Utility of CD4 count measurement in the era of universal antiretroviral therapy: an analysis of routine laboratory data in Botswana.

OBJECTIVES: National guidelines in Botswana recommend baseline CD4 count measurement and both CD4 and HIV viral load (VL) monitoring post-antiretroviral therapy (ART) initiation. We evaluated the utility of CD4 count measurement in Botswana in the era of universal ART. METHODS: CD4 and VL data were analysed for HIV-infected adults undergoing CD4 count measurement in 2015-2017 at the Botswana Harvard HIV-Reference Laboratory. We determined (1) the proportion of individuals with advanced HIV disease (CD4 count < 200 cells/µL) at initial CD4 assessment, (2) the proportion with an initial CD4 count ≥ 200 cells/µL experiencing a subsequent decline in CD4 count to < 200 cells/µL, and (3) the proportion of these immunologically failing individuals who had virological failure. Logistic regression modelling examined factors associated with advanced HIV disease. CD4 count trajectories were assessed using locally weighted scatterplot smoothing (LOWESS) regression. RESULTS: Twenty-five per cent (3571/14 423) of individuals with an initial CD4 assessment during the study period had advanced HIV disease at baseline. Older age [≥ 35 years; adjusted odds ratio (aOR) 1.9; 95% confidence interval (CI) 1.8-2.1] and male sex were associated with advanced HIV disease. Fifty per cent (7163/14 423) of individuals had at least two CD4 counts during the study period. Of those with an initial CD4 count ≥ 200 cells/µL, 4% (180/5061) experienced a decline in CD4 count to < 200 cells/µL; the majority of CD4 count declines were in virologically suppressed individuals and transient. CONCLUSIONS: One-quarter of HIV-positive individuals in Botswana still present with advanced HIV disease, highlighting the importance of baseline CD4 count measurement to identify this at-risk population. Few with a baseline CD4 count ≥ 200 cells/µL experienced a drop below 200 cells/µL, suggesting limited utility for ongoing CD4 monitoring.

Ablation of Stat3 by siRNA alters gene expression profiles in JEG-3 cells: a systems biology approach.

Control of inflammation at the maternal-fetal interface is a critical element in mammalian pregnancy. Previous work from our laboratory has shown that Stat3 may be a placental mediator involved in maintaining immunologic homeostasis at the maternal-fetal interface. The aim of the current study is to further elucidate the role of Stat3 in response to inflammation. As ablation of Stat3 in mice results in embryonic lethality, we evaluated the role of Stat3 in vitro using an siRNA approach. Trophoblast-like JEG-3 cells were transfected with an siRNA construct specific to Stat3. Experimental and control cells were exposed to conditioned medium from PHA-activated peripheral blood mononuclear cells and incubated for 45 min. Cells were then collected and RNA isolated for transcriptional profiling using human Affymetrix U133 plus 2.0 GeneChips. Differences in gene expression between control and Stat3-ablated cells were evaluated using conventional statistical methods. Fifty-two genes were detected as up-regulated in conditioned medium in both mock transfected and in Stat3 siRNA transfected JEG-3 cells. Two genes (EPAS1 and RASGEF1B) were up-regulated only in cells transfected with negative control siRNA, while 36 genes were up-regulated only in cells transfected with Stat3 siRNA. Sixty genes were differentially expressed between Stat3 siRNA transfected cells relative to mock transfected cells both in basal and conditioned medium. These included 31 genes up-regulated with Stat3 siRNA transfected cells and 29 genes down-regulated with Stat3 siRNA. Eleven genes were differentially expressed only in basal medium. Seven of these were up-regulated in the presence of Stat3 siRNA and four were down-regulated. Nine genes were differentially expressed only in conditioned medium. Six of these were up-regulated and three down-regulated in the presence of Stat3 siRNA. Off-target effects were excluded in a second set of experiments in which Stat3 mRNA was targeted at a different site and quantitative real-time PCR performed on selected genes derived from the microarray analysis. While some of the genes that showed differential expression between Stat3-ablated cells and mock transfected controls were genes typically associated with immune response (e.g., CCR7 and IRAK1), in silico modeling of the microarray data also revealed complex networks of signaling molecules and molecules associated with cellular metabolism previously seen in transcription factor ablation in model organisms. We conclude thus: Stat3 controls a specific gene set in trophoblast-like JEG-3 cells. While some differentially expressed genes and in silico models of their functions are consistent with the hypothesis that Stat3 plays a role in regulating inflammation, Stat3-mediated response to inflammation appears to also involve complex homeostatic adaptations of a non-immunologic nature.

Soluble factors from LPS- and PHA-activated PBMC induce MAPK, Stat1 and Stat3 phosphorylation in primary cultures of human term placental trophoblasts: implications for infection and prematurity.

Infection of the maternal vaginal tract is one of the single most important antecedents of premature labor. We have hypothesized that the abundant local synthesis of pro-inflammatory cytokines that occurs during infection may disrupt the delicate "immunological cross-talk" that must occur between maternal and fetal tissues in order to carry pregnancy to term. These experiments were undertaken as part of a larger study directed at testing that hypothesis. We prepared primary cultures of human trophoblasts from term placentas. Cell cultures were stimulated with conditioned medium from resting, PHA or LPS-activated peripheral blood mononuclear cells (PBMC). Medium with LPS or PHA at the same concentration as that used to stimulate the PBMC was used as an additional control. Lysates were subjected to western blotting for activated forms of the mitogen-activated protein kinases (MAPK), Stat1, and Stat3. Western blotting showed phosphorylation of the Jun kinase (JNK), p38, and Erk1/Erk2 MAPK in trophoblasts incubated with conditioned medium from LPS or PHA-activated PBMC but not from medium from resting PBMC, or with PHA or LPS alone. Phosphorylation could be detected as early as 5 min and was still observable by 10 min, the latest time point tested. Similarly, Stat1 and Stat3 phosphorylation was observed within 10 min of exposure to conditioned medium and was still observable 10 min after exposure. Immunohistochemistry also demonstrated nuclear translocation of both Stat1 and Stat3 after stimulation of trophoblasts with medium from activated PBMC. These findings are compatible with the hypothesis that immunologic balance at the maternal-fetal interface is maintained by ongoing "cross-talk" between the fetus (and fetally-derived tissues) and the maternal immune system. Infection of the maternal vaginal tract may disrupt this delicate immunologic balance, initiating inflammatory events that ultimately result in preterm labor.

Circulating cytokines in Norwegian patients with psoriatic arthritis determined by a multiplex cytokine array system.

OBJECTIVES: Serum cytokines play an important role in the pathogenesis of psoriatic arthritis (PsA) by initiating and perpetuating various cellular and humoral autoimmune processes. The aim of this study was to describe a broad spectrum of T- and B-cell cytokines, growth factors and chemokines in patients with PsA and healthy individuals. METHODS: A novel protein array system, denoted as multiplex cytokine assay was utilized to measure simultaneously the levels of 23 circulating cytokines of patients with PsA and healthy individuals. Additionally, correlational clustering and discriminant function analysis (DFA), two multivariate, supervised analysis methods were employed to identify a subset of biomarkers in order to describe potential functional inter-relationships among these pathological cytokines and identify biomarkers with prognostic and diagnostic utility. RESULTS: Univariate analysis demonstrated that serum levels of a complex set of immune and inflammatory modulating cytokines are significantly up-regulated in patients with PsA relative to unaffected controls including interleukin (IL)-10, IL-13, interferen (IFN)-alpha, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor [CCL3 macrophage inflammatory protein (MIP)-1alpha], CCL4 (MIP-1beta) and CCL11 (Eotaxin), while granulocyte-colony stimulating factor was significantly reduced in PsA patients. Correlational clustering was able to discriminate among, and hence subclassify, patients with varying levels of disease activity, which may prove useful in guiding therapy in these apparently phenotypically distinct disease subsets. DFA identified EGF, IFN-alpha, VEGF, CCL3 (MIP-1alpha) and IL-12p40 as analytes with the strongest discriminatory power among various PsA patients and controls. CONCLUSIONS: Our findings suggest that these factors modulate PsA pathology and the articular involvement in a synergistic manner. Identifying factors could be used in the development of clinical diagnostic tests, which are valuable to guide evidence-based diagnosis and disease management of PsA.

Lactic acidosis in Gabonese children with severe malaria is unrelated to dehydration.

BACKGROUND: Hyperlactatemia is an important and common complication of severe malaria. We investigated changes in fluid compartment volumes in patients with severe malaria and control patients with the use of bioimpedence analysis. METHODS: We estimated extracellular water and total body water volumes in a total of 180 children: 56 with severe malaria, 94 with moderate malaria, 24 with respiratory tract infection, and 6 with severe diarrhea. RESULTS: There was a mean (+/-SD) decrease in total body water volume of 17+/-24 mL/kg (or 3% of total body water volume) in patients with severe malaria. This compares with a mean (+/-SD) decrease in total body water volume of 33+/-28 mL/kg (or 6% of total body water volume) in patients with severe diarrhea. There was no increase in extracellular water volume in patients with severe malaria, suggesting no significant intravascular volume depletion in patients with severe malaria. There was no relationship between lactatemia and any changes in fluid compartment volumes. CONCLUSIONS: The changes in fluid volumes that were observed are unlikely to be of physiological significance in the pathophysiology of severe malaria.

hCG Secretion in human choriocarcinoma JAR cells is MAPK but not Stat3 dependent: contributions of TNFalpha and IL-1beta to inflammation-induced hCG secretion.

Inflammatory mediators play critical physiologic roles throughout the course of normal pregnancy. At the same time, uncontrolled inflammation appears to have an adverse affect on pregnancy outcome. Thus, a crucial task of reproductive immunology is to define the mechanisms through which inflammation is controlled at the maternal-fetal interface. We examined the signaling pathways activated in the human choriocarcinoma cell line, JAR, in response to an in vitro model of an inflammatory challenge. We incubated JAR cells with medium from peripheral blood mononuclear cells (PBMC) that had been activated with either LPS or PHA. Conditioned medium from each experimental model induced MAP kinase and Stat3 phosphorylation as well as human chorionic gonadotropin (hCG) secretion from JAR cells. hCG secretion could be blocked by pharmacologic inhibition of MAP kinase but not by inhibition of Stat3 using an siRNA approach. The MAPK activators IL-1beta and TNFalpha both induced hCG secretion from JAR cells, but not the Stat3 activators IFN-gamma and IL-6. Furthermore, hCG secretion induced by conditioned media could be blocked by IL-1 receptor antagonist. We conclude that inflammation at the maternal-fetal interface may involve complex webs of regulatory and counter-regulatory mechanisms that involve multiple cytokines and at least two major, independent cell-signaling systems.

T cell activation by soluble C1q-bearing immune complexes: implications for the pathogenesis of rheumatoid arthritis.

Both innate and adaptive immune systems are thought to participate in the pathogenesis of rheumatoid arthritis in adults and children. The experiments reported here were undertaken to examine how immune complexes, potent stimulators of inflammation, may regulate cells of the adaptive immune system. Human T cells were prepared from peripheral blood by negative selection and incubated with bovine serum albumin (BSA)-anti-BSA immune complexes that were formed in the presence or absence of human C1q. C1q-bearing immune complexes, but not unopsonized complexes, elicited both TNF-alpha and IFN-gamma secretion from human T cells. Secretion of both cytokines was time- and dose-dependent. Cross-linking C1q on the cell surface of T cells produced the same results. Cytokine secretion was not inhibited by blocking the C3b receptor (CR1, CD35) on T cells prior to incubation with immune complexes. Reverse transcriptase polymerase chain reaction (RT-PCR) of immune complex-stimulated cells revealed accumulation of both TNF-alpha and IFN-gamma mRNA within 2 h post-stimulation. IL-2 was not detected in cell culture supernatants, but IL-2 receptor alpha chain (CD25) was detected in low density on a small proportion of T cells activated by C1q-bearing immune complexes. Secretion of both cytokines was inhibited partially, but not completely, by IL-10. These experiments show that immune complexes, potent inflammatory mediators, may activate T cells through a novel mechanism. These findings have implications for chronic inflammatory diseases in humans.

C1q-bearing immune complexes induce IL-8 secretion in human umbilical vein endothelial cells (HUVEC) through protein tyrosine kinase- and mitogen-activated protein kinase-dependent mechanisms: evidence that the 126 kD phagocytic C1q receptor mediates immune complex activation of HUVEC.

Endothelial cells play a pivotal role in the initiation and perpetuation of inflammation. C1q, the first component of the classical pathway of complement, is a potent stimulus leading to endothelial cell activation and cytokine production. The specific cellular mechanisms through which endothelial cells are stimulated by C1q are not known. We stimulated human umbilical vein endothelial cells (HUVEC) with either monomeric C1q or C1q-bearing immune complexes (C1q-IC) in the presence or absence of inhibitors of protein tyrosine kinases (PTK) or mitogen-activated protein kinases (MAPK). C1q-IC, but not monomeric C1q, induced IL-8 production in dose- and time-dependent fashion. R3, a cross-linking monoclonal IgM antibody against the 126 kD phagocytic C1q receptor (C1qR), also stimulated IL-8 production. IL-8 mRNA accumulation was detected by Northern blot analysis within 2 h of stimulation by the immune complexes and was enhanced by the addition of cycloheximide. Secretion of IL-8 by C1q-IC stimulated HUVEC was completely blocked by the PTK inhibitor, genistein or the MAPK inhibitor, UO126. These experiments demonstrate that C1q-IC-induced production of IL-8 in HUVEC is dependent upon the activation of PTK and MAPK. These findings also support a role for the phagocytic C1qR as an important activator of HUVEC by immune complexes.

Subspecialty referrals for pauciarticular juvenile rheumatoid arthritis.

OBJECTIVES: To examine referral patterns from primary care physicians for children with pauciarticular juvenile rheumatoid arthritis (JRA) and to determine whether children with pauciarticular JRA referred to pediatric rheumatologists differ in clinical presentation from children referred to other specialists. DESIGN: A retrospective records review of 49 patients with pauciarticular jRA was performed. Records were reviewed to determine the specialty of the referring physician and whether the children referred had symptoms and signs compatible with a synovitis at the time primary care was sought. SETTING: Inner-city tertiary pediatric rheumatology referral center. PARTICIPANTS: Children with pauciarticular JRA. MAIN OUTCOME MEASURES: Identification of referral patterns of primary care physicians. Associated morbidity owing to JRA was ascertained at the time of referral. RESULTS: Most children with pauciarticular JRA (62%) were referred to orthopedic surgeons prior to referral for pediatric rheumatology care. No differences in clinical symptoms were seen between children referred to pediatric rheumatologists and those referred to orthopedic surgeons. Children referred initially to orthopedic surgeons were younger than those referred to pediatric rheumatologists. CONCLUSION: A notable number of children with pauciarticular JRA are referred to orthopedic surgeons prior to the establishment of that diagnosis, even when such children present with unequivocal signs of synovitis. This may be owing to the misconception that arthritis is rare in preschool-aged children or to the difficulty of ascertaining the presence of synovitis in younger children.

Immune complex size and complement regulate cytokine production by peripheral blood mononuclear cells.

We have previously shown that immune complexes isolated from children with juvenile rheumatoid arthritis are heterogeneous in their size, composition, and proinflammatory capacities. The experiments described here were undertaken to clarify further the roles of size and composition in determining the proinflammatory effects of immune complexes. We incubated peripheral blood mononuclear cells (PBMCs) with different soluble immune complex preparations: opsonized complexes, which were formed in the presence of serum, unopsonized complexes, which were formed in the absence of serum, and immune precipitates solubilized by complement after their formation. ELISA assays showed that immune complexes formed in the presence of complement were less efficient than unopsonized complexes in inducing IL-1beta and IL-8 secretion from leukocytes. Solubilized immune precipitates showed intermediate capacity to stimulate the release of both cytokines. Complexes formed in heat-inactivated serum were as efficient as unopsonized complexes in eliciting cytokine secretion from the cells. The capacity of complement to regulate cytokine secretion from leukocytes was related, at least in part, to immune complex size. Sucrose density gradients showed unopsonized complexes and solubilized immune precipitates were larger than opsonized immune complexes. In contrast, fluid-phase binding of C4 to immune complexes, which did not appreciably change immune complex size, substantially increased IL-1beta secretion from PBMC.

Complement deposition on immune complexes reduces the frequencies of metabolic, proteolytic, and superoxide oscillations of migrating neutrophils.

Neutrophils exhibit intrinsic sinusoidal metabolite concentration oscillations of 3 min in resting cells and an additional approximately 10- or 20-s oscillation in migrating/adhering cells. To better understand immune complex (IC)-mediated leukocyte activation, we have studied neutrophil metabolic oscillations in the presence of ICs either with or without fixed complement. Using a microscope photometer we quantitated NAD(P)H autofluorescence oscillations. Cells exposed to ICs exhibited metabolic oscillation periods of approximately 12 s in the absence of complement and approximately 22 s in the presence of complement opsonization. To determine if the effects could be associated with C3 deposition, we used ICs opsonized with only C3 or only C1 and C4. Untreated ICs, heat-inactivated complement-treated ICs, and C1,C4-treated ICs trigger rapid metabolic oscillations, as do fMLP and yeast; in contrast, ICs treated with full complement or C3 alone did not affect NAD(P)H oscillations in comparison to controls. The induction of higher frequency (approximately 10 s) NAD(P)H oscillations by ICs could be blocked by addition of anti-FcgammaRII, but not FcgammaRIII mAb fragments, suggesting the participation of FcgammaRII in cellular metabolic responses to ICs. Parallel changes in the frequencies of oxidant release and pericellular proteolysis were found for all of these stimuli. Thus, immune complex composition affects both intracellular metabolic signals and extracellular functional oscillations. We suggest that complement attenuates the phlogistic potential of ICs by reducing the frequency of cytoplasmic NAD(P)H oscillations.

Pathogenesis and mechanisms of inflammation in the childhood rheumatic diseases.

The presence of CD4+ cells in the synovium of children with juvenile rheumatoid arthritis has led to the generally accepted hypothesis that aberrant activation or regulation of acquired immunity is central to the pathogenesis of this family of diseases; however, this hypothesis remains unproven, and, indeed, a specific role for T cells in the process of chronic synovitis in rheumatoid disease has yet to be identified for either adults or children. In contrast, processes associated with innate immunity are undeniably involved in the pathophysiology of chronic synovitis in both rheumatoid arthritis and juvenile rheumatoid arthritis. The presence of neutrophils in the synovial fluid, complement activation, and immune complex accumulation in the synovial fluid and serum all indicate an active inflammatory process. It is reasonable to hypothesize, therefore, that there are important clues to the cause of juvenile rheumatoid arthritis to be gleaned from a more careful study of inflammation or innate immunity. This review will focus on the roles of complement, immune complexes, and the vascular endothelium in the pathogenesis of juvenile rheumatoid arthritis. In so doing, it will reexamine the dogma of the central role of the T cell in juvenile rheumatoid arthritis disease pathogenesis and offer new paradigms with which to understand this and other rheumatic diseases of childhood.

Human choriocarcinoma JAR cells constitutively express pro-interleukin-1beta that can be released with Fc(gamma) receptor engagement.

Some authors have suggested that fetally derived syncytiotrophoblasts, which form the barrier between mother and the fetus, are an integral part of a complex macrophage-cytokine network involving maternal leukocytes, decidual cells, placental tissues, and even the fetus itself. We report here that syncytiotrophoblast-like JAR cells, a human choriocarcinoma cell line, share another feature common to cells of the monocyte-macrophage lineage, the ability to secrete IL-1beta when stimulated through their Fc(gamma) receptors. We incubated JAR cells with physiologically relevant concentrations of model BSA-rabbit IgG-anti-BSA immune complexes or monomeric rabbit IgG for periods of up to 72 h. Both monomeric IgG and immune complexes induced IL-1beta from JAR cells, although levels produced by immune complexes were approximately twice those induced by monomeric IgG. IL-1beta secretion was not inhibited by cycloheximide, and Western blots of JAR cell lysates using pro-IL-1beta MAb revealed constitutive expression of a 31-kD protein, whose levels declined within 2 h of stimulation by either IgG or immune complexes, but returned to baseline within 18 h.

In vitro induction of proinflammatory cytokine secretion by juvenile rheumatoid arthritis synovial fluid immune complexes.

OBJECTIVE: To characterize juvenile rheumatoid arthritis synovial fluid (SF) immune complexes and to examine their interaction with leukocytes. METHODS: SF immunoglobulin-containing fractions were prepared by sequential chromatography on protein A and Sephacryl 300. Fractions were subdivided according to molecular weight, characterized for immunoglobulin and complement content, and incubated with either promonocytic U937 cells or normal human peripheral blood mononuclear cells (PBMC). RESULTS: High molecular weight SF immunoglobulin-containing fractions stimulated the release of interleukin-1beta (IL-1beta) from U937 cells. These same complexes stimulated tumor necrosis factor alpha (TNFalpha), IL-1beta, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) from PBMC. Lower molecular weight material was less efficient in inducing any of the cytokines. TNFalpha and IL-1beta were the earliest of the messenger RNAs examined to be induced by the high molecular weight complexes. However, the secretion of IL-6, IL-8, and GM-CSF stimulated by the complexes was not completely dependent upon the secretion of IL-1beta. Addition of IL-1 receptor antagonist to the cell cultures reduced GM-CSF and IL-6 production by 40% and IL-8 production by 25% in PBMC. CONCLUSION: SF immunoglobulin fractions contain immune complexes that vary in size, composition, and phlogistic potential. High molecular weight complexes are capable of inducing a spectrum of proinflammatory cytokines, all of which have been implicated in the pathogenesis of rheumatic disease.

Juvenile rheumatoid arthritis in African Americans.

OBJECTIVE: To examine racial differences in disease expression in African American and Caucasian children with pauciarticular and polyarticular juvenile rheumatoid arthritis (JRA). METHODS: A retrospective chart review was conducted of 35 African American and 137 Caucasian children with pauciarticular and polyarticular JRA. RESULTS: African American children were significantly older than Caucasian children at the time of presentation. This was true both for the group as a whole and for each of the disease onset subtypes. African American children were less likely to have positive antinuclear antibody tests than Caucasian children. This finding paralleled a low incidence of uveitis in African American children. African American children were also more likely to have IgM rheumatoid factors (detected by latex agglutination) than Caucasian children. This was true even for African American children with pauciarticular JRA. CONCLUSION: There are significant phenotypic differences between African American and Caucasian children with JRA.

Constitutive expression c-fos, c-jun, and NF kappa B mRNA is in nucleated fetal blood cells and up-regulation of c-fos and c-jun with anti-CD3 stimulation.

Fetal and neonatal lymphocytes are relatively resistant to activation and cytokine production when stimulated either via their T-cell antigen receptors or lectins. The molecular mechanism(s) responsible for this phenomenon have not been clearly elucidated. We have hypothesized that such defects in fetal/neonatal T-cell activation may be due to lack of expression of the transcriptional regulatory elements required for T-cell activation. We used reverse transcriptase-polymerase chain reaction to examine both fetal and term neonatal cord bloods for mRNA expression of three transcription factors implicated in T-cell activation: c-jun, c-fos, and NF kappa B (p50 subunit). We demonstrate that mRNAs for all three of these regulatory factors are expressed in fetal blood cells by the 27th week of gestation and in term cord bloods. Activation of term infant cord blood mononuclear cells with anti-CD3 monoclonal antibodies resulted in up-regulation of both c-jun and c-fos mRNAs within 15 min of stimulation. However, secretion of IL-2 by anti-CD3-stimulated cord blood mononuclear cells was still blunted compared with control cells from adults. We conclude that fetal nucleated blood cells constitutively express important genes for cytokine regulation and are able to increase intracellular accumulation of the mRNAs for these factors in response to anti-CD3 stimulation. Thus, qualitative differences in the capacity to regulate these factors could not be shown in fetal blood cells. Quantitative experiments comparing binding of these transcription factors to the IL-2 promoter are currently under investigation.

Regulation of cytokine mRNA expression in activated lymphocytes by human choriocarcinoma JAR cells.

Because the fetus is semiallogenic to the mother, considerable modulation of the maternal immune response must occur in order for pregnancy to be successfully carried to term. Some authors have hypothesized that the immunomodulation of pregnancy includes an adjustment of cytokine responses away from the Th1 paradigm and toward the Th2 pattern. In vivo data from murine pregnancy support this hypothesis. However, in humans, the Th1/Th2 model appears to be more complex than that in mice, and cytokine expression of mRNA in human decidual tissue does not reflect a clear-cut Th2 bias. The experiments described here were undertaken to determine whether and how trophoblastic cells modulate cytokine expression in activated lymphocytes, and whether there is a trend toward the use of the Th2 pattern in an experimental model of the maternal-fetal interface. We used reverse transcriptase polymerase chain reaction (rtPCR) to detect cytokine mRNA expression in human peripheral blood mononuclear cells cocultivated with human choriocarcinoma JAR cells. We found that although IL-2 (a paradigmatic Th1 cytokine) was significantly down-regulated by JAR cells at the mRNA level, similar decreases were also seen in IL-10, which participates in the Th2 paradigm. We were unable to detect changes in either interferon-gamma (IFN-gamma, a Th1 cytokine) or IL-4 (a Th2 cytokine) mRNA's or in IL-2R expression by fluorescence-activated cell sorting. These studies indicate that human choriocarcinoma JAR cells are capable of modifying cytokines in activated lymphocytes other than those involved in the Th1 paradigm. While it may be useful to view human responses against the background of these patterns established from murine systems, it is reasonable to conclude that human pregnancy may not involve regulation of Th1 immune responses exclusively.

Expression of complement regulatory proteins on fetal blood cells in utero.

Since the fetus is semiallogenic to the mother, mechanisms have evolved to protect fetal tissue from the maternal immune response. Among these mechanisms is the expression of cell-surface complement regulatory proteins at the maternal-fetal interface. However, beginning in the third trimester, fetal blood cells are exposed to actively-transported IgG antibody. Thus, we speculated that fetal blood cells would require expression of one or more complement regulators by the early third trimester. Using flow cytometry and Western blots, we have demonstrated the presence of three important complement regulatory proteins in the circulating blood cells of human fetuses. These findings are consistent with the putative biological role of the cell-surface complement regulatory proteins.

Diminished expression of cell-surface complement regulatory proteins in HIV-infected children and with HIV infection of peripheral blood mononuclear cells in vitro.

Experimental data have established that HIV-infected lymphocytes activate the complement system. However, because mammalian lymphocytes possess a series of cell-surface complement regulators that inhibit amplification on autologous cells, complement-mediated destruction of host cells is usually inhibited. These studies were performed to examine whether alterations in the cell-surface complement regulatory proteins decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) may occur during HIV infection in vitro or in vivo. The physiologic significance of these alterations were assessed by radiolabeled chromium release experiments. We show that MCP fluorescent intensity is significantly lessened in HIV-infected children and that DAF intensity is similarly lessened in infected children with advanced disease. These findings could be duplicated with HIV infection of peripheral blood mononuclear cells in vitro.