An imbalance in interleukin-17-producing T and Foxp3⁺ regulatory T cells in women with idiopathic recurrent pregnancy loss.

BACKGROUND: T cells which produce interleukin (IL)-17 are involved in chronic inflammatory processes and regulatory T (Treg) cells are possibly the most important immune regulators. We aimed to investigate peripheral blood IL-17(+) T and Foxp3(+) Treg cells in women with idiopathic recurrent pregnancy loss (RPL). METHODS: The study design is a cross-sectional evaluation of Th1, Th2, IL-17(+) T and Treg cells in women with idiopathic RPL (n = 42) and age-matched parous controls (n = 24). Flow cytometric analysis was performed to measure IL-17(+) T and Foxp3(+) Treg cells, and ratios of Th1/Th2 cells using anti-IL-17A and anti-Foxp3 antibodies, and monoclonal antibodies to tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Student's t-test and partial correlations were applied for statistical analysis. RESULTS: TNF-α-/IL-10-producing CD3(+)CD4(+) T cell ratio was higher in women with RPL than controls (P = 0.048). Levels of IL-17(+) T cells (P = 0.021) and the IL-17(+) T/CD4(+)Foxp3(+) Treg cell ratio (P = 0.001) were increased, whereas Foxp3(+) (P = 0.035), Foxp3(low) (P = 0.032) and CD4(+)Foxp3(+) T cell (P = 0.037) levels were decreased in women with RPL, compared with controls. Levels of IL-17(+) T cells were correlated with TNF-α-producing CD3(+)CD4(+) T cells (r = 0.269, P = 0.033), and with ratios of TNF-α/IL-10 (r = 0.276, P = 0.027) and IFN-γ/IL-10 (r = 0.266, P = 0.035)-producing CD3(+)CD4(+) cells. Furthermore, the ratio of IL-17(+) T cells to CD4(+)Foxp3(+) Treg cells showed a positive correlation with TNF-α-producing CD3(+)CD4(+) T cells (P = 0.047) and IFN-γ-producing CD3(+)CD4(+) T cells (P = 0.048) as well as a ratio of IFN-γ/IL-10-producing CD3(+)CD4(+) T cells (P = 0.037). CONCLUSIONS: Enhanced pro-inflammatory immune responses with suppressed immune regulation may be an important immune mechanism involved in RPL.

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.

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.

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.

Allotypic and isotypic specificities of rabbit IgM: localization to the Fab mu or Fc mu fragments.

IgM from trypanosome-infected rabbits was digested with trypsin under different conditions to obtain Fab mu or Fc5 mu fragments suitable for analysis with anti-allotype and anti-isotype antibodies. The Fab mu but not the Fc5 mu fragment was shown to have the n-locus allotypic specificities, n80, n81, n82, n83 and n87, characteristic of the IgM class of immunoglobulins. Thus, the n82 and n83 allotypic specificities, conformationally dependent on the a VH locus for expression, and the n80, n81 and n87 allotypic specificities, independent of the a VH locus for expression, are in either the CH1 or CH2 domain of IgM heavy chains. In addition, two high-affinity mouse monoclonal antibodies (MoAbs) specific for IgM and able to bind IgM in direct-binding radioimmunoassays were produced and characterized. One MoAb (3C1) was specific for an isotypic determinant (epitope) in the Fab mu fragment, presumably in the CH1 or CH2 domain, whereas another MoAb (8C2) was specific for an isotypic epitope in the Fc5 mu fragment, presumably in the CH3 or CH4 domain. The proximity of the n-locus allotypic specificities (CH1 or CH2 domain) to the VH domain is consistent with the finding that some IgM allotypic specificities are expressed only in conjunction with certain a VH locus allotypic specificities.

The common idiotypic specificity of anti-a2 allotype antibodies: contribution of H and L chains to idiotype expression.

Previously an idiotypic specificity common to anti-a2 allotype Ab from 15 rabbits was detected and characterized. The contribution of H and L chains to the expression of this common idiotypic specificity was determined. Two types of recombinant IgG were prepared: (1) H chains from anti-a2 Ab with L chains from normal a3b4 IgG, and (2) L chains from anti-a2 Ab with H chains from normal a3b4 IgG. By inhibition of binding radioimmunoassay, recombinant IgG having either H or L chains from anti-a2 did not inhibit the binding between anti-a2 Ab and anti-idiotype Ab. By direct binding radioimmunoassay, recombinant IgG of H chains from anti-a2 Ab with L chains from a3b4 IgG reacted with anti-idiotype Ab whereas recombinant IgG with H chains from normal a3b4 IgG and L anti-a2 Ab is determined primarily by the H chain; however, the full expression of the common idiotypic specificity requires the interaction of both H and L chains from anti-a2 Ab.

An increase in the number of recombinant molecules and other effects of the simultaneous allotype suppression of trans-chromosomal a VH and n Cmu Ig gene products.

The concomitant effects of trans-chromosomal allotype suppression of both an a VH and an n Cmu locus allotype in multiheterozygous rabbits were investigated. For example of the expression of the a2 VH and n81 Cmu allotypes were suppressed in a multiheterozygous rabbit having the a1 chi-y-n81de12,15f73g74/a2 chi 32y33n80de12.14f69g77 genotype. This trans-chromosomal allotype suppression led to the concomitant suppression of other CH allotypes in the same parental haplotype as the suppressed-n81 allotype (i.e. the e15, f73 and g74 allotype) and the partial suppression of the a1 VH allotype (from the normal level of 70% of the total Ig to 10%), and also led to compensation by other VH allotypes from the same parental haplotype as the suppressed-a2 allotype (i.e. the x32 and y33 allotypes). The x32 and y33 allotypes were expressed on Ig molecules with the CH allotypes coded by the same haplotype (i.e. the cis molecules). In a further analysis of the IgG molecules having the partially-suppressed-a1 allotype, one-half (5%) of these molecules were trans-chromosomal recombinant molecules (i.e. a1e14 IgG) and the other half (5%) were cis-chromosomal molecules (i.e. a1e15 IgG). The trans-chromosomal a1e14 IgG molecules probably were derived from the expansion of a limited number of lymphoid clones that normally produce only 1.5% trans-chromosomal recombinant molecules. The cis-chromosomal a1e15 IgG molecules were probably derived either from lymphoid clones that survived the suppression by the anti-n81 Ab, or from lymphoid clones that bore a different subclass of IgM (i.e. n-negative IgM).

Comparison of the immune response to Ars-BGG in germfree or conventional piglets.

Neonatal germfree (GF) colostrum-deprived and conventional (CV) colostrum-fed piglets were immunized IP with p-azo-phenyl-arsonate-bovine gamma globulin (Ars-BGG) in Freund's adjuvant to study the development of the immune response in the absence or presence of maternal antibodies and environmental antigens. Overall, the immune response varied greatly within each group but did not differ in GF from CV piglets statistically. Affinity immunoblot analysis suggested that anti-Ars antibody was more restricted in GF than CV piglets and clonotype shifts occurred more in GF than CV piglets after each antigenic stimulation. In contrast, the clonotype pattern of the anti-BGG antibody was similarly heterogeneous in the two groups. Based on the affinity immunoblot data the antibodies generated to the Ars-haptenic group in CV piglets are more heterogeneous than GF piglets and suggest that clonotype generation is influenced by maternal antibodies and environmental antigens.

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.

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.

Presence of multiple anti-phospholipid antibody specificities in a pediatric population.

Thrombotic related events are thought to be associated with the presence of anti-phospholipid antibodies (APA). However, the association of anti-cardiolipin antibody is much weaker than the association with antibodies to other phospholipids. Much of the literature equates antiphospholipid antibodies and anticardiolipin antibodies because of the relationship of APA and false positive tests for syphilis. However, recently the presence of antibodies to naturally occurring phospholipids other than cardiolipin have been reported. In fact, some investigators report that antibodies to phosphatidylserine appear to correlate more closely to disease processes than anti-cardiolipin antibodies. We describe here the presence of non-anti-cardiolipin antiphospholipid antibodies in a pediatric population that lack anti-cardiolipin antibodies and demonstrate the association of these antibodies with thrombotic disease. Antibodies to phosphatidic acid were the most prevalent and correlated (p < .001) with thrombotic disease and idiopathic thrombocytopenia purpura. The rank order of prevalence of antibodies to phospholipids was phosphatidic acid, phosphatidylglycerol, phosphatidylinosital, phosphatidylserine, cardiolipin and phosphatidylethanolamine. Antiphospholipid antibodies of the three major sera isotypes were present in the positive sera examined. These descriptive findings suggest that the significance of APA other than anti-cardiolipin antibodies in pediatric patients should be further investigated.

Inhibition of binding of anti-CD3 antibodies to paternal lymphocytes correlates with failure of immunotherapy for treatment of recurrent spontaneous abortions.

A two color flow cytometry crossmatch (FCXM) was used to evaluate the induction of anti-lymphocyte antibodies in 34 women undergoing immunotherapy for recurrent spontaneous abortions. All women had anti-lymphocyte antibodies that reacted with T-cells when analyzed by FCXM. However, inhibition of the binding of anti-CD3 to paternal CD3 lymphocytes in the presence of maternal antipaternal lymphocyte antiserum was found for some couples following lymphocyte immunotherapy for spontaneous recurrent abortions. Ten couples who had another spontaneous abortion following immunotherapy showed inhibition. In contrast, eight couples who did not show inhibition of the binding of anti-CD3 T lymphocytes to paternal lymphocytes by maternal anti-lymphocyte antiserum had live births. Women of the remaining 16 couples were either pregnant and awaiting birth or not pregnant. Thus, by FCXM it may be possible to predict those couples who will have successful pregnancies following this treatment.

Autoantibodies in women with primary recurrent spontaneous abortion of unknown etiology.

A group of 153 women with 3 or more recurrent spontaneous abortions (RSAs) with unknown etiology and 90 normal multigravida controls were evaluated for antibodies to phospholipids and nuclear antigens. We demonstrate that women with recurrent spontaneous abortions showed significantly higher incidence of antibodies to phospholipids than normal multigravida controls. In contrast, the incidence of antibodies to polynucleotides and histones was not different between these two groups. These findings suggest that antiphospholipid antibodies are either epiphenomena or causally related to recurrent spontaneous abortions.

Intravenous immunoglobulin infusion therapy in women with recurrent spontaneous abortions of immune etiologies.

We have investigated clinical effectiveness of intravenous immunoglobulin G infusion (IVIg) on antiphospholipid antibody titers in five women with evidence of antiphospholipid antibody-associated recurrent spontaneous abortions and one with antinuclear antibody who became refractory to conventional autoimmune treatment during pregnancy and experienced pregnancy complications. Three women developed intrauterine growth retardation and three had complicated twin pregnancies with rising autoantibody titers. Antiphospholipid antibody and antinuclear antibody titers were tested pre and 2 weeks after each IVIg infusion. We report that: (i) IgG antiphospholipid antibody titers were significantly suppressed after each IVIg infusion (P < 0.05); (ii) IgM antiphospholipid antibody titers were also significantly suppressed after each IVIg infusion (P < 0.0001); (iii) decreased titers of autoantibodies paralleled increased levels of maternal IgG which lasted for at least 30 days; the autoantibodies showed a definite rise again prior to the next infusion; (iv) antinuclear antibody titers were effectively suppressed; and (v) rising autoantibody titers combined clinical manifestation of intrauterine growth retardation and women with complicated twin pregnancies. We conclude that IVIg infusion effectively suppresses IgM and IgG autoantibodies to phospholipids and antinuclear antibody in autoimmune women with a history of recurrent spontaneous abortions and refractory to conventional anticoagulation or immunosuppressive treatment.

Characterization of antibodies induced by paternal lymphocyte immunization in couples with recurrent spontaneous abortion.

This study was designed to identify and characterize the allo- and autoantibodies induced following successful paternal lymphocyte immunization to prevent recurrent spontaneous abortion. Firstly the titers of maternal anti-paternal antibodies in women with successful pregnancies as determined by the flow cytometry crossmatch (FCXM) were highly variable; however, in all cases, the initial pre-immunization titers were negative and the post-immunization titers were positive by the FCXM in successfully treated women. Secondly, the specificities of maternal alloantibodies to paternal HLA antigens (immunogen) were evaluated. No all predicted antibodies to mismatched paternal HLA antigens were found by microlymphocytotoxicity (MCX) assays and the specificities varied. Thirdly, antibodies in post- but not preimmunization sera reacted with two lymphoid cell lines, SupT1 and SB; in addition, the rise and fall of the titers of these sera with paternal cells seemed to be reflected with the cell lines by the FCXM. Fourthly, autoantibodies to activated lymphocytes were detected and seemed to correlate with successful immunization since women who had another abortion following immunotherapy lacked these autoantibodies. These findings suggest that the antibody response following successful immunotherapy is complex and needs to be studied further to understand the mechanism of this treatment.

Antibodies to phospholipids and nuclear antigens in non-pregnant women with unexplained spontaneous recurrent abortions.

In a collaborative study of 73 non-pregnant Kuwaiti women with unexplained spontaneous recurrent abortion (RSA), 30 control healthy non-pregnant multiparous Kuwaiti women and 20 North American women who received elective abortion(s), autoantibodies to 6 phospholipids and 9 nuclear antigens were measured. Women with recurrent spontaneous abortions demonstrated 3 times higher incidence of antibodies to phospholipids (30.1%) than controls (10% each) (P = 0.029). The incidence of both IgM and IgA class antiphospholipid antibodies were significantly higher than those of controls. The incidence of antibodies to cardiolipin in women with recurrent spontaneous abortions (12.3%) was significantly higher than those of controls (P = 0.035) and incidence of IgM but not IgG anticardiolipin antibody was significantly higher in women with RSAs than in controls (P = 0.053). The incidences of anti-polyinosinic acid (P = 0.035) and anti-histone 1 antibody (P = 0.052) were significantly higher in women with recurrent spontaneous abortions than controls. There was no significant difference in the incidence of autoantibodies between primary and secondary aborters. However, women with a history of second trimester abortions showed a higher incidence of antiphospholipid antibodies than women with first trimester abortions only. Recurrent spontaneous abortion is associated with autoantibodies to phospholipid epitopes including IgA antiphospholipid antibodies.

Effect of intravenous immunoglobulin G on natural killer cell cytotoxicity in vitro in women with recurrent spontaneous abortion.

Intravenous immunoglobulin (IVIg) has been used to treat women with recurrent spontaneous abortion (RSA), particularly for women with elevated natural killer (NK) cells. We investigated the effect of IVIg on peripheral blood NK cell activity in vitro in women with RSA. 51Cr-release assays using K562 in the presence of varying concentrations of IVIg were performed using PBL from 16 women with RSA. Antibody dependent cellular cytotoxicity (ADCC) was evaluated using Daudi cells. Effectors and targets were preincubated with IVIg. Binding of IVIg to K562 and Daudi was evaluated by flow cytometry. The effect of K562 absorbed IVIg on NK activity was compared to that of non-absorbed IVIg. NK cytotoxicity and ADCC in the presence of F(ab')2 fragments were compared with those in the presence of intact IVIg. IVIg produced a significant, dose dependent inhibition of NK activity in vitro. Inhibition of NK activity occurred when effectors but not targets were preincubated with IVIg. IVIg binds to K562 and Daudi. IVIg increased ADCC when targets but not effectors were incubated with IVIg. K562 absorbed IVIg produced more inhibition of NK cytotoxicity than non-absorbed IVIg. Suppression of NK cytotoxicity by F(ab')2 was as effective as that of IVIg. However, F(ab')2 did not increase ADCC. IVIg effectively reduces peripheral blood NK cytotoxicity in vitro. Inhibition of NK cytotoxicity is mediated at the effector cell level through the antigen binding portion of the immunoglobulins. Women with RSA and elevated NK cells may benefit from IVIg treatment.

Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey.

Infantile Mediterranean visceral leishmaniasis (IVL) and anthroponotic cutaneous leishmaniasis (ACL) have long been known to exist in the western and southeastern Turkey, respectively. To further study these and other related diseases, a recombinant antigen (rK39) specific to VL was used in an ELISA for serodiagnosis of selected patients and for screening dog reservoir populations in several endemic sites. Among 24 confirmed VL cases from western Turkey, the rK39 ELISA proved to be more sensitive than a combination of cultivation and microscopy of bone marrow aspirates. The specificity of rK39 for leishmaniasis was demonstrated by its lack of cross-reactivity with sera from other human diseases in the same sites. Interestingly, six of the 83 parasitologically proven ACL cases from southeast Turkey were also rK39 positive. The end point titers of the positive VL and CL cases vary from 10(-2) to 10(-5) and from 10(-2) to 10(-3), respectively. The rK39 ELISA was also used to screen 494 apparently healthy dogs from Urfa in southeast Turkey, Manisa/Alasehir near the Aegean Sea, and Karabuk near the Black Sea. Eighteen rK39-positive cases (3.6%), all from the latter two areas, were found to have varying endpoint titers (10(-2)-10(-4)). The high titers predicted increased severity and frequency of the clinical symptoms (i.e., lymphadenopathy, depilation, skin lesion, weight loss and/or death), which were manifested subsequently in 16 of these 18 cases. In addition, more positive canine cases were diagnosed by the rK39 ELISA preclinically than the procedures to detect parasites postsymptomatically in the lymph node aspirates. The use of the rK39 ELISA as a sensitive tool makes it possible to demonstrate coendemicity of canine and human VL, as expected in the case of IVL. The results also point to the possible presence of additional VL types in western Turkey and cutanovisceral type in the southeast part of this country.

Serodiagnosis of Asian leishmaniasis with a recombinant antigen from the repetitive domain of a Leishmania kinesin.

rK39 is a recombinant product of the 39 amino acid repeats found in a kinesin-like gene of visceral Leishmania spp. This and other antigens were compared for immunodiagnostic potential by enzyme-linked immunosorbent assay with sera from confirmed cases of Asian cutaneous and visceral leishmaniasis. In preliminary trials, rK39 proved superior to 2 purified Leishmania antigens, a cytosolic protein (p36) and a membrane protein (gp63), for immunodiagnosis of visceral leishmaniasis. Of the 53 visceral cases from China and Pakistan assayed, 52 were seropositive (98%) at a 10(-1) dilution with 36 ng of rK39. End point titrations of 27 highly positive samples yielded anti-rK39 antibody titres ranging from c. 10(-3) to beyond 10(-4). Antigen titrations with one positive serum further revealed that rK39 was 25-fold more sensitive than Leishmania whole cell soluble lysates. 31 cutaneous leishmaniasis cases from Turkey assayed for anti-rK39 antibody gave reactions ranging from negative or marginally positive to positive. In Brazil, all cutaneous and mucocutaneous leishmaniasis cases gave negative results in this assay.

Intestinal absorption of immunologically intact macromolecules in germfree colostrum-deprived piglets maintained on total parenteral nutrition.

We have compared the neonatal absorption of anti-bovine gamma-globulin (BGG) antibody supplied in colostrum or saline in three groups of piglets born and maintained under different environmental conditions to determine the effect of these conditions on the cessation of intestinal absorption of macromolecules (anti-BGG antibody), termed "closure." An enzyme-linked immunosorbent assay was used to estimate the concentration of anti-BGG antibody in sera from each group of piglets. Three stages of macromolecular absorption through the piglet's intestine could be detected. The first stage is a nonselective massive absorption of macromolecules (in milligram levels) that lasts up to 3 days in germfree (GF) colostrum-deprived or conventional colostrum-fed piglets but up to 5 days in GF piglets maintained on total parenteral nutrition. In this stage, absorption was significantly (r = .05) higher in piglets fed anti-BGG serum with colostrum than in piglets fed anti-BGG serum without colostrum on GF day 0 (31.28% vs 15.59%) and GF-total parenteral nutrition day 3 (3.08% vs 0.11%). Thus, whenever there was the ability to absorb a massive amount of macromolecules, the sow colostrum had an enhancing affect. Although there was a minor effect of environmental or orally received stimuli in delaying closure, absorption of macromolecules decreased in all piglets maintained either parenterally or enterally after day 3. Thus, intestinal closure to massive absorption of macromolecules in piglets is primarily time (age)-dependent. The second stage is a selective absorption of immunoglobulins in much smaller quantities (microgram levels), inasmuch as absorption of 0.02% to 0.1% was determined in all 5-day-old piglets.(ABSTRACT TRUNCATED AT 250 WORDS)