We show here that PLC-γ activated by the insulin pathway participates broadly and positively during cell growth modulating EGF pathway activity, whereas in cell differentiation PLC-γ activated by the insulin receptor negatively regulates the EGF pathway. These roles require different SH2 domains of PLC-γ, and act via classic PLC-γ signaling and EGF ligand processing. By means of PLC-γ, the insulin receptor therefore modulates differentiation as well as growth. Overall, our results provide evidence that PLC-γ acts during development at a time when growth ends and differentiation begins, and is important for proper coordination of these two processes.Coordination between growth and patterning/differentiation is critical if appropriate final organ structure and size is to be achieved. Understanding how these two processes are regulated is therefore a fundamental and as yet incompletely answered question. Here we show through genetic analysis that the phospholipase C-γ (PLC-γ) encoded by  IP3 promotes the release of Ca2+ from intracellular stores, via its association with the inositol triphosphate receptor (IP3R), while DAG, together with Ca2+, activates protein kinase C (PKC) During development, proper coordination of growth, proliferation, and cellular differentiation is critical for appropriate final organ structure and size. These processes involve signal transduction cascades that determine the spatial and temporal pattern of cellular instructions. Many of these signaling pathways are activated by growth factors via receptor tyrosine kinases (RTKs). In vertebrates, phospholipase C-Γ (PLC-Γ) is required by some RTKs to regulate these cellular processes. PLC-Γ is an intracellular enzyme activated by a multistep process; the initial step involves membrane association via RTKs (or non-receptor tyrosine kinases) through an SH2 (Src-homology 2) domain in PLC-γ with a phosphotyrosine residue in the intracellular domain of the activated receptor. Following this binding, PLC-γ is phosphorylated by the RTK on one or more tyrosines and then catalyzes the hydrolysis of the membrane phospholipid phosphatidylinositol  bisphosphate  sl null mutants are recovered, and are viable and fertile. Yet, sl homozygotes do have developmental defects: their eyes have a mildly rough phenotype, due to some ommatidia containing one or more extra R7 photoreceptor cells, and their wings are smaller than wild type and contain patches of ectopic veins In sl was also identified in a cell-based RNAi screen for loss-of-function phenotypes that inhibit endoplasmic reticulum (ER) accumulation of processed Spitz (Spi), which is an EGF receptor ligand sl-encoded PLC-γ participates in a Spi retention mechanism in the ER. Genetic rescue experiments indicate that Sl is required specifically in cells processing the ligand (a non-cell autonomous effect), as demonstrated by the rescuing capacity in sl mutant flies of wild type Sl expression in R8 photoreceptor cells only Genetic experiments suggest that the extra R7 photoreceptor phenotype results from over activation of the MAPK cascade, showing that Sl is a negative regulator of this pathway during photoreceptor development DrosophilaNevertheless, other questions remain. What is the mechanism by which Sl regulates these different processes , and how is Sl itself activated? Do other RTKs activate Sl, as is the case for PLC-γ in vertebrates? Besides the EGF receptor, an interesting additional RTK candidate for Sl activation is the insulin receptor, because the insulin pathway regulates growth and cellular proliferation in all tissues in 3R and Pkc53E, Sl stimulates cellular growth. Also, in the developing wing and eye, Sl activated by the insulin receptor inhibits MAPK signaling and thus cellular differentiation in a manner again dependent on PLC-γ signaling downstream components, such as the IP3R. We also show that PLC-γ genetically interacts with Star and Rhomboid, suggesting modulation of the processing of the EGF ligand Spitz as an avenue of Sl function in all these different phenotypes.Here, we show that Sl stimulates growth. We use the wing to show through genetic experiments that together with the insulin and EGF/MAPK pathways and in a manner dependent on components downstream of Sl such as the IPFinally, we also show that the C-SH2 and N-SH2 domains individually are not required for cellular growth, whereas only the N-SH2 domain is required for patterning/differentiation.sl1 and sl2 mutants were previously shown to be reduced in length by about 20% sl mutant wings and found that the area of the wing blade is also reduced by 20%  showed a similar effect to mutations in genes of the MAPK pathway  binds to the cytoplasmic substrate Chico (a Drosophila homolog of IRS), which leads to the activation of the lipid kinase phosphoinositide 3-kinase (PI3K) and, further downstream, the ser/thr kinase protein kinase B   pathway . In cont3 and DAG by PIP2 hydrolysis, leading to modulation of Ca2+ release by IP3 receptors and PKC activation. In order to study PKC involvement, we characterized a mutation in one of the fly homologs of PKC, PKC53E. PKC53E mutants are homozygous viable and fertile, yet have a reduction in wing size of 11.5%, similar to sl mutants; by contrast, PKC53E heterozygotes have wild-type wings , which encodes a Golgi membrane protease that cleaves the inactive, membrane bound Spi precursor to generate mature, secretable Spi Star (S), which encodes a type II transmembrane protein involved in trafficking Spi from the ER to the Golgi spi itself. Flies heterozygous for mutations in these three genes in an sl2 mutant background indeed showed greater reductions in wing size than solely sl2 mutant flies . We also evaluated pupae using the same genotypes, measuring both pupal length 9sl homozygotes to 3.14±0.009 mm for 9sl heterozygotes measuring pupal longest axis, and 1.446±0.012 mg for 9sl homozygotes, to 1.464±0.012 mg for 9sl heterozygotes. n = 73 for 9sl homozygotes, and n = 79 for 9sl heterozygotes; mean ± SEM). These analyses show that Sl plays a more general role in the promotion of cell growth, beyond the one classically described in the wing.If Sl is involved more globally in growth control we might expect to see a change in the overall size of the fly, as observed for mutations in the insulin signaling pathway sl2 mutant flies sometimes show small ectopic wing veins, indicating an excess of cellular differentiation sl2 mutant wings, indicating that the ectopic veins are a result of over activation of MAPK signaling, the exceptions being aos and spi. Consistent with its role as an EGFR inhibitory ligand, heterozygosity for aos significantly augmented the ectopic vein sl phenotype, strongly implying Aos in the regulation of vein differentiation. In contrast, heterozygosity for spi had no significant effect, indicating that for vein differentiation Vn, rather than Spi, is critical , one shows a significant decrease (Dp110) and two others (chico and PKB) are unchanged . In each case we observed low percentages of wings with small ectopic vein-like patches , confirming their participation in vein differentiation  for sibling 2sl males with a copy of GC25InR. We found the same result with another hypomorphic allele of PLC-γ: 1sl  for sibling 1sl males with a copy of GC25InR. By contrast, in an 9sl background, the InR mutation did not increase the severity of the R7 phenotype . A similar effect was observed for the same 3RIP mutation in an sl9 mutant background . We examined whether this dominant phenotype could be modified in an sl mutant background, and found that in S2 heterozygotes the R7 phenotype is not changed if also mutant for sl2 , C-SH2 mutant (C-SH2) or N-SH2 mutant (N-SH2) constructs rescued the wing size defect in sl2 mutants (In order to study in more detail the relationship between Sl and receptor tyrosine kinases (insulin and EGF receptors) in the wing and eye, we examined the role of the Sl SH2 domains, which are known to be involved in the binding of PLC-γ to RTKs in vertebrates. We generated  systems  [21], [2 mutants . However mutants . These rsl mutant wings have a reduction in cell growth but not cell proliferation. This defect is qualitatively similar to mutations in MAPK signaling; cells with homozygous mutations for members of this pathway have higher cell densities, suggesting smaller cells Drosophila wing growth We show, by measuring cell density, that sl mutant eyes very likely contain fewer cells, despite the fact that some ommatidia sport one or two extra R7 cells, as the number of cells missing due to reduced numbers of ommatidia is bigger than the number of extra R7 cells present. This suggests either reduced proliferation or increased cell death in differentiating sl mutant eyes, and is different from the growth defect found in wings, yet consistent with a moderate requirement of MAPK output to promote growth and cellular survival.We found that Sl regulates cellular growth in the eye. Whole eyes are smaller, and the difference in size can be largely explained by the presence of fewer ommatidia. This means that sl loss-of-function clones in the eye, we determined that sl mutant cells are smaller than neighboring wild-type cells. This growth defect is similar to that seen in wings, suggesting a more general growth regulation by Sl in different tissues, albeit in a situation where competition between cells of different genotype does take place.We also surveyed whether mutant eye cells are smaller, in a situation where a direct comparison could be made between mutant and wild-type cells. By studying sl homozygotes; we found that the adult animal as a whole has reduced mass. Given that the reduction in mass (8%) is of a similar magnitude to the reduction in cell size in the eye (15%) and wing (20%), the most parsimonious explanation for this change in mass is that the same Sl functions found in the eye and wing are required more generally throughout the animal, suggesting that cell size may be reduced in many tissues. However, we found that the reduced growth observed in the adult was not reflected by a reduction in length of sl mutant pupae. This is in contrast to mutations of other genes involved in growth control, such as the neurofibromin 1 gene sl has a relatively small effect on growth, varying between 5% and 20% in different contexts, so our sample may not have been large enough to observe a small change in mean length. Given that Sl does not appear to affect the length of appendages other than the wing , it may be that there are other compensatory effects resulting from lost Sl function that maintain the pupal case at an approximately wild-type length.Not only is cell size reduced to a similar extent in both the eye and wing of DrosophilaAnother complementary explanation for the reduction in adult mass is via a role for Sl on nutrient sensing. As Sl is clearly involved in insulin signaling, and as insulin is required for integrating nutrient sensation in Drosophila and PC12 cells.We propose that the overall role for Sl is to act as a pro-growth agent, allowing cells and tissues to attain normal numbers and sizes. This is achieved by dampening MAPK output in growth control in a non-cell autonomous manner, by restricting processing of EGFR ligand(s), as shown previously for R7 cell differentiation sl mutant flies show ectopic vein patches indicating an excess of vein differentiation. Here we show that heterozygosity for MAPK pathway genes, in a sensitized sl mutant background, caused a partial rescue of the sl vein differentiation defect, indicating that the sl phenotype is a consequence of MAPK over activation. In this case, Sl acts as a negative regulator of MAPK signaling.As opposed to promoting wing cell growth, Sl inhibits wing vein formation. Positioning and differentiation of provein and intervein territories in the wing are determined by at least five different signaling pathways: Hedgehog (Hh), Decapentaplegic , epidermal growth factor (EGF)/MAPK, Wingless , and Notch The dual roles played by Sl in the wing via the same MAPK pathway – promotion of growth and subsequently, promotion of differentiation –– can be explained if Sl is helping to establish appropriate MAPK activity at each developmental stage. Alternatively, given that growth and differentiation tend to be temporally separated sl mutant backgrounds. These results clearly indicate a requirement for the insulin receptor in Sl-mediated inhibition of vein differentiation, suggesting that Sl is activated by this receptor. In mammalian cells there is some evidence for this type of association Drosophila. Such an interaction might be direct via one of the Sl SH2 domains, or indirectly through an adaptor such as Lnk.Strikingly, there appears to be no role for the insulin pathway in vein differentiation, save for the insulin receptor itself; however, this finding is consistent with studies showing that the insulin pathway as a whole is generally not involved in differentiation sl mutant eyes indicates that Sl normally inhibits R7 differentiation, resulting in only one R7 cell per ommatidium sl eye defect sl phenotype in an sl mutant background. Thus, in this instance as well, the insulin receptor appears to activate Sl to enable it to regulate R7 differentiation. This eye phenotype therefore follows the same logic as vein differentiation, constituting another case of close association between the insulin receptor and Sl. As Sl does not show the same interaction with Chico as with InR, activation of Sl via InR may require either direct binding of Sl to InR or another insulin pathway adaptor molecule, such as Lnk Photoreceptor differentiation begins in the third larval instar with the passage of the morphogenetic furrow across the eye imaginal disc. The R8 photoreceptor differentiates first, nucleating developing ommatidia without requiring EGF/MAPK activation. In contrast, the R7 photoreceptor is the last to differentiate and requires MAPK-mediated activation involving both the EGF and Sevenless receptors 2. In this study we showed that regulation of growth and differentiation by Sl must depend on PIP2 hydrolysis to some extent, because of the interaction between sl and mutations in 3RIP, PKC53E and Rack1. Also, by means of genetic tests, we found that Sl requires the Spi processing machinery  to regulate growth and differentiation. It has previously been shown that Sl acts on Spi processing during R7 differentiation, by favoring Spi retention in the endoplasmic reticulum sl alleles affecting the wing but not the eye is strong evidence for this assertion A central function of all phospholipase C enzymes is hydrolysis of PIPsl lead to ectopic differentiation at the expense of growth.In general, during growth, Sl activated by the insulin pathway acts as a liaison regulating MAPK pathway ligand processing, to promote MAPK activation to a level permitting growth. In agreement with a well-characterized case in mammalian cells sl constructs carrying either a mutant C-SH2 or N-SH2 domain rescued the wing size defect of sl mutants, to an extent equivalent to a wild type sl construct. The amino acid sequence of Sl is well conserved compared to mammals, suggesting that the mode of enzyme activation has not changed since flies and mammals diverged; if this is so, our results imply that PIP2 hydrolysis is not required for Sl's role regulating cell growth. This is consistent with previous reports showing that mitogenic activity triggered by PLC-γ in mammals does not depend on its phospholipase function We found that genomic sl mutants are driven by distinct and mutationally separable pathways In contrast to the results for cell growth, we found that the N-SH2 domain is necessary for Sl to regulate proper differentiation of R7 photoreceptors and wing veins. In mammals, the PLC-γ N-SH2 domain binds to a phosphotyrosine either on the activated receptor or an adaptor; whereas the PLC-γ1 C-SH2 domain is thought to be necessary for activation of phospholipase activity, binding to a phosphotyrosine within PLC-γ itself, this site having been phosphorylated before by the activated RTK after PLC-γ binding Taken together, our results indicate that Sl participates in fine coordination of growth and differentiation during development. Although Sl is not essential for wing or eye growth and development, it is necessary to achieve appropriate final structure and size. In the absence of Sl function, these tissues arrest growth prematurely and probably initiate differentiation earlier, resulting in ectopic differentiation while attaining smaller cellular sizes. As such, Sl can be seen as exerting a kind of “parental control” that protects cells from differentiating before attaining a normal size. This function requires Sl to change cellular behavior from growth (or possibly inhibition of differentiation) to differentiation in a short period of time.2 hydrolysis PLC-γ1 has been demonstrated to be a phosphorylation target of MAPK sl1/FM7c and sl2/FM7c obtained from J. Carlson . The 1sl allele is produced by a insertion of a 412 transposable element in the exon encoding the C-terminal end of the split PH domain, resulting in loss of the region Y catalytic domain as well as the C2 domain; 2sl carries a 13 bp deletion just before the exon encoding the N-terminal SH2 domain, resulting in the loss of both SH2 domains, the SH3 domain, region Y and the C2 domain. Both alleles are strong hypomorphs 9/slFM7c was generated in a Canton S background and characterized by one of us (J. Thackeray); 9sl is a point mutation that changes a tryptophan to a stop codon at the beginning of the open reading frame, and thus, creates a null allele InRE19/TM3, InRGC25/TM3, InR3T5/TM3, chico1/CyO, Dp1105W3/TM6, PKB1/TM3 and PKB3/TM3 were obtained from E. Hafen ; aos7Δ/TM3, Egfrf2/CyO, DosR31/TM3, drke0A/CyO, rl1/CyO, Ras85De2f/CyO, Sos34/CyO, Sosdm7/CyO, EE/CyORack1, ve-1rho/TM3, S2/In(2LR)bwV1ds33K, 1/CyOspi, Ip3RB4/TM3, PKC53EEY14093/C221/TM3CyO, vn and P{ey-FLP.N}6, 506ry were from the Bloomington Drosophila stock center at Indiana University, USA.Flies were raised at 25°C under standard conditions. Fly stocks used were: sl1, sl2, or sl9 homozygous virgin females with males of the following genotypes: InRE19/TM3, InRGC25/TM3, chico1/CyO, Dp1105W3/TM6, PKB1/TM3, aos7Δ/TM3, Egfrf2/CyO, DosR31/TM3, drke0A/CyO, rl1/CyO, Ras85De2f/CyO, Sos34/CyO, Sosdm7/CyO, EE/CyORack1, ve-1rho/TM3, S2/In(2LR)bwV1ds33K, 1/CyOspi, Ip3RB4/TM3, C221/TM3vn and PKC53EEY14093/CyO. We selected male progeny hemizygous for sl1, sl2 or sl9 and heterozygous for mutations under study by the absence of the marked balancer chromosomes. For rescue experiments, we likewise crossed males with the different rescue constructs to 2sl mutant females, and selected male progeny hemizygous for 2sl with the rescue construct.Genetic interactions were made by crossing 9sl homozygotes and 9/FM7sl females were collected and aged for two days in groups of ten in fresh vials before use. Individual cold-killed flies were measured in a microbalance  with 0.1 µg sensitivity and a range of 0.1 µg–25 mg. n = 70 flies/genotype. For pupal measurements, we crossed 9sl males to 9/FM7sl females in non-crowded conditions in fly bottles. Third instar larvae were recovered and sexed, and females, in groups of twenty, were housed in food vials. From these, female pupae from stages P12–P15 For adult body weight measurements, sibling 2 and wings were dissected, placed in absolute ethanol and mounted in a mixture of lactic acid:ethanol (6∶5) 2). This measurement was validated as representative of total wing area by counting the total number of cells in one wing surface in a few experiments.Flies were anesthetized with COFlies were cold anesthetized, mounted and oriented sideways unto stubs by means of carbon paint. They were then viewed with a JEOL 1010 SEM under high vacuum conditions, with 20 KeV accelerating voltage, and whole eyes positioned perpendicular to the electron beam were digitized at 230x. Only one eye was accessible per fly. Digitized images were used to measure eye area and number of ommatidia using ImageJ.2 and decapitated. Heads were placed on slides and cut in half by a medial sagittal section. The eyes were then fixed in 2% glutaraldehyde, 1% OsO4 in 1x cacodylate buffer  on ice for 30 minutes. The eyes were post-fixed in 2% OsO4 in 1x cacodylate buffer on ice for 2 hours. The OsO4 solution was then removed and the eyes were dehydrated through a graded acetone series. Absolute acetone was replaced with a mixture of Spurr's resin:acetone (1∶1) overnight. This mixture was replaced with 100% fresh Spurr's for at least 4 hours to allow the resin to infiltrate the tissue. The eyes were placed in plastic molds in 100% Spurr's and oriented using a fine needle. The molds were incubated at 60–65°C for 24–70 hours. Eyes were sectioned tangentially with a microtome (Leica RM2265) using glass knives. Sections of 1 µm thick were obtained, placed on slides, and stained with toluidine blue for approximately 30 seconds Histology was performed according to Riesgo-Escovar, et al. sl function were generated by the FLP-FRT system as described y w sl9 FRT19A/FRT19A;hsFLP/+ to heat shock. Heads containing at least one w- clone were embedded in resin, sectioned and stained with toluidine blue as described w+ ommatidium surrounded by all w+ ommatidia, and one w- ommatidium surrounded by w- ommatidia. Each member of the pair was separated from the other member by no more than 3 other ommatidia, so that differences that occur due to the angle of section were minimized. The area of each rhabdomere was determined using the wand tool within ImageJ version 1.43u .Patches of tissue lacking sl transcription unit. The X10 fragment was subcloned into pBluescript KS, reisolated as a KpnI/NotI digest and ligated into the KpnI and NotI sites of pCaSpeR-4. Modified versions of X10 were produced by site-directed mutagenesis using the Quikchange kit (Stratagene). The X10 fragment was modified in R619 to A  and independently in R726 to A . The mutations were generated in a 1.7 kb BamH1 fragment subcloned from X10 and confirmed by sequence analysis. The fragments were reconstructed in pBluescript and recloned into the KpnI and NotI sites of pCaSpeR-4. The plasmids were injected into syncytial blastoderm embryos at a final concentration of 1 µg/ µl together with 0.1 µg/ µl Δ2–3 helper plasmid in a buffer containing 0.1 mM sodium phosphate (pH 7.8) and 5 mM KCl. The plasmids were detected by the presence of the w+ gene marker of pCaSpeR-4 in transformed progeny A series of constructs were prepared from a 10 kb genomic DNA fragment (X10), that includes the entire t test.Data are presented as mean ± SEM. The mean values were compared using Student's Figure S1PKC53E mutant flies have wing phenotypes. Homozygous mutant EY14093PKC53E flies are viable and fertile, but have reduced wings (A) and ectopic wing veins (B), yet normal numbers of R7 photoreceptors in the eye (C). Each test is accompanied by corresponding control siblings. *p<0.001; error bars represent SEM, n = 100 for (A) and (B), and n = 4 eyes for each genotype, ≤150 ommatidia examined per eye.(TIF)Click here for additional data file.Figure S2Reduced gene dosage of signaling genes on the sl9 ectopic wing vein phenotype. n = 100. *p<0.001; error bars represent SEM.(TIF)Click here for additional data file.Figure S3aos acts as a strong enhancer of sl in the eye. (A) Shows a scanning EM of an eye from an aos heterozyote male fly, with normal morphology. (B) Shows a scanning EM of an eye from a male 2sl fly, sibling to the fly in (C) that also carries a mutant copy of aos (aos7Δ). Note slight roughness of the eye on (B), enhanced in the eye in (C). Scale bar in (B) is 100 micrometers.(TIF)Click here for additional data file.Figure S4Reduced gene dosage of signaling genes on sl1 and sl9 extra R7 phenotypes.  show histograms with the effects of heterozygosity for different signaling genes on the number of ommatidia with extra R7 cells in sl1 (A) and sl9 (B) mutants. n = 50–100 ommatidia per eye, from 4–7 eyes. *p<0.001; error bars represent SEM.(TIF)Click here for additional data file.Figure S5Homozygosity for sl2 does not modify the dominant S2 R7 phenotype in the eye. Histogram showing the average number of R7 cells per ommatidium in eyes from heterozygous S2 flies, or from flies also mutant for sl2. n = 5 eyes each with ≤150 ommatidia per eye. Error bars represent SEM.(TIF)Click here for additional data file.


In the history of public health, HIV/AIDS is unique; it has widespread and long-lasting demographic, social, economic and political impacts. The global response has been unprecedented. AIDS exceptionalism - the idea that the disease requires a response above and beyond "normal" health interventions - began as a Western response to the originally terrifying and lethal nature of the virus. More recently, AIDS exceptionalism came to refer to the disease-specific global response and the resources dedicated to addressing the epidemic. There has been a backlash against this exceptionalism, with critics claiming that HIV/AIDS receives a disproportionate amount of international aid and health funding.This paper situations this debate in historical perspective. By reviewing histories of the disease, policy developments and funding patterns, it charts how the meaning of AIDS exceptionalism has shifted over three decades. It argues that while the connotation of the term has changed, the epidemic has maintained its course, and therefore some of the justifications for exceptionalism remain. In the 30 years since it was first recognized, HIV has spread globally. An estimated 25 million people have died, and about 33 million people are currently living with HIV/AIDS. The epidemic is not homogenous; the global picture is diverse. In wealthy countries, most of Latin America, Asia, north Africa and the Middle East, infections are concentrated in particular geographical locations and among specific population groups. These are often socially and politically marginalized populations, including injecting drug users, men who have sex with men, and commercial sex workers.Generalized epidemics are found in eastern, central and southern Africa, where between 5% and 30% of adults are infected. However, even here, specific groups, such as women, remain disproportionately at risk. The development of HIV "risk environments"  has beenIn the history of public health, HIV/AIDS is unique in terms of how it is spread and attacks the body and because of its widespread and long-lasting demographic, social, economic and political impacts. HIV/AIDS is a long-wave event: an epidemic that, where it is most prevalent, will have consequences that will be felt for generations . Just asAIDS exceptionalism began as a Western response to the originally terrifying and lethal nature of the virus, which disproportionately affected specific groups. The first activists argued that HIV/AIDS required an exceptional response in order to protect the rights of those infected, to generate resources to assist them and to curb a then mysterious epidemic. More recently, AIDS exceptionalism came to refer to the disease-specific global response. This international response was unprecedented, as the commitment of resources exceeded any other health cause.International organizations, such as the Joint United Nations Programme on HIV/AIDS (UNAIDS), the Global Fund to Fight AIDS, Tuberculosis and Malaria  and the US President's Emergency Plan for AIDS Relief (PEPFAR), were formed to specifically address HIV/AIDS. In the past few years, there has been a backlash against this exceptionalism, with critics claiming that HIV/AIDS receives a disproportionate amount of international aid and health funding, and that this has implications for other health issues.This paper reviews the histories of the disease, policy developments and funding patterns to chart how the meaning of AIDS exceptionalism has shifted over three decades. It argues that, while the connotation of the term has changed, the epidemic has maintained its course, and therefore some of the justifications for exceptionalism remain.In the early 1980s, when previously healthy, mostly homosexual young men began dying, the unknown cause and rising numbers of deaths combined with homophobia to generate a response of blame and fear. Extreme religious right-wing advocates spoke of divine punishment for "sinful" lifestyles . The disAs haemophiliacs, women and children in the West increasingly presented with the same symptoms, it became clear that the cause of illness was not related to sexual orientation. In 1983, the human immunodeficiency virus (HIV) was identified as the cause of AIDS. The realization that HIV could spread to the general public, and that it was linked to the life-giving and pleasurable acts of sexual intercourse, resulted in increased hysteria. Governments, the media and scientists sought a quick response: "A sense of urgency defined the problem, and the public information materials developed in this period often emphasized danger at the expense of clear information about prevention measures" . Even asThe gay rights movement, building on the momentum it had gained in the preceding decades, began campaigning for HIV/AIDS to be viewed as a human rights issue. Advocates argued that infection was not the only risk; if found positive, individuals also faced harmful discrimination . In thisAvoiding compulsory measures such as isolation and quarantine, which were so much a part of the public health tradition, was all the more crucial, since the people with increased risk -- gay and bisexual men, drug users, and their sexual partners -- were already socially vulnerable ... Policies and practices that appeared to threaten such persons could only drive the epidemic underground and make it more difficult to work with the populations within which HIV was spreading .Recognizing the unique needs of populations at risk of HIV infection, an exceptionalist alliance, including the gay community, liberal and left-wing parties, and the healthcare and psychosocial professions, was formed to advocate for a unique response. Bayer writes, "The embrace of exceptionalism must be understood in broad political terms, as representing in large measure, a singular victory on the part of gay men, their community-based organisations and their allies" . The allDuring the 1980s, public health adopted a human rights framework that took societal-based vulnerability into consideration and increasingly became involved in societal transformation efforts . HIV/AIDDescriptively, exceptionalism posited that in the early years of the HIV epidemic, HIV was considered so different, so "exceptional" in comparison to other communicable diseases that advocates and public health officials agreed that HIV policy should cater to the uniqueness of the epidemic rather than treat it like all other communicable diseases. Supposedly, the argument goes, public fear was so great, the political power of gay men so substantial, and concern over stigmatization so real, that public health authorities abandoned "traditional" approaches to communicable disease control in favor of a civil liberties approach .This public health approach helped contain the epidemic among those groups most at risk, and meet, to varying degrees, their specific needs. New infections in the United States fell from approximately 130,000 in 1984 to about 60,000 in 1991 . The feaThe hysteria surrounding HIV/AIDS faded; the media and public policy lost interest. The human rights approach that had previously been revolutionary was integrated into existing public health traditions that included education, technical solutions and regular testing. In 1991, Bayer wrote, "As AIDS has become less threatening, the claims of those who argued that the exceptional threat would require exceptional policies have begun to lose their force" . Highly When antiretroviral treatment (ART) was unveiled at the 1996 International AIDS Conference in Vancouver, Canada, AIDS was transformed into a treatable disease. The advent of treatment shifted Western priorities of response: "The availability of more advanced antiretroviral therapies has made it possible to treat effectively those with HIV infection, thereby increasing the importance of early identification and tracking. These developments establish a strong case for moving beyond HIV exceptionalism and treating HIV antibody tests like other blood tests" .As technical solutions of testing and treatment gained priority, the social movement that had spurred the early HIV/AIDS response continued to fade from public awareness. This shift was assisted by a rapid fall in the price of the drugs. Had they remained expensive, AIDS exceptionalism would have been perpetuated. As Casaratt and Lantos noted, "Medical therapy has become more effective but also prohibitively expensive. A medical tragedy has been transformed into a financial crisis and society has responded by establishing special programs and sources of funding for AIDS. These manoeuvres parallel earlier approaches to HIV testing and reporting that have collectively come to be known as exceptionalism" . The mobThroughout the rise and fall of Western AIDS exceptionalism, the growing global epidemic remained largely ignored by the international community . During The Global Programme on AIDS in the World Health Organization (WHO) lacked both the funding and capacity to respond to the epidemic. Outside of WHO, HIV/AIDS was not on the agenda of other United Nations (UN) agencies. International responses between 1986 and 1996 were characterized by denial, underestimation and over-simplification . AIDS exIn 1996, UNAIDS was formed as a joint programme of UN agencies engaged in the AIDS response . In 1998The Global Infectious Disease Threat and Its Implications for the United States  threatens not just individual citizens, but the very institutions that define and defend the character of a society ... It strikes at the military, and subverts the forces of order and peacekeeping" [d States . Six mond States . In 2001d States .The Pretoria-based Institute for Security Studies wrote, "The severe social and economic impact of HIV/AIDS, and the infiltration of the epidemic into the ruling political and military elites and middle classes of developing countries may intensify the struggle for political power to control scarce state resources. Such dynamics, even singularly, have the potential to lead to political instability" .It was argued that HIV/AIDS could hinder processes of democratization by undermining social development and intensifying the struggle for resources. It was further suggested that AIDS orphans could contribute to social unrest as they made likely recruits for terror and rebel groups: "Bluntly put, those who are orphaned may be indifferent to prevailing norms and values, may look for salvation to millenarian and fundamentalist beliefs of one kind or another, and may ultimately do this with assistance from a Kalashnikov or a bomb" .While there was little evidence to actually link HIV/AIDS prevalence and security issues , the disHowever, as policy and activists spoke of a global HIV/AIDS epidemic, it became apparent that, like many diseases that are expensive to treat, how this epidemic was experienced differed drastically by region. ART, available from 1996, proved effective in curbing AIDS deaths in those regions that could afford the high costs of medications . In those countries with mid to high HIV/AIDS prevalence, ART remained unaffordable, even while costs dropped to about $10,000 per patient per year by 2000 and to less than $100 by 2010. A mid to high disease burden combined with lack of health resources in much of the developing world, making the benefits of ART beyond the reach of most domestic health budgets. While AIDS had become a chronic disease in the West, in most of the developing world. it was still a death sentence.What made this discrepancy unique was the international mobilization that occurred around it, positioning the inequity of treatment access for people living with HIV and AIDS (PLHIV) as an international human rights cause. The International AIDS Conference in Durban in 2000 called for treatment to be rolled out in the developing world and for prices of ART be cut. Activists in South Africa demanded that their government provide universal access to ART, despite political resistance and denial. The governments of India and Brazil took on the World Trade Organization, arguing for compulsory licensing that would enable them to manufacture cheaper generic medications. Activists in the United States, largely led by the PLHIV organization, ActUp, began a sustained campaign against the US government's support for pharmaceutical companies' patent legislation .Arguments that ART could not be provided in the developing world due to limited capacity and poverty were challenged by the successful implementation of such programmes by health organizations like Médecins Sans Frontières . ResearcIn 2002, Botswana implemented the first universal access programmes in sub-Sahara Africa. In 2003, the South African Government gave in to local and international pressure and announced its public treatment programme. The World Health Organization launched the "3 × 5" campaign, aiming to place 3 million people on treatment by 2005. In 2006, 111 countries committed to achieving universal access to prevention, treatment, care and support by 2010 .The development of international AIDS programmes combined with a favourable political environment to create a new discourse of AIDS exceptionalism. At the 2001 UN General Assembly Special Session on AIDS, 189 nations agreed that HIV/AIDS was a national and international development issue of the highest priority . This trIn 2004, at the Copenhagen Consensus, a policy think tank in Denmark, a panel of eight prominent economists ranked controlling HIV/AIDS as the number one economic priority in terms of a cost-benefit analysis in health and nutrition . UNAIDS The Invisible Cure focused on the African epidemic, suggesting that one of the main drivers of the epidemic in hyper-epidemic countries was concurrent sexual partnering, but that such sensitive social issues had not been openly or adequately addressed, despite the resources mobilized for curbing the epidemic [Arguments against exceptionalism began to gain attention in 2007. The amount of funding allocated to HIV/AIDS was called into question, as were the types of programmes being implemented. Three important books put forward new arguments. Chin and Pisani focused on the Asian experience, suggesting that scientists, UNAIDS and AIDS activists accept certain myths about HIV epidemiology to maintain the political profile of AIDS, and their own jobs . Pisani epidemic .British Medical Journal, Roger England argued that HIV/AIDS was not the "global catastrophe" claimed by "AIDS exceptionalists", and that donor aid for HIV/AIDS was disproportionate to the contribution of HIV/AIDS to the global disease burden [The strongest argument against AIDS exceptionalism has centred on the claim that responses undermined health systems in developing countries. In a series of opinion pieces in the e burden . Englande burden . He wrotIt is no longer heresy to point out that far too much is spent on HIV relative to other needs and that this is damaging health systems. Although HIV causes 3.7 percent of mortality, it receives 25 percent of international healthcare aid and a big chunk of domestic expenditure ... Until we put HIV in its place, countries will not get the delivery systems they need .England and others' commentaries highlighted that many diseases and health issues  resulted in more deaths than those related to AIDS in many parts of the world, but were receiving less funding. Whether or not this neglect was because of the prioritization of the AIDS response or due to other factors was hotly contested.et al argued that HIV/AIDS funding actually increased access to other health resources in many underserved areas [UNAIDS and WHO responded defensively, noting that those against AIDS exceptionalism attributed the UN agencies more power than they had to address the epidemic . Researced areas . A 2007 ed areas . These ded areas ; here, tThe emergence of the most recent exceptionalism debate is concurrent to a shift in donor country spending and priorities. Some donor countries have begun redirecting HIV/AIDS funding; in 2009, the UK Department for International Development reassigned a portion of its AIDS funds to maternal and child mortality programmes and health system strengthening . Médecinet al argue for transforming the Global Fund from a disease-specific fund into a "Global Health Fund" [As these changes take place, proposals are emerging to reorganize disease-specific funds in new ways. Sachs and Pronyk argue that the Global Fund should increase its mandate to include health systems, maternal and child mortality and neglected tropical diseases . Similarth Fund" . HIV/AIDth Fund" ; here, tWhile the debate on the global exceptionality of AIDS continues, HIV/AIDS will prevent many sub-Saharan African countries from achieving the Millennium Development Goals (MDGs) . In highThe number of new HIV infections each year remains unacceptably high, and models of effective prevention programmes are few and far between. In the endemic countries, mass behaviour-change campaigns, such as the ABC  strategy, have failed to achieve their intended impacts. Advocates are stressing the need to address the systemic contributors to risk and vulnerability.In South Africa, where one of the largest epidemics continues to unfold, Venter writes that prevention efforts are failing: "500 000 South Africans are infected each year, and there is no sign that this is letting up. In 8 to 10 years' time, as they enter the AIDS stage, these 500 000 South Africans will need ARVs, and this will continue forever until a prevention strategy that works is implemented" .et al write from the American perspective:In Western countries, though the epidemic is concentrated in specific population groups, infection rates among these groups are stable (but not declining). El-Sadre For the past decade, however, progress has been stalled. It had been anticipated that effective antiretroviral therapy, with its suppressive effect on viral replication, would reduce the overall rate of new infections, but this expectation has not been realized. More than half a million Americans became infected with HIV in the past decade, including about 56,000 in the past year .The authors advocate for interventions that "address the socioeconomic milieu in which HIV transmission occurs" . The socMeanwhile, more than half of those in need still do not have access to treatment, and treatment is posing new challenges for sustainable funding. Most countries with mid to high prevalence cannot afford the cost of treatment without international aid; as aid is reduced or cancelled, treatment programmes are threatened, drug resistance develops and large numbers of PLHIV die. In countries like Uganda, patients are being turned away from treatment clinics due to lack of resources; 300,000 Ugandans in need of treatment are denied their right to health . These nThe gap between resources required to implement HIV/AIDS programmes and those available has continued to grow over the past three years . In 2009De Waal writes, "Normalization in the sense of adjusting reality to take account of the miseries of AIDS can be found in many places, when it is looked for. Even the statistics become numbing, and when lower-than-expected HIV prevalence is reported, as recently in Kenya and Zimbabwe, it can give the impression that things are 'not so bad' - when 7 to 10 percent of an adult population is living with HIV" . While iThe concept of AIDS exceptionalism developed as a Western response to an epidemic that threatened the lives and rights of specific populations in the developed world. As that epidemic was contained and effective treatment became available, the case for exceptionalism shifted to the international stage, where resources and organizations were mobilized to respond to the extreme need in developing countries. As a result, the numbers of PLHIV on treatment has increased each year. Infection rates in much of the world have stabilized.These gains have been accompanied by criticisms of the type and size of response. It is argued that the HIV/AIDS response has done harm, as well as good, particularly by creating vertical programmes for a single disease, which may have diverted resources. As donor countries shift priorities, and in the context of the economic recession, the urgency around the HIV/AIDS response is once again declining.This shift in policy and international priorities does not change the reality of an epidemic that, after three decades, is still unfolding. In southern Africa, the demographic effects of the generalized epidemic will shape societies for generations. In other parts of the world, HIV/AIDS continues to mark inequalities: one in 40 blacks, one in 10 men who have sex with men, and one in eight injection drug users in New York City are HIV positive [The Lancet, "We are not overspending on AIDS but underspending on the rest ... The choice is not between AIDS, health systems, and other Millennium Development Goals. We can and must support them all" [Both the human rights approach, originally adopted by the HIV/AIDS response, and the more recent demands for universal access to treatment, remain relevant to the 33 million people living with HIV/AIDS and to their communities; these issues should also remain pertinent within global health policy. Meanwhile new challenges are developing, not the least of which is the need to successfully integrate the HIV/AIDS response within broader public health responses to the benefit of all. As Sachs notes in a commentary in hem all" .As how to best approach such challenges is debated, we must not lose sight of the approximate 2 million AIDS-related deaths that occur each year. Defining these deaths as either exceptional or unexceptional seems both callous and arbitrary.The authors declare that they have no competing interests.Both authors contributed to the research and writing of this paper, and read and approved the final manuscript.


Introduction. Preeclampsia (PE) is an important cause of mortality and morbidity for mothers, fetuses, and the newborns. Placenta plays a pivotal role in pathogenesis of PE. Hepatic growth factor (HGF) is a cytokine expressed by the mesenchymal stalk of placental villi during pregnancy and assumes a paracrine role in trophoblasts which express its receptor (c-MET). In the present study, we investigate the diagnostic value of s-Met (the soluble form of the receptor) in the first and second trimesters of pregnancy for early diagnosis of preeclampsia. Method and Materials. This is a case-control study conducted on 95 pregnant women. The serum level of s-Met was measured in the first and second trimesters, and the participants were followed until delivery. 44 individuals with preeclampsia (the case group) and 51 individuals without preeclampsia (the control group) were evaluated. Results. Serum level of s-Met in preeclamptic participants was lower than that of the control group in both the first and the second trimesters (P < 0.0001). In addition, serum levels of s-Met were significantly lower during the first and second trimesters in patients with early, severe preeclampsia compared to those with late, mild preeclampsia (P < 0.0001). The sensitivity and specificity of s-Met in the first and second trimesters were, respectively,  and  for early preeclampsia and  and  for severe preeclampsia. Conclusion. Considering our findings, serum level of s-Met may be used as a predictive factor for early detection of preeclampsia. Further research is required to corroborate the functional and therapeutic value of s-Met in preeclampsia. Subsequently, they were referred to the central laboratory of Sari to give 5cc blood sample in serum separated tube (SST) in the 11–13th weeks (as blood sample of the first trimester) and again in the 24–28th weeks (as blood sample of the second trimester). Serum was separated by centrifugation for 15 minutes at 1000 ×g and was preserved at −70°C.These women were followed during the period of their pregnancy. Those developing preeclampsia were labeled as the case group and then were further classified as early or late PE, as well as mild or severe PE. The control group was selected randomly from the initial population of pregnant women without preeclampsia who were similar to those in the case group in terms of age, gestational age, body mass index, diabetes, and history of preeclampsia. Serum level of s-Met in both groups was measured by technicians who did not have access to the outcome of pregnancy, using CUSABIO kit to perform ELISA.t-test, and nonparametric variables were analyzed using sum of squares. P values below 0.05 were considered significant. Statistical tests for comparing the serum levels of s-Met in the above groups were completed on SPSS software. We calculated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for this marker. Using the ROC curve, we determined the cutoff point for s-Met as the marker. Other parametric variables were analyzed using student's The present study was conducted on 95 pregnant women, consisting of 44 individuals with preeclampsia (case group) and 51 individuals without preeclampsia (control group). P < 0.0001).The mean serum level of s-Met in women with preeclampsia was significantly lower than that of the normal women in both the first and the second trimesters  required C-section, while it was only required for 20 cases (39.2%) in the control group.P < 0.0001 in both cases). Out of 44 cases of preeclampsia, 13 (29.5%) were early onset (<34 weeks) and 31 (70.5%) were late onset (>34 weeks). The mean serum level of s-Met in the first trimester was 177.69 ± 9.26 ng/mL in patients with early preeclampsia and 263.13 ± 28.9 ng/mL in patients with late preeclampsia. Furthermore, the mean serum level of s-Met in the second trimester was 187.79 ± 14.32 ng/mL in patients with early preeclampsia and 241.9 ± 36.18 ng/mL in patients with late preeclampsia (P < 0.0001 for both cases). This is illustrated in Out of 44 cases of preeclampsia, 25 cases (56.8%) were mild and 19 cases (43.2%) were severe. The mean serum levels of s-Met in cases with mild preeclampsia were 242.8 ± 25.08 ng/mL and 253.60 ± 29.84 ng/mL in the first and second trimesters, respectively. For severe preeclampsia, the mean serum levels of s-Met in the first and second trimesters were 187.37 ± 22.56 ng/mL and 189.47 ± 12.68 ng/mL, respectively , regeneration of epithelial renal cells, and suppression of fibrogenic cytokines in fibroblasts . HGF maypoptosis . Also itpoptosis . PreeclaIdentifying the source of s-Met production in pregnancy may help to clarify the mechanisms disrupting serum levels of s-Met in PE. Both HGF and c-Met have been detected in human placenta, and c-Met has been localized mostly in trophoblasts and endothelial cells , 13.Wajih et al.  demonstrTherefore, considering the findings of this study and others, as well as the pathophysiological mechanisms of HGF/s-Met, it is possible that s-Met can serve as an appropriate marker for early detection of preeclampsia. Nonetheless, further research is required to corroborate the functional and therapeutic value of s-Met in preeclampsia.


Statistics on prevalence and incidence indicate that the HIV/AIDS pandemic has increasingly affected women during the past decade. The mechanisms, techniques, and data sources used to monitor and evaluate global AIDS prevention and treatment services may vary according to gender. The Joint United Nations Programme on HIV/AIDS has been charged with tracking the response to the pandemic by using a set of indicators developed as part of the Declaration of Commitment endorsed at the U.N. General Assembly Special Session on AIDS in 2001. Statistics on prevalence and incidence indicate that the pandemic has increasingly affected women during the past decade. Women's biologic, cultural, economic, and social status can increase their likelihood of becoming infected with HIV. Since 2000, global financial resources have increased to allow expansion of both prevention and treatment services through a number of new initiatives, such as the Global Fund to Fight AIDS, TB and Malaria; the U.S. President's Emergency Plan for AIDS Relief; and the World Bank MAP program. Programs should be monitored and evaluated to ensure these investments are used to maximum effect. Different types of data should be included when assessing the status of the HIV/AIDS epidemic and effectiveness of the response. Each of these "data streams" provides information to enhance program planning and implementation.These types of data include the prevalence of HIV infection in the general adult population and in persons at high risk, as well as the actual estimated number of persons who are infected, who have symptoms of AIDS, and who died of the disease. These data are generally obtained from a combination of sentinel surveillance methods and biologic testing as part of community population surveys.These types of data include establishing policies that increase access to services, protect the rights of vulnerable groups, and provide adequate resources. These data are primarily obtained from a set of discrete tools that include the National Composite Index and the AIDS Programme Effort Index; both tools review legislation, policies, and key interviews of relevant groups, including government officials, clinical providers, and persons affected by or living with HIV/AIDS.These types of data measure levels of risk for HIV transmission and changes in risk levels over time. Major population survey questionnaires, such as demographic health surveys, gather this information, along with targeted behavioral surveillance, such as the Behavioral Sentinel Surveillance Surveys, pioneered by Family Health International.These types of data include tracking the contributions from external donors  as well as national expenditures and "out of pocket" spending from families and persons affected by HIV/AIDS. These data are generally obtained from global resource tracking databases  and at the country level from subanalyses of National Health Accounts and through the use of a new tool, National AIDS Accounts.These types of data provide useful proxy information on program implementation. They are often only available from the donor community that procures these commodities  and are rarely routinely collected as part of a country's health information system.These types of data are generally measured through health information systems. However, because such systems are often inadequate in many developing counties, these data remain poor. Tracking services, for prevention or treatment, also necessitates monitoring the quality of these services. Much of this information can be obtained from health facility surveys, but these surveys are staff- and resource-intensive and are generally not conducted routinely.These types of data are frequently obtained from vital events registration, including estimated births, deaths, and causes of death. Again, in many developing countries, the collection of this type of information tends to be deficient.The Joint United Nations Programme on HIV/AIDS (UNAIDS) has been charged with tracking the response to the pandemic by using a set of indicators developed as part of the Declaration of Commitment, which was endorsed at the U.N. General Assembly Special Session on AIDS in 2001. When these indicators were first measured in 2003, differences in the comprehensiveness and quality of available data became clear. The biologic surveillance data are improving over time, and now the policy environment can be assessed to determine levels of commitment. However, we currently obtain very poor data on commodities, resource flows, and the coverage and quality of prevention and treatment services. This lack of credible data is made worse by the inability to disaggregate these activities by gender.Until recently, most of this information has been gathered from sentinel surveys of general adult populations and populations at risk. These data are now being complemented by the increasing use of population surveys that include seroprevalence testing. These new data collection methods deepen our understanding of the pandemic. Statistics on prevalence, incidence, and estimated numbers of persons infected with HIV and those with HIV disease have gender implications and point to the increased feminization of the pandemic over the past decade. Almost 60% of HIV-infected persons in sub-Saharan Africa are female. Nearly 20 million HIV-infected persons in the developing world are girls and women . BiologiA major focus of The U.N. General Assembly Declaration of Commitment is to examine whether countries themselves are creating an environment with a positive and effective response. Are the appropriate policies in place? Is there real commitment by government and civil society to implementing programs that will make a difference? UNAIDS attempts to examine stigma and discrimination by using the National Composite Policy Index (NCI), the AIDS Program Index (API), and a series of media analyses. Globally, funds made available by various sources, HIV/AIDS policies within multinational corporations, and the value of global advocacy efforts are measured. At the national level, funds from national governments, existence of policies to promote programs for risk reduction, and access to care and treatment are measured.NCI assesses legislation and policy related to national strategic plans, prevention efforts, maintenance of human rights, and provision of care and support. For instance, if a national strategy exists for HIV/AIDS, does it address those at risk in the uniformed services, youth, migrants, and other risk groups? Does the strategy address human rights, particularly access to services for women? However, this approach does not assess the public's awareness of this legislation, how the policy is enforced and implemented, or the quality of these policies. For example, in sub-Saharan Africa, 80% of developing countries have a policy in place to provide antiretroviral therapy, but only 1% of HIV-infected persons in this region actually have access to these lifesaving treatments.In addition to NCI, which quantitatively measures the policy environment, we are also using API, which is a more qualitative, subjective index that examines a range of issues, including stigma, discrimination, and access to services. API is measured by using a Delphi approach. Relevant groups  are brought together and asked a series of ≈100 standardized questions. This instrument allows us to assess whether policies are actually being implemented and whether they are making a difference. This tool has become increasingly useful to measure subtle and complex changes that occur in a country response, and it is now being applied to other interventions, such as the scope and quality of orphan care, which is difficult to track quantitatively.One downside to the API is that it does not allow for cross-country comparison. The reason is that citizens' expectations about their government vary widely across countries. Some countries may be assessed poorly because the citizens expect more; however, a country with no policy may do well because the expectations from the public are much lower. However, API is being revised to make some of the responses to the structured interview questions less subjective.Behavioral data fall into three areas, which include data on vulnerability, knowledge and awareness, and risk behavior over time. Women have biologic, cultural, economic, and social vulnerabilities that increase their likelihood of acquiring an infectious disease. They also face hostile justice systems, are exposed to sexual violence, lose inheritance rights and property, lack access to reproductive services, and have few support systemsAnalysis of data can lead to a better understanding of inherent vulnerabilities. For example, marriage does not necessarily protect against sexually transmitted infections. Marriage at a very young age can actually increase vulnerability, initiating a young woman into sexual activity earlier than if she were not married. While we believe these findings could be a pathway for future potential interventions, exactly what these programs would look like is being debated.The age of first sexual activity can be closely correlated with HIV prevalence . HoweverCross-generational sex is a controversial, but important, area when considering vulnerability. Recently, efforts have been increased to better understand what is happening in this area. While exploitation is a reality, not all persons involved perceive these acts as exploitation. Most young women participate in these relationships to survive. Older men think that they reduce their chance of being infected by being with a younger girl. Addressing these complex and culturally sensitive issues will require innovative thinking.Violence is another major issue that makes women vulnerable. Data from South Africa indicate that 33% of women are afraid of saying no to sex, 29% have been forced to have sex, and 55% have had sex because their boyfriend insisted .While not traditionally part of monitoring and evaluation, tracking financial resource flows is now considered a key element for monitoring program implementation and effectiveness. How much money comes from the U.S. government or other international donors into a country versus how much comes from the national government itself? Do you see economies of scale as service coverage goes up? Do commodities, such as antiretroviral drugs, become more inexpensive, or as we move from first-line to second-line antiretroviral treatments, will the costs actually go up over time. At a global level, we rely on the Organization for Economic, Cooperation, and Development, which was created by the major industrialized countries to track financial resources and by the Netherlands Interdisciplinary Demographic Institute, which is under contract to UNAIDS to provide information on funding levels from bilateral donors, multilateral organizations, foundations, and the private sector. Resource flows are often tracked at the country level through National Health Accounts and National AIDS Accounts. While spending is increasing, so are unmet needs. The estimated need represented by the top portions of the bar chart  does notTo examine services and coverage, we use key informant coverage surveys, health facility surveys, and donor data. In the UNAIDS 2003 Progress Report on the Global Response to the HIV/AIDS Epidemic, the available data on the delivery of preventive and treatment services demonstrated a lack of access. Only 1 in 100 pregnant women receives services to prevent mother-to-child transmission of HIV. One in 100 eligible HIV-infected persons (men and women) has access to antiretroviral therapy. One in 10 has access to testing for HIV infection. One in four has access to basic information on AIDS and protection against AIDS. Much of these data cannot currently be disaggregated by gender. When we look at antiretroviral therapy, ≈400,000 persons in the developing world are being treated, of which ≈80,000 are in sub-Saharan Africa, but the ratio of women to men receiving these services is difficult to break down.We face a number of challenges in containing the AIDS pandemic and increasing prevention and treatment programs, particularly as they relate to gender. Setting standards and targets for equity of access and then monitoring progress will be critical. For example, UNAIDS advocates that of the 3 million infected persons who should be started on antiretroviral therapy by the end of 2005, an appropriate number should be women, depending on the ratio of infected men to women in that specific country.Measuring sectorwide effects of these new resources will be a major issue. If a massive investment in infrastructure is focused on the three major infectious diseases , will the health sector be positively affected as a whole or will we see staff, commodities, and resources diverted from other areas of public health, such as immunization of children or treatment of childhood diarrhea? How are we going to measure this?A new initiative, the Global Coalition on Women and AIDS, was launched February 2004 by UNAIDS and other partners. The coalition focuses on selected key areas: preventing HIV infection in girls and women, reducing violence against women, protecting property and inheritance rights, ensuring equal access by women and girls to care and treatment, and supporting efforts for universal education for girls. We need to find ways to accurately measure progress in all these areas, while recognizing that quantitative methods may not be the most appropriate way to get a true picture of progress or lack thereof.We are making progress in monitoring and evaluating prevention measures, but we have a long way to go. As we collect appropriate biologic, policy, behavioral, and service coverage data, patterns of gender dynamics and inequities begin to emerge. We must use this information to address these inequities.


Bacterial responses to antibiotics are concentration-dependent. At high concentrations, antibiotics exhibit antimicrobial activities on susceptible cells, while subinhibitory concentrations induce diverse biological responses in bacteria. At non-lethal concentrations, bacteria may sense antibiotics as extracellular chemicals to trigger different cellular responses, which may include an altered antibiotic resistance/tolerance profile. In natural settings, microbes are typically in polymicrobial communities and antibiotic-mediated interactions between species may play a significant role in bacterial community structure and function. However, these aspects have not yet fully been explored at the community level. Here we discuss the different types of interactions mediated by antibiotics and non-antibiotic metabolites as a function of their concentrations and speculate on how these may amplify the overall antibiotic resistance/tolerance and the spread of antibiotic resistance determinants in a context of polymicrobial community. Their dAntibiotic resistance in microbes is widespread and it has been demonstrated that soil bacteria are rich in resistance determinants to both natural and synthetic antibiotics commonly used in clinics . The colDespite a better understanding of the different mechanisms leading to resistance, exposure to antibiotics is still considered the major driver in the selection for antibiotic-resistant bacteria  and the The focus on the medical use of antibiotics has limited fundamental research regarding the other potential activities of these compounds in their natural settings, including the environment  and hosts such as humans, animals, and plants. In complex communities containing antibiotic-producing microorganisms, bacteria are naturally exposed to lethal and non-lethal antibiotics making them trained at responding to these compounds. Non-lethal levels of antibiotics can alter the expression of genes involved in a variety of bacterial functions like metabolism, regulation, virulence, DNA repair, and stress response . SubinhiAntibiotics are, like other bioactive small molecules, low molecular weight metabolites produced by secondary metabolism of microorganisms, i.e., are not considered essential for growth and viability. Microorganisms such as bacteria and fungi produce a wealth of small molecules that has been called the parvome . SecondaThe explosion of research in cell–cell interactions mediated by bioactive small molecules in microbiology has led to the general assumption that most interspecies cell–cell interactions could be labeled as “communication” or “signaling.” However, it is important to note that the demonstration of a biological response upon exposure to a chemical does not necessarily imply communication. The improper use of terms like signal, signaling, or communication in microbiology has created confusion since most interspecies metabolite-mediated interactions labeled as “signaling/communication” are often in conflict with evolutionary theories. A detailed analysis of the appropriate terminology is beyond the scope of this review; therefore we refer the reader to recent reviews that have thoroughly discussed the topic . A chemiTable 1, a signal is defined when both partners take advantage of the interaction , while cues or coercions have unidirectional benefits for receivers or emitters, respectively. In true signaling interactions, the production and detection of the signal have co-evolved specifically for that purpose and from an evolutionary perspective these events will only be maintained when both partners benefit from the information conveyed by the signal for which they evolved  from the oropharyngeal microbiota  or inhibit bacterial growth (bacteriostatic). Their concentrations are highly variable in natural communities and bacteria have evolved mechanisms to respond accordingly. Although their antimicrobial properties have been demonstrated in both Figure 2). From high to low concentrations, antibiotics act as either toxins, stress inducers, or as cues/coercions, respectively. Among interactions mediated by subinhibitory antibiotics, receiver bacteria can interestingly induce mechanisms leading to antibiotic resistance or tolerance . How antibiotics become stress inducers or cues/coercions will further be discussed in the following sections, but before we will briefly describe the toxin-like behavior of antibiotics and the impact of antibiotic resistance on the biological response exhibited by bacteria upon antibiotic exposure.To first differentiate the biological responses induced by antibiotics, here we represent their bioactivities from a receiver bacterium’s perspective on a large concentration spectrum , antibiotics behave like toxins on susceptible bacterial cells. However, although the understanding of resistance mechanisms is well-characterized, molecular mechanism(s) induced by lethal concentrations of antibiotics is an area of research where our fundamental understanding is still very limited . Recent Figure 2), antibiotic resistance would also impact receiver bacteria in an antibiotic dose-dependent manner. The presence of an antibiotic resistance mechanism would shift the spectrum of responses to an antibiotic to higher concentrations. The resistance would lower the effective concentration of antibiotics at the target site . Exceptions to this would occur if there were secondary target sites for the antibiotics that mediate other responses. At toxic concentrations, resistance would function in the conventional protective role and allow receiver bacteria to avoid the antibiotic toxicity by blocking death or growth arrest. At subinhibitory concentrations, antibiotic resistance genes would shift the effective antibiotic concentration required for inducing the biological responses  of receiver bacteria  could therefore establish a chemical “arms race” between producer and receiver bacteria independent of lethal effect. In the context of bacterial communities, the displacement of the response curve for a single strain will modify its own behavior, which may in return shift the community composition or activity. Stress induction, coercion, and detection of cues are selectable phenotypes that can be tuned to meet the conditions of natural environments. Selections that reduce stress induction and coercion could be contributing factors in the evolution of the “cryptic resistome” . In the context of chemical interactions within natural communities, antibiotics released by emitter bacteria at lethal concentrations for the receiver cells would not be considered as a signaling event, but rather coercion at the extreme. Coercion can occur at subinhibitory concentrations and one bacterial cell may use chemicals to manipulate another. If the receiver cell possesses the corresponding antibiotic resistance determinant, receiver bacteria will be less susceptible to chemical manipulation and the overall benefit or harm of the interaction will therefore be reduced.In natural environments, antibiotics are likely present in a gradient of concentrations while receiver organisms may also be heterogeneous. Even in a homogeneous clonal population, cells may be present in different physiological states and the presence of antibiotic tolerance and resistance mechanisms will shift the response curves for individual cells within the population  and the active concentration for a particular response will be shifted higher by resistance mechanisms . In natural environments, a population will be expected to exhibit a heterogeneous response because of gradients of antibiotic concentration and heterogeneity in the responsiveness of different cells in the population . These differences in cellular responsiveness may be due to genetic heterogeneity and/or through differences in physiological states of different cells in the population.Outside of the well-mixed homogeneous environments of laboratory cultures, antibiotic effects on bacterial populations will be heterogeneous. For any bacteria, the response to a specific antibiotic will be concentration-dependent . When behaving as stress inducers, antibiotics often induce the SOS stress response, which is also associated with various antibiotic resistance mechanisms. The following section will mainly highlight some of the main resistance mechanisms impacted by the induction of the SOS response in bacteria upon antibiotic exposure.Antibiotics at subinhibitory concentrations can act as stress inducers or cues/coercion on receiver bacteria , and chemical inactivation TetX; . The regEnterococcus species  is the primary mechanism of resistance . Six typ species . Interes species . VancomyStaphylococcus aureus. This affects biofilm formation and autoaggregation (P. aeruginosa biofilms induces tolerance against aminoglycosides by chelating cations (P. aeruginosa virulence-associated genes leading to increased secretion of phenazines and rhamnolipids (P. aeruginosa phenazines (P. aeruginosa biofilms through hydrogen peroxide (H2O2) mediating cell lysis .Although subinhibitory concentrations of antibiotics like tetracycline and vancomycin can induce their own resistance mechanisms, other biological responses have the potential to indirectly impact antibiotic tolerance as well. It was recently demonstrated that subinhibitory concentrations of β-lactam antibiotics induce the autolysin-dependent release of extracellular DNA (eDNA) by regation , two gro cations . Additionolipids . Pyocyanenazines  was recell lysis . These eThese are a few examples demonstrating that sensing of subinhibitory concentrations of antibiotics as cues can trigger direct or indirect mechanisms, for which receiver bacteria will subsequently have the ability to resist or tolerate lethal concentrations of antibiotics. In microbial communities, the response of one organism may lead to induction of antibiotic resistance/tolerance in other bacteria.A significant portion of the antibiotic resistome is made of mobile resistance genes that are horizontally transferable between bacterial cells . Mobile E. coli were demonstrated to display some level of protein translation  or more general mechanisms like biofilms induction. However, the induction of antibiotic resistance/tolerance via cues is not limited to antibiotics. Within natural communities, bacteria are continuously exposed to a variety of small molecules other than antibiotics. Among these bioactive metabolites, some have been shown to induce biological responses in bacteria leading to a change in the overall antibiotic tolerance profile.2S), and volatile ammonia were recently shown to impact the antibiotic tolerance profile of receiver bacteria  patients .For this exercise, we chose a simple polymicrobial community including the pathogens patients , 2011 anirulence . In the S. aureus in an autolysin-dependent manner and triggering biofilm formation as a growth protective mechanism  restored their sensitivity to aminoglycosides to a level comparable to non-persister cells (in vivo and in vitro suggesting that synergistic drug combinations have therapeutic potentials (The nature of the effect of volatile ammonia on antibiotic resistance is dependent on the class of antibiotic. While bacterial exposure to volatile ammonia induces tolerance against β-lactam and tetracycline antibiotics it increases the efficacy of aminoglycosides . Intereser cells . These ttentials .in vitro susceptibilities to isolated organisms has many limitations clinically and in polymicrobial infections, many community interactions can reduce antibiotic efficacy in addition to traditional resistance mechanisms. Understanding chemical interactions within microbial communities should include the role of antibiotics in these communities and mechanisms of resistance. This will provide new opportunities and strategies to accelerate the discovery of bioactive molecules. Finally, we have to continue our efforts toward our global understanding of these non-classical views of antibiotic-mediated resistance mechanisms as complementary and potential strategies to new drug development programs in order to control and adequately manage the spread of antibiotic resistance in the future.Furthering our understanding of bacterial interactions mediated by antibiotics and non-antibiotic molecules will be valuable in the development of new strategies to combat antibiotic resistance. Although significant findings have been made in this emerging area of research, we need to further expand our views of these dynamic interactions in microbial communities. The conventional approach of determining The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio cholerae was converted into a culturable state by coculture with several eukaryotic cell lines including HT-29 cells. In this study, we found that a factor converting VBNC V. cholerae into a culturable state (FCVC) existed in cell extracts of eukaryotic cells. FCVC was nondialyzable, proteinase K-sensitive, and stable to heating at <60°C for 5 min. We prepared thiosulfate citrate bile salts sucrose (TCBS) plates with FCVC (F-TCBS plates). After confirming that VBNC V. cholerae O1 and O139 formed typical yellow colonies on F-TCBS plates, we tried to isolate cholera toxin gene-positive VBNC V. cholerae from environmental water samples collected in urban slum areas of Kolkata, India and succeeded in isolating V. cholerae O1 El Tor variant strains harboring a gene for the cholera toxin. The possible importance of VBNC V. cholerae O1 as a source of cholera outbreaks is discussed.Previously, we reported that viable but nonculturable (VBNC)  Vibrio cholerae O1 and O139 continues to be a major cause of severe diarrhea, especially in developing countries  V. cholerae O1 (N16961) and V. cholerae O139 (VC-280) were taken from stock cultures in the laboratory repository at the National Institute of Cholera and Enteric Diseases. The culture media used were alkaline peptone water, pH 8.8 (APW) , nutrient agar  supplemented with 1% NaCl (NA) and thiosulfate citrate bile salts sucrose agar (TCBS) (Eiken).−1 NaHCO3 , 3.56 g L−1 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Sigma), and 10% fetal bovine serum . CHO cell (Chinese hamster ovary cells) and HeLa cells  were cultured in minimum essential medium  supplemented with 10% FBS, while Caco-2 cells  were cultured in MEM supplemented with 20% FBS. T84 cell (human colonic adenocarcinoma cells) and Intestine 407 cells (human jejunum and ileum cells) were cultured in DMEM supplemented with 10% FBS. All media were supplemented with 100 μg mL−1 streptomycin and 100 UmL−1 penicillin. All cell cultures were carried out in a CO2 incubator at 37°C under 5% CO2.HT-29 cells (human colon adenocarcinoma grade II cells) were cultured in Dulbecco's modified Eagle's medium  supplemented with 1.5 g LV. cholerae O1 (N16961) and O139 (VC-280) were prepared as described previously , washed twice with a VBNC microcosm buffer, comprising 1% sterile solution of artificial seawater , and suspended in 200 mL of VBNC microcosm buffer in a 1-L flask to a final concentration of ∼1 × 108 cells mL−1. The cells in VBNC microcosm buffer were incubated at 4°C in the dark without shaking. After incubation for 11 weeks, no culturable cells were observed following incubation of each VBNC microcosm buffer in APW at 37°C for 16 h. Thus, the VBNC microcosm buffer after incubation for 11 weeks contained VBNC V. cholerae O1 (N16961) and O139 (VC-280).VBNC V. cholerae was serially diluted by twofold, and 0.1 mL of each dilution was inoculated into a mixture of 0.3 mL of 1.5-fold-condensed APW and 0.05 mL of phosphate-buffered saline, pH 7.0 (PBS). After incubation at 37°C for 16 h without shaking, the turbidity of each APW was observed by the naked eye and the highest dilution of the microcosm buffer that became turbid was recorded. Assuming that one culturable cell produced turbidity by growth, the number of culturable cells per milliliter of the original microcosm buffer was calculated.A 0.1 mL aliquot of VBNC microcosm buffer containing VBNC g for 5 min at 25°C. The collected cells were resuspended in 0.5 mL of PBS, mixed with 0.1-mm-diameter glass beads, and disrupted by shaking for 90 sec using a ShakeMaster . After centrifugation of each mixture at 20,000g for 5 min at 4°C, the supernatant was passed through a 0.20-μm membrane filter . The obtained cell extract without further dilution (∼0.5 mL) was designated a solution of a factor converting VBNC to a culturable state (FCVC).Eukaryotic cells were cultured as described above. Confluent cells in 10-cm-diameter petri dishes were washed with PBS, scrapped off, and centrifuged at 3000V. cholerae preparation and 0.05 mL of FCVC were added to 0.3 mL of 1.5-fold-condensed APW and incubated at 37°C for 16 h without shaking. When the APW became turbid, a 0.1-mL aliquot was inoculated onto TCBS plates and incubated at 37°C for 16 h. The yellow colonies that appeared were inoculated onto NA plates and incubated at 37°C for 16 h. The serotypes of the colonies on NA plates were confirmed by appropriate typing sera .A 0.1-mL aliquot of a VBNC A 0.5-mL aliquot of FCVC was placed in a dialysis bag  and dialysis was carried out against 2 L of 20 mmol/L Tris-HCl (PH 8.5) at 4°C for 16 h.μL aliquot of 25 mg mL−1 of Proteinase K (Sigma) in PBS was added to 98 μL of FCVC and incubated at 37°C for 1 h. To inactivate the proteinase K activity for further assays with VBNC V. cholerae, 1 μL of 50 mg mL−1 proteinase K inhibitor  in PBS was added to 99 μL of proteinase K-treated FCVC and incubated at 37°C for 1 h.A 1-μL of 25 mg mL−1 of Proteinase K in PBS and 1 μL of 50 mg mL−1 Pefabloc SC in PBS was incubated at 37°C for 1 h, followed by addition of the mixture to 98 μL of FCVC, and incubated at 37°C for 1 h.A mixture of 1 V. cholerae preparation were added to 0.3 mL of 1.5-fold-condensed APW. After incubation at 37°C for 16 h, the turbidity of the APW was observed by the naked eye and confirmation of V. cholerae was carried out as described above.Aliquots (0.1 mL) of FCVC were treated at different temperatures for various time intervals and then subjected to 10-fold serial dilution. A 0.05-mL aliquot of each FCVC dilution and 0.1 mL of VBNC TCBS plates with FCVC (F-TCBS plates) were prepared as follows. TCBS medium was dissolved in water and boiled in accordance with the manufacturer's instructions. When the TCBS had cooled to about 60°C, ∼20 mL was poured into a 9-cm-diameter petri dish together with 1.0 mL of previously added nondiluted FCVC from HT-29 cells. The solutions were mixed thoroughly and allowed to solidify.2. Point C was at a backwater in another slum area. Point D was at a riverside. At each of the sampling points, 50 mL of water was collected twice a month, with at least 2-week intervals, for 1 year (October 2010–September 2011).Four sampling points were identified in urban slum areas of Kolkata Fig. . Points Aliquots (0.1 mL) of the collected environmental water samples were inoculated directly on both F-TCBS and TCBS plates and incubated at 37°C for 16 h. When the total numbers of colonies formed on the plates were <10, the water samples were condensed by 10-fold. Aliquots (0.1 mL) of the condensed samples were then used for direct plating on F-TCBS and TCBS plates.V. cholerae polymerase chain reaction (PCR) for a V. cholerae-specific gene, ompW and a cholera toxin-specific gene, ctx was carried out as described previously  or V. cholerae O139 (VC-280) was incubated at 4°C in the dark without shaking. Culturable cells in each VBNC microcosm buffer were enumerated periodically as described in the Material and Methods. As shown in Figure V. cholerae O1 (N16961) or O139 (VC-280) were observed after incubation for 11 weeks. Furthermore, 0.1-mL aliquots of these VBNC microcosm buffers did not produce any colonies on either NA or TCBS plates. In further experiments, these VBNC microcosm buffers were used as VBNC V. cholerae O1 (N16961) and V. cholerae O139 (VC-280).A VBNC microcosm buffer containing ∼1 × 10 O139 VC-80 was inV. cholerae O139 (VC-280) to a culturable state (Table V. cholerae O139 (VC-280) into the APW, incubation was carried out at 37°C for 16 h without shaking. The conversion was confirmed by turbidity of the APW. Growth of VBNC V. cholerae O139 (VC-280) was observed after addition of the cell extracts of various eukaryotic cells, while no growth of VBNC V. cholerae O139 (VC-280) was observed without addition of the cell extracts. Among the eukaryotic cells examined, the cell extract of HT-29 cells showed the highest conversion activity. These results indicate that the cell extracts of eukaryotic cells contained a factor that converts VBNC V. cholrae into a culturable state. We named this factor FCVC.Cell extracts of various eukaryotic cells converted VBNC V. cholerae conversion activity of FCVC from HT-29 are presented in Table V. cholerae conversion activity of FCVC from HT-29 cells, FCVC lost its conversion activity after the proteinase treatment, and this effect was inhibited by treatment with proteinase K inhibitor.Some properties of the VBNC V. cholerae conversion activity of FCVC from HT-29 cells was examined and the results are shown in Figure The heat stability of the VBNC These results suggest that the FCVC is a heat-stable protein with a molecular weight of more than 10,000.V. cholerae O139 (VC-280) was converted to a culturable form on F-TCBS plates. As shown in Figure V. cholerae O139 formed yellow colonies on F-TCBS plates, while no colonies appeared on TCBS plates without FCVC  compared with the number from TCBS plates without FCVC . All ompW-positive strains were analyzed for whether they harbored a cholera toxin gene, ctx. A total of 120 ctx-positive strains were identified from F-TCBS plates, while only six were identified from TCBS plates without FCVC. These ctx-positive V. cholerae strains were only detected at sampling points A and B. All ompW-positive, ctx-positive V. cholerae strains were confirmed to be serotype O1, biotype El Tor variant by their polymyxin B sensitivity and to harbor classical ctxB by MAMA-PCR.Using F-TCBS plates, we tried to isolate VBNC V. cholerae strains isolated from environmental water samples is shown in Figure ompW-positive VBNC V. cholerae strains were isolated throughout the year, although there was a peak of isolation from April to June, with the highest isolation in May. On the other hand, most of the ompW-positive, ctx-positive VBNC V. cholerae strains were isolated in April. In September, only one ompW-positive, ctx-positive VBNC V. cholerae strain was isolated.The seasonal distribution of VBNC Escherichia coli and V. cholerae, the existence of VBNC states in the environment of various bacteria, such as V. cholerae  was inoculated on F-TCBS plates, colonies of V. cholerae O139 were formed, suggesting that the VBNC bacteria were converted into a culturable state. By using F-TCBS plates, we successfully isolated VBNC V. cholerae from environmental water samples in urban slum areas of Kolkata, India. Quite large numbers of V. cholerae O1 strains carrying the ctx gene formed typical yellowish colonies on F-TCBS plates. Not only during the cholera epidemic season (April), but also between epidemic seasons (September and October), VBNC V. cholerae O1 was isolated from environmental water samples, although the frequency of its isolation between epidemic seasons was much lower than that during the epidemic season.Previously, we reported the conversion of VBNC ctx-positive VBNC V. cholerae O1 exists in environmental water within cholera endemic areas strongly supports the hypothesis that the aquatic environment of cholera endemic areas is the source of cholera outbreaks. Further systematic epidemiology of the geographical and seasonal distributions of ctx-positive VBNC V. cholerae O1 and O139 in the environment of cholera endemic areas like Kolkata using F-TCBS plates will strengthen the above hypothesis.The evidence presented in this study that