Early pregnancy immune biomarkers in peripheral blood may predict preeclampsia.

We performed a prospective cohort study in 197 pregnant women. Peripheral blood was collected between 5 and 16 weeks of gestation. Intracellular cytokine analysis and immunophenotype were performed by flow-cytometry. Serum levels of cytokines and chemokines were analyzed by multiplex assay. 86 patients were eligible for the analysis and 10.5% (n=9) developed preeclampsia. Patients with preeclampsia had significantly higher percentage of CD3+CD4+TNFα+ T helper (Th) 1 cells (45.4±10.3 vs 37.1±8.5, P=0.032) and CD3+CD4+IL17+ Th 17 cells (2.4±1.3 vs 1.6±1.1, P=0.029) when compared to those of patients without preeclampsia. CD3+CD4+CD25+CD127dim/- T regulatory cells (Treg) cells (5.7±1.2% vs 7.0±1.6%, P=0.015) were significantly lower in patients with preeclampsia when compared to those without preeclampsia. Patients with preeclampsia had significantly higher TNFα/IL-10 cell ratio (43.8±10.3 vs 34.3±7.9, P=0.005) and Th17/Treg cell ratio (0.5±0.3 vs 0.2±0.2, P=0.011) when compared to those of patients without preeclampsia. IL-8 and Macrophage inflammatory protein (MIP)-1α serum levels were significantly higher in patients with preeclampsia when compared with patients without preeclampsia (Median=341.0 vs 87.6, U=152, P=0.020 and Median=35.7 vs 17.7, U=120, P=0.029 respectively). Serum MCP-1 levels were significantly lower in patients with preeclampsia when compared with patients without preeclampsia (Median=233.8 vs 390.9, U=183, P=0.021). The logistic regression predictive model combining TNFα/IL-10 ratios, IL-8 and MCP-1 serum levels had the best performance (AUC=0.886, 95%CI 0.8-0.9). We concluded that elevated Th1 and Th17 cell percentages, elevated TNFα/IL-10 and Th17/Treg cell ratios and decreased Treg cell percentages in early pregnancy are associated with preeclampsia.

Deficiency in memory B cell compartment in a patient with infertility and recurrent pregnancy losses.

Alterations in normal balance of B cell subsets have been reported in various rheumatic diseases. In this study, we report a woman with a history of recurrent pregnancy losses (RPL) and infertility who had low levels of memory B cells. A 35-year-old woman with a history of RPL and infertility was demonstrated to have increased peripheral blood CD19+ B cells with persistently low levels of memory B cell subsets. Prior to the frozen donor egg transfer cycle, prednisone and intravenous immunoglobulin G (IVIg) treatment was initiated and patient achieved dichorionic diamniotic twin pregnancies. During pregnancy, proportion (%) of switched memory B cells CD27+IgD- increased, while percent of total CD19+ B cells and CD27-IgD+ naive B cells were gradually decreased with a high dose IVIg treatment. She developed cervical incompetence at 20 weeks of gestation, received a Cesarean section at 32 weeks of gestation due to preterm labor, and delivered twin babies. B cell subset abnormalities may be associated with infertility, RPL and preterm labor, and further investigation is needed.

Inhibition of vacuolar ATPase subunit in tumor cells delays tumor growth by decreasing the essential macrophage population in the tumor microenvironment.

In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. The soluble, cleaved N-terminal domain of V-ATPase a2 isoform is associated with in vitro induction of tumorigenic characteristics in macrophages. This activity led us to further investigate its in vivo role in cancer progression by inhibition of a2 isoform (a2V) in tumor cells and the concomitant effect on tumor microenvironment in the mouse 4T-1 breast cancer model. Results showed that macrophages cocultivated with a2V knockdown (sh-a2) 4T-1 cells produce lower amounts of tumorigenic factors in vitro and have reduced ability to suppress T-cell activation and proliferation compared with control 4T-1 cells. Data analysis showed a delayed mammary tumor growth in Balb/c mice inoculated with sh-a2 4T-1 cells compared with control. The purified CD11b(+) macrophages from sh-a2 tumors showed a reduced expression of mannose receptor-1 (CD206), interleukin-10, transforming growth factor-ß, arginase-1, matrix metalloproteinase and vascular endothelial growth factor. Flow cytometric analysis of tumor-infiltrated macrophages showed a significantly low number of F4/80(+)CD11c(+)CD206(+) macrophages in sh-a2 tumors compared with control. In sh-a2 tumors, most of the macrophages were F4/80(+)CD11c(+) (antitumor M1 macrophages) suggesting it to be the reason behind delayed tumor growth. Additionally, tumor-infiltrating macrophages from sh-a2 tumors showed a reduced expression of CD206 compared with control whereas CD11c expression was unaffected. These findings demonstrate that in the absence of a2V in tumor cells, the resident macrophage population in the tumor microenvironment is altered which affects in vivo tumor growth. We suggest that by involving the host immune system, tumor growth can be controlled through targeting of a2V on tumor cells.

Tumor-associated vacuolar ATPase subunit promotes tumorigenic characteristics in macrophages.

Macrophage polarization contributes to distinct human pathologies. In tumors, a polarized M2 phenotype called tumor-associated macrophages (TAMs) are associated with promotion of invasion and angiogenesis. In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma/vesicular membranes and critically influences the metastatic behavior. In addition, the soluble, cleaved N-terminal domain of a2 isoform of V-ATPase (a2NTD) is associated with in vitro induction of pro-tumorigenic properties in monocytes. This activity of a2 isoform of V-ATPase (a2V) caused us to investigate its role in cancer progression through the evaluation of the immunomodulatory properties of a2NTD. Here, we present direct evidence that surface expression of V-ATPase is associated with macrophage polarization in tumor tissue. Macrophages from BALB/c mice (peritoneal/bone marrow derived) were stimulated with recombinant a2NTD in both ex vivo and in vivo systems and evaluated for TAM characteristics. a2V was highly expressed in tumor tissues (breast and skin) as well as on the surface of tumor cell lines. The a2NTD-stimulated macrophages (a2MΦ) acquired TAM phenotype, which was characterized by elevated expression of mannose receptor-1, Arginase-1, interleukin-10 and transforming growth factor-ß. a2MΦ also exhibited increased production of other tumorigenic factors including matrix metalloproteinase-9 and vascular endothelial growth factor. Further, a2MΦ were cocultured with mouse B-16F0 melanoma cells for their functional characterization. The coculture of these a2MΦ subsequently increased the invasion and angiogenesis of less invasive B-16F0 cells. When cocultured with naive T cells, a2MΦ significantly inhibited T-cell activation. The present data establish the role of V-ATPase in modulating a macrophage phenotype towards TAMs through the action of a2NTD, suggesting it to be a potential therapeutic target in cancer.

Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF.

BACKGROUND: We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. METHODS: Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RESULTS: RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. CONCLUSIONS: The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.

Coagulation changes in individuals with sickle cell trait.

Sickle cell disorders, such as Hb SS and Hb SC, are associated with a hypercoagulable state that may contribute to the vaso-occlusive episodes observed in the disorders. To what extent increased coagulation activity occurs in individuals with sickle cell trait has had limited study. Because such information may help clarify clinical and pathologic findings that may occur in these individuals and may be useful in clarifying the hypercoagulable state in sickle cell disease, we have examined individuals with Hb AS to determine the extent that increased coagulation activity does occur. We measured d-dimers, thrombin-antithrombin (TAT) complexes, prothrombin fragment 1.2 (F1.2), absolute blood monocyte levels, proteins C and S, and isotypes of antiphospholipid antibodies in individuals with Hb AS and in matched controls (Hb AA). Results showed that d-dimers, TAT, and F1.2 were increased significantly above normal levels. Absolute blood monocyte levels were increased. The d-dimers, TAT, F1.2, and monocyte counts showed significant increasing trends through groups of increasing severity (Hb AA, Hb AS, Hb SC, and Hb SS). Our study shows that individuals with Hb AS have increased coagulation activity, with d-dimers, TAT, and F1.2 being consistent indicators. The measures of coagulation activity in Hb AS are lower than in patients with Hb SC and Hb SS disease. These results extend our previous observation that the degree of coagulation activation parallels the degree of disease severity among sickle cell genotypes. The findings suggest that monocytosis, with the possible expression of monocyte-derived tissue factor, and the associated hypercoagulable state are driven by disease severity.

Expression of a novel protein by regenerating hepatocytes and peripheral blood lymphocytes.

Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis.

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.

Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology.

The aim of this study was to investigate the functional status and immunophenotypic characteristics of natural killer (NK) cells in women who suffer recurrent spontaneous abortions (RSA) or have infertility of unknown aetiology. Peripheral blood mononuclear cells (PBMC) were obtained from 40 study patients and 13 normal healthy multiparous controls. NK cells were identified using anti-CD56 and anti-CD16 monoclonal antibodies (mAb). The expression of CD69, CD25, CD122, CD30, CD154, CD128 and CD94 on NK cells was detected using specific mAb and analysed by flow cytometry. CD69 expression on NK cells after ED(27) human trophoblast cell line co-culture with PBMC was also investigated. A significant increase in CD69 expression on CD56(+) NK cells was demonstrated in women with RSA (P < 0.005) and infertility (P < 0.05) as compared with that of normal controls. Conversely, CD94 expression was significantly decreased in women with RSA (P < 0.005) and infertility (P < 0.05) in comparison with that of controls. Increased CD69 expression on NK cells was induced after 24 h co-culture with ED(27). In conclusion, peripheral blood NK cells of women with RSA and infertility of unknown aetiology have higher proportions of activated NK cells in vivo. Unbalanced CD69 and CD94 expression may explain the underlying pathology.

Regeneration and tolerance factor of the human placenta induces IL-10 production.

Regeneration and tolerance factor (RTF) was originally identified in the placenta of mice and the isolated protein shown to have suppressive effects. In these studies, the gene cloned from thymus tissue was mapped to human chromosome 12. The role of recombinant RTF on cytokines was examined. In addition, we examined the human placenta by immunohistochemistry for RTF expression. RTF was expressed at the peripheral layer of cytotrophoblast in 7-9-week-old placentas. Using the RTF gene sequence, a recombinant protein was prepared and shown to induce IL-10 production. These data indicate that RTF is expressed by the tissues most intimately involved at the maternal-fetal interface, and its biological activity is capable of producing the necessary immune response for initiating and maintaining the maternal-fetal relationship.

Regeneration and tolerance factor's potential role in T-cell activation and apoptosis.

Regeneration and tolerance factor (RTF) is a novel membrane protein that has a diverse expression pattern and immunoregulatory properties. RTF is expressed in vivo on the surface of individuals with B cell chronic lymphocytic leukemia and on activated T lymphocytes of HIV infected individuals as determined by their coexpression with CD38 and HLA-DR. The unique expression patterns of this protein in vivo lead us to investigate its expression in vitro. The activation of human PBMCs through the TCR, using anti-CD3 antibody and PMA, upregulated cell surface expression of RTF from 2. 3% to 91.2% (mean channel fluorescence [MCF] increased threefold). The activation of Jurkat T cells through the TCR upregulated surface expression of RTF from 8.3% (MCF-1.3) to 58.7% (MCF-13.1). The Jurkat T-cell line was used as a model system to explore RTF's role in cellular activation. Using the Jurkat T-cell model, we found anti-RTF antibody induces apoptosis. The addition of anti-RTF antibody increased annexin V binding by threefold compared with the IgG1 kappa isotype control antibody (p < 0.00002) and activated caspase 3. These data indicate that RTF is expressed during T-cell activation and may be associated with apoptosis.

Immune modulation following aerobic exercise in children with cystic fibrosis.

Previous studies have demonstrated altered immune response following exercise in healthy adults and children. As data are lacking in children with cystic fibrosis, we evaluated the immune response following acute exercise and hypothesized that acute increases in cellular changes would be seen but would be blunted in subjects with CF. Leukocytes, lymphocytes, and their subsets as well as natural killer cell number and activity were determined before, immediately after, and one hour post exhaustive exercise in 15 children with cystic fibrosis (8-21 yrs, FEV1 69.5+/-18.0%, colonized with P aeruginosa) and 15 healthy controls (8-18 yrs, FEV1 107.5+/-10.7%). At baseline the cystic fibrosis group had greater leukocytes (9.25+/-2.83 vs. 5.17+/-0.96 x 10(9) cells/liter). Immediately post exercise, the cystic fibrosis group demonstrated increases in cell counts for leukocytes (32.4%), lymphocytes (61.8%), granulocytes (36.4%), monocytes (76.2%), and natural killer cells (315%). Similar percentage increases were seen in cell counts for the controls (leukocytes: 39.5%, lymphocytes: 78.5%, granulocytes: 32.0%, monocytes: 75.9%, and NK cells: 442%). Natural killer cell activity also increased by 57.9% in the group with cystic fibrosis and by 43.6% in the healthy controls. Except for elevated leukocyte and granulocyte counts, values returned to baseline at one hour post-exercise. In conclusion, the cellular immune response to acute exercise in children with mild to moderate cystic fibrosis appears normal.

Expression of regeneration and tolerance factor correlates directly with human immunodeficiency virus infection and inversely with hepatitis C virus infection.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3(+) T cells of HIV-seropositive (HIV(+)) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV(+) HCV-seropositive (HCV(+)), HIV- and HCV-seropositive (HIV(+) HCV(+)), and HIV- and HCV-seronegative (HIV(-) HCV(-)) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV(+) individuals is upregulated and that its expression on T cells from HCV(+) individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3(+) T cells was seen in both HIV(+) and HIV(+) HCV(+) individuals compared to HIV(-) HCV(-) individuals. HCV(+) individuals had lower levels of RTF expression than HIV(-) HCV(-) individuals (P < 0.005 for CD4(+); P < 0.0005 for CD8(+)). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV(+) > HIV(+) HCV(+) > HIV(-) HCV(-) > HCV(+). The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.

Immunoglobulin G infusion treatment for women with recurrent spontaneous abortions and elevated CD56+ natural killer cells.

We aimed to investigate the clinical effect of intravenous immunoglobulin G (IVIg) treatment in recurrent aborters with elevated peripheral blood CD56+ NK cell levels while on lymphocyte immunization, anticoagulation and prednisone treatment, with respect to subsequent live birth and reproductive outcome. Thirty-three women with recurrent abortions achieved alloimmune recognition after lymphocyte immunizations. All had autoimmune abnormalities and received preconception anticoagulation and prednisone treatment. At the time of positive pregnancy testing, 18 women with normal NK cell levels (<12%) and 6 with elevated NK cell levels (>12%) continued anticoagulation and prednisone treatment, and 9 with elevated NK cell level initiated additional IVIg treatment. The live birth rates of women with elevated NK cell level (>12%) who initiated post-conception IVIg treatment in addition to anticoagulation and prednisone (100.0%), women with normal NK cell levels (<12%) who continued anticoagulation and prednisone (83.3%) and women with elevated NK cell level (>12%) who continued anticoagulation and prednisone (33.3%) are significantly different (P=0.0065). Prevalence of intrauterine growth retardation and preterm delivery among 3 study groups were not different. In conclusion, post-conception IVIg treatment significantly improves reproductive outcome in women with elevated CD56+ NK cells with pregnancy who received preconception lymphocyte immunization, anticoagulation and prednisone treatment.

Clarithromycin-induced alterations in vaginal flora.

Many antibiotics have been shown to alter both the bacterial and the fungal flora of the vagina, in some cases potentially increasing a woman's propensity toward urinary tract infections and vaginal candidiasis. The effects of some of the newer macrolide antibiotics on women's vaginal flora have not been previously studied, and almost none of the previous studies specifically cultured for effects on vaginal lactobacillus. Young women (ages 18-45 years) who were about to go onto therapy with clarithromycin, who did not have any conditions known to affect the vaginal flora (eg, diabetes mellitus, spermicide use, menopausal status without hormone replacement therapy), and who agreed to participate in the study were cultured with aerobic and anaerobic and fungal vaginal cultures before starting the antibiotic. These same women were then retested about 4 to 6 weeks after the start of their antibiotic course, and the results of their preantibiotic and postantibiotic cultures were compared. Lactobacillus was present in 33% of patients by vaginal culture before treatment, but this decreased to 0% after treatment. Escherichia coli was present in only 8% of patients before treatment, but this increased to 17% of patients after treatment. Enterococcus was present in 25% of patients before treatment but in only 8% of patients after treatment. The incidence of Gardnerella vaginalis was not affected by the treatment. Candida species incidence increased from 17% to 33% with treatment. The overall effects of clarithromycin on the vaginal flora are similar to other older antibiotics that have been tested.

Regeneration and tolerance factor: a correlate of human immunodeficiency virus-associated T-cell activation.

Human immunodeficiency virus (HIV) infection causes extensive phenotypic alterations in lymphocytes. Cellular markers that are normally absent or expressed at low levels on quiescent cells are upregulated throughout the disease course. The transmembrane form of regeneration and tolerance factor (RTF) is expressed at negligible levels on resting T cells but is quickly upregulated following in vitro stimulation and activation. Recently, we reported that expression of RTF was significantly higher in cells from HIV-seropositive (HIV(+)) individuals than in cells from HIV-seronegative (HIV(-)) individuals. Because T cells from HIV(+) individuals express markers reflecting chronic activation, we hypothesized that these in vivo-activated cells would coexpress RTF. Flow cytometry was used to assess RTF expression on activated (CD38(+) and HLA-DR(+)) CD4(+) and CD8(+) T cells. HIV(+) individuals had higher percentages of RTF(+) CD38(+) (P < 0.0001) or RTF(+) HLA-DR(+) (P = 0.0001) CD4(+) T cells than HIV(-) individuals. In HIV(+) individuals, increased percentages of CD4(+) T cells that were RTF(+), RTF(+) CD38(+), and RTF(+) HLA-DR(+) correlated inversely with the absolute number and percentage of CD4(+) T cells and correlated positively with plasma beta(2)-microglobulin concentrations. HIV(+) individuals had higher percentages of CD8(+) T cells that were RTF(+) CD38(+) (P = 0.0001) or RTF(+) HLA-DR(+) (P = 0.0010). In HIV(+) individuals, increased percentages of CD8(+) T cells that were RTF(+) HLA-DR(+) correlated inversely with the percentage of CD4(+) T cells, and high percentages of CD8(+) T cells that were RTF(+) CD38(+) correlated positively with plasma beta(2)-microglobulin levels. These findings strongly suggest that increased RTF expression is a correlate of HIV-associated immune system activation.

Antiphospholipid antibodies, proteins C and S, and coagulation changes in sickle cell disease.

The significance, interactions, and sources of coagulation abnormalities and their relationship to clinical severity and painful episodes in sickle cell disease are not clear. To evaluate this, we have examined various measures of coagulation in 37 patients with sickle cell disease (20 patients with HbSS disease and 17 patients with HbSC disease). Measurements have included isotypes of antiphospholipid antibodies (IgG, IgM, IgA) to specific phospholipids; proteins C (activity, total antigen) and S (activity, total and free antigen); measures of coagulation activation (prothrombin fragment 1.2, thrombin-antithrombin, fibrinopeptide A, d-dimers); indicators of clinical severity; and studies obtained during steady states and painful episodes. Results in HbSS disease showed that antiphospholipid antibodies were increased, with IgG phosphatidylserine showing the highest and most frequently increased levels (37% of patients). Protein C (activity) and protein S (activity, total, free antigen) were decreased (P<.01), and all measures of coagulation activation were increased (P<.001). In HbSC disease, antiphospholipid antibodies were normal, protein C (activity) and protein S (free antigen) were decreased (P<.001), and all measures of coagulation activation were increased (P<.02). A strong correlation was observed in HbSS disease between IgG-PS and d-dimers. Moderate correlations occurred between protein C activity and thrombin-antithrombin and fibrinopeptide A, between protein S activity and prothrombin fragment 1.2 and d-dimers, and between protein C and protein S activity. In HbSC disease, moderate and fewer correlations occurred. Significant differences between HbSS disease and HbSC disease were observed in aPLs, proteins C and S, and measures of coagulation activation. Measurements during steady states and during painful episodes were not significantly different. We conclude that the antiphospholipid antibody IgG-PS may contribute to coagulation activation in HbSS disease and that IgG-PS, protein C, and protein S relate to each other and jointly to measures of coagulation activation. The increased level of IgG-PS in HbSS disease most likely reflects exposure of the procoagulant phosphatidylserine on the surfaces of red cell-shed vesicles and sickle red cells, which would further affect coagulation activation. The significant differences in coagulation measures between HbSS disease and HbSC disease are consistent with differences in clinical severity between the diseases. The development of painful episodes does not appear to be related to the coagulation changes.

Expression of regeneration and tolerance factor on B cell chronic lymphocytic leukemias: a possible mechanism for escaping immune surveillance.

Regeneration and tolerance factor (RTF) is a protein expressed on developing tissue such as the thymus and the placenta. RTF has been reported to down-regulate cell-mediated immune responses. To examine the potential role of tumor-derived RTF to suppressing antitumor responses, we analyzed a panel of seven B cell tumor lines for the membrane RTF using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody, which reacts with membrane RTF. All the B cell tumor lines we examined express RTF on the cell surface. We also tested conditioned media from these B cell lines for their ability to suppress IL-2R expression on activated cells. Conditioned media from each B cell line suppressed IL-2R expression on activated Jurkat T cells and activated peripheral blood mononuclear cells. A monoclonal antibody to the biologically active portion of RTF reversed this suppressive activity. Finally, the tumor cell population from patients with chronic lymphocytic leukemia was found to express cell surface RTF. Thus, RTF expression could be a new mechanism used by tumor cells to escape immune surveillance.

Natural killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions.

PROBLEM: Natural Killer (NK) cell measurement and NK cytotoxicity are two measurements for assessing the cellular immune response. Both of the techniques have been reported to be prognostic for women with recurrent spontaneous abortion (RSA). We evaluated the two methods to determine the relationship of the two assays. Because both methods portend to evaluate the same process, the previous clinical data suggested that the methods evaluate the same phenomena. We undertook these studies to determine whether simple NK cell counts may be sufficient in the evaluation of NK activity in RSA. METHOD OF STUDY: The NK cell cytotoxicity at effector-to-target ratios of 50:1 and 25:1 was determined using a flow cytometric NK cell cytotoxicity assay. These values were then correlated with the percentages and absolute counts of three peripheral blood NK cell subsets. RESULTS: The data indicate that the flow cytometric assay is reproducible and precise and can be successfully used to evaluate patient samples. Linear regression analysis indicated a lack of correlation between peripheral blood NK cell cytotoxicity and percentages or absolute counts of CD56+CD16+, CD56+CD16- or CD3+CD56+ lymphocyte subsets (range of correlation coefficients, 0.1-0.3). CONCLUSIONS: NK cell cytotoxicity and peripheral blood NK cell values measure different aspects of NK cells and do not correlate. These data indicate that simple enumeration of NK cells may not be sufficient in the evaluation of NK cells in RSA.

Increased expression of regeneration and tolerance factor in individuals with human immunodeficiency virus infection.

Regeneration and tolerance factor (RTF) plays a pivotal role in successful pregnancy outcome and has potent immunomodulating properties. During pregnancy, it is abundantly expressed in the placenta and on peripheral B lymphocytes. Several lines of evidence suggest that both successful pregnancy outcome and progression from human immunodeficiency virus (HIV) infection to AIDS are associated with a Th2-type response. As a result, we hypothesized that the cellular expression of RTF may also be increased during infection with HIV. Using flow cytometric analysis, we showed a significantly (P < 0.01) increased expression of RTF on CD3(+) cells obtained from individuals with HIV over that for individuals without HIV. On average, 32.1% of the CD3(+) cells from individuals with HIV expressed high levels of RTF. In contrast, an average of only 6.7% of the CD3(+) cells from individuals without HIV expressed high levels of RTF. Similar results were obtained when CD19(+) cells from individuals with (mean, 44.1%) and without (mean, 25.8%) HIV were evaluated. Linear regression analysis suggested that high levels of RTF expression by CD3(+) cells correlated better with viral load (r value, 0.46) than with absolute CD4 count (r value, 0.09). While additional experiments are necessary to delineate the precise immunologic role of RTF, our current data suggest that RTF expression during HIV infection may be a useful marker of immune activation.