 nM, at least 75 nM, and at least 100 nM. 17. The method of claim 16, wherein the amount of the agent is at least 1 nM, at least 10 nM, or at least 100 nM. 18. The method of claim 17, wherein the amount of the agent is at least 10 nM. 19. A method for treating an IFN-gamma-mediated condition or disease in a mammal, comprising administering to a mammalian subject in need thereof an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in one or more lymphoid cells of the subject. 20. The method of claim 19, wherein the lymphoid cells comprise one or more cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. 21. The method of claim 20, wherein the lymphoid cells comprise one or more peripheral blood leukocytes (PBL). 22. The method of claim 19, wherein the condition or disease comprises inflammation. 23. The method of claim 19, wherein the mammal is human.

24. The method of claim 23, wherein the condition or disease comprises HIV/AIDS or an HTV-related cellular condition or disease. 25. The method of claim 19, wherein the subject is female. 26. The method of claim 19, wherein the agent is 17β-estradiol. 27. The method of claim 19, wherein the amount of the agent is sufficient to raise the serum concentration of the agent to within a range selected from the group consisting of 30 pg/ml to 1000 pg/ml, 50 pg/ml to 500 pg/ml, and 100 pg/ml to 250 pg/ml. 28. The method of claim 27, wherein the amount of the agent is sufficient to raise the serum concentration of the agent to within 100 pg/ml to 250 pg/ml. 29. The method of claim 19, wherein the amount of the agent is sufficient to raise the serum concentration of the agent to a value greater than 250 pg/ml. 30. The method of claim 24, further comprising administration of an anti-HTV agent. 31. The method of claim 30, wherein the anti-HTV agent is selected from the group consisting of: nucleoside reverse transcriptase inhibitors (NRTIs); non-nucleoside reverse transcriptase inhibitors (NNRTIs); protease inhibitors (Pis); fusion inhibitors (FI), and combinations thereof. 32. The method of claim 31, wherein the nucleoside reverse transcriptase inhibitor (NRTI) is selected from the group consisting of: lamivudine and zidovudine; FTC, emtricitabine; lamivudine, 3TC; abacavir/ lamivudine; zalcitabine, ddC, dideoxycytidine; zidovudine, AZT, azidothymidine, ZDV; abacavir, zidovudine, and lamivudine; tenofovir disoproxil/emtricitabine; enteric coated didanosine; didanosine, ddl, dideoxyinosine; Didanosine (ddl) delayed release capsules; tenofovir disoproxil fumarate; stavudine, d4T; abacavir, and combinations thereof. 33. The method of claim 31, wherein the non-nucleoside reverse transcriptase inhibitor (NNRTI) is selected from the group consisting of: delavirdine, DLV; efavirenz; nevirapine, BI-RG-587, and combinations thereof. 34. The method of claim 31, wherein the protease inhibitor (PI) is selected from the group consisting of: Amprenavir; indinavir, JDY, MK-639; saquinavir mesylate, SQV; saquinavir; lopinavir and ritonavir; Fosamprenavir Calcium; ritonavir, ABT-538; atazanavir sulfate; nelfinavir mesylate, NFV, and combinations thereof. 35. The method of claim 31, wherein the fusion inhibitor (FI) comprises enfuvirtide, T-20. 36. The method of claim 24, wherein the HTV-related cellular effect, condition or disease is selected from the group consisting of: AIDS; Bacillary angiomatosis; Candidiasis of bronchi, trachea, or lungs; Candidiasis, esophageal; Candidiasis, oropharyngeal (thrush); Candidiasis, vulvovaginal; persistent, frequent, or poorly responsive to therapy; Cervical dysplasia (moderate or severe)/cervical carcinoma in situ; Cervical cancer; Coccidioidomycosis, disseminated or extrapulmonary; Constitutional symptoms, such as fever (38.5°C) or diarrhea lasting greater than 1 month; Cryptococcosis, extrapulmonary; Cryptosporidiosis, chronic intestinal (greater than 1 month's duration); Cytomegalovirus disease (other than liver, spleen, or nodes); Cytomegalovirus retinitis (with loss of vision); Encephalopathy, HTV-related; Herpes simplex: chronic ulcer(s) (greater than 1 month's duration); or bronchitis, pneumonitis, or esophagitis; Hairy leukoplakia, oral; Herpes zoster (shingles), involving at least two distinct episodes or more than one dermatome; Histoplasmosis, disseminated or extrapulmonary; Idiopathic thrombocytopenic purpura; Isosporiasis, chronic intestinal (greater than 1-month's duration); Kaposi's sarcoma; Listeriosis; Lymphoma, Burkitt's (or equivalent tenn); Lymphoma, immunoblastic (or equivalent term); Lymphoma, primary, of brain; Mycobacterium avium complex or M kansasii, disseminated or extrapulmonary; Mycobacterium tuberculosis, any site (pulmonary or extrapulmonary); Mycobacterium, other species or unidentified species, disseminated or extrapulmonary; Peripheral neuropathy; Pelvic inflammatory disease, particularly if complicated by tubo-ovarian abscess; Pneumocystis carinii pneumonia; Pneumonia, recurrent; Progressive multifocal leukoencephalopathy; Salmonella septicemia, recurrent; Toxoplasmosis of brain; and Wasting syndrome due to HTV, and combinations thereof 37. A method for treating an IFN-gamma-mediated condition or disorder in a gender- dependent manner, comprising administering to a mammalian subject in need thereof an amount of at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to reduce IFN-gamma expression or secretion, at least to some extent, in one or more lymphoid cells of the subject, wherein the sensitivity or extent of reduction of IFN-gamma expression or secretion is greater for females than for males. 38. The method of claim 37, wherein the lymphoid cells comprise one or more cells selected from the group consisting of T cells, peripheral blood leukocytes (PBL), lamina propria mononuclear cells (LPMC), and combinations thereof. 39. The method of claim 38, wherein the lymphoid cells comprise one or more peripheral blood leukocytes (PBL). 40. The method of claim 37, wherein the condition or disease comprises inflammation. 41. The method of claim 37, wherein the mammal is human. 42. The method of claim 41, wherein the condition or disease comprises HTV/AIDS or an HTV-related cellular condition or disease. 43. The method of claim 37, wherein the agent is 17β-estradiol. 44. The method of claim 37, wherein the amount of the agent is sufficient to raise the serum concentration of the agent to within a range selected from the group consisting of 30 pg/ml to 1000 pg/ml, 50 pg/ml to 500 pg/ml, and 100 pg/ml to 250 pg/ml. 45. The method of claim 44, wherein the amount of the agent is sufficient to raise the serum concentration of the agent to within 100 pg/ml to 250 pg/ml. 46. The method of claim 37, wherein the amount of the agent is sufficient to raise the serum concentration of the agent to a value greater than 250 pg/ml. 47. A method for conferring estrogen responsive expression of a desired sequence within a mammalian cell, comprising: operably-linked placement, in an expression vector, of a sequence downstream from a SNP IFN-γ- 179G/T promoter; and introduction of the expression vector into a cell; wherein transcription of the downstream sequence is driven by the estrogen responsive SNP IFN-γ- 179G/T promoter. 48. The method of claim 47, wherein the SNP IFN-γ- 179G/T promoter comprises SEQ ID NO: 15. 49. The method of claim 47, further comprising contacting the cell with at least one agent selected from the group consisting of estrogen, estriol, estrone and a selective estrogen receptor modulator (SERM), the amount sufficient to modulate SNP IFN-γ- 179G/T promoter- driven expression in the cell. 50. The method of claim 47, wherein introduction of the expression vector is by gene therapy, into a subject in need thereof. 51. The method of claim 47, wherein the mammal is human.