Pro-inflammatory and anti-inflammatory cytokine profiles in fetal growth restriction.

The purpose of this investigation was to measure cytokine production by maternal peripheral blood lymphocytes from women with intrauterine growth restriction (IUGR) and from healthy pregnant women, and to investigate the relationship between cytokine profiles and IUGR. Thirty-six women with IUGR and 22 control healthy pregnant women with normal fetal growth were studied. Levels of pro-inflammatory cytokines (IFNy, TNFa, IL-8, IL-12, IL-18, IL-23) and anti-inflammatory cytokines (IL-4, IL- 10, IL-13) produced by mitogen-stimulated peripheral blood mononuclear cells were measured by ELISA. Levels of the anti-inflammatory cytokine IL-4 were higher in normal pregnancy compared to IUGR, indicating an anti-inflammatory bias. Levels of the pro-inflammatory cytokines IL-6, TNFα, and IL-12 were significantly higher and levels of the anti-inflammatory cytokine IL- 10 lower in IUGR with placental insufficiency than in IUGR without placental insufficiency, suggesting a stronger pro-inflammatory bias in IUGR with placental insufficiency. Ratios of pro- to anti-inflammatory cytokines suggest a dominance of pro-inflammatory cytokines. The authors conclude that an increased pro-inflammatory cytokine bias is observed in IUGR compared to normal pregnancy, and an increased pro-inflammatory cytokine dominance is seen in IUGR with placental insufficiency compared to IUGR without placental insufficiency.

IL-10 and pregnancy complications.

PURPOSE OF INVESTIGATION: Successful pregnancy depends on the ability of the mother's immune system to undergo a process of immunoregulation in order to tolerate the fetus, and also to create and sustain a nurturing environment during all the stages of pregnancy. Several reports point to interleukin 10 (IL-10) as being vital for normal pregnancy, and low IL-10 levels as being associated with preg- nancy complications. This study aimed to compare IL-10 levels in normal and complicated pregnancy conditions. MATERIAL AND METHODS: The authors compared levels of IL-10 produced upon stimulation of maternal peripheral blood mononuclear cells (PBMC) from women at different stages of normal gestation with those produced by women with pregnancy complications, such as recurrent spontaneous miscarriage (RSM), preterm delivery (PTD), premature rupture of fetal membranes (PROM), pre-eclampsia, and intrauterine fetal growth retardation (IUGR). RESULTS: Median levels of IL-10 are statistically significantly lower in pathological conditions as com- pared to matching gestational ages of normal pregnancy. CONCLUSION: Healthy pregnancy is associated with higher levels of IL-10, while pathologic pregnancies are associated with lower levels of IL-10.

Cytokines as diagnostic markers of pulpal inflammation.

AIM: To measure and compare the levels of the cytokines IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ in pulpal blood from irreversible pulpitis, asymptomatic caries exposure and normal pulps. METHODOLOGY: Blood was obtained from pulp exposure sites using cotton pellets. Twenty-five samples were obtained from normal teeth, 40 from asymptomatic caries-exposed pulps and 43 from irreversible pulpitis teeth. Cytokine levels were determined by high-sensitivity ELISA. Data were statistically analysed using Kruskal-Wallis and Mann-Whitney U-tests. RESULTS: Significantly higher levels (P < 0.05) of IL-6, IL-8, IL-10, TNF-α and IFN-γ were detected in caries-exposed pulps and irreversible pulpitis as compared to normal teeth. IL-2 and IL-10 levels were significantly higher (P < 0.05) in caries-exposed pulps as compared to irreversible pulpitis, whilst IL-8 was significantly higher (P < 0.001) in irreversible pulpitis as compared to caries-exposed teeth. Most interestingly, IL-6/IL-10 and IL-8/IL-10 ratios were significantly higher (P < 0.001) in irreversible pulpitis compared with both caries-exposed and normal teeth. CONCLUSION: Levels of IL-8 and the ratios of IL-6/IL-10 and IL-8/IL-10 have the potential to be indicators of pulpal inflammation in caries exposure cases. Cytokine estimation in pulpal blood may help in the diagnosis of pulpal inflammation.

Is combined pretransplantation seropositivity of kidney transplant recipients for cytomegalovirus antigens (pp150 and pp28) a predictor for protection against infection?

OBJECTIVE: This study was aimed at detecting antibodies to the antigens which may contribute to protection against cytomegalovirus (CMV) infection after organ transplantation. MATERIALS AND METHODS: A total of 203 kidney transplant patients were enrolled in the study. Based on CMV antigenemia assay, 23 patients were antigen-positive and of the remaining 180 antigen-negative patients, 46 were selected as controls matched for age, gender and source of kidney. The 69 kidney recipients (KR) had CMV antibody due to previous infection and were followed up for a period of 6 months after transplantation for the development of active CMV infections by the antigenemia assay. Antibody responses to five CMV-related peptide antigens (pp65, gB, pp150, pp28 and pp38) were investigated by enzyme immunoassay and their presence was correlated with the results of the CMV antigenemia assay. RESULTS: Of the five CMV-related peptide antigens, only gB antigen showed response to the antibody in 10/23 (43.5%) antigen-positive patients and 9/46 antigen-negative patients and the difference was statistically significant (p = 0.048). On the other hand, there was no significant difference in antibody responses between the antigen-positive and antigen-negative KR to the other four CMV peptide antigens (p > 0.05). However, among the antigen-positive KR there was only 1 patient who had antibodies to both pp150 and pp28 antigen, while among the antigen-negative KR, 22 of 46 (47.8%) had the antibodies (p < 0.001). CONCLUSION: The findings suggest that the combined presence of antibodies against the pp150 and pp28 antigens may indicate a lower risk of CMV reactivation after kidney transplantation.

C24 Sphingolipids Govern the Transbilayer Asymmetry of Cholesterol and Lateral Organization of Model and Live-Cell Plasma Membranes.

Mammalian sphingolipids, primarily with C24 or C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we present evidence that C24 sphingomyelin, when placed in the outer leaflet, suppresses microdomains in giant unilamellar vesicles and also suppresses submicron domains in the plasma membrane of HeLa cells. Free energy calculations suggested that cholesterol has a preference for the inner leaflet if C24 sphingomyelin is in the outer leaflet. We indeed observe that cholesterol enriches in the inner leaflet (80%) if C24 sphingomyelin is in the outer leaflet. Similarly, cholesterol primarily resides in the cytoplasmic leaflet (80%) in the plasma membrane of human erythrocytes where C24 sphingolipids are naturally abundant in the outer leaflet. We conclude that C24 sphingomyelin uniquely interacts with cholesterol and regulates the lateral organization in asymmetric membranes, potentially by generating cholesterol asymmetry.

Vaccines against fertility.

The first evidence for the efficacy of a birth control vaccine in humans is now available from the Phase II trials on the human chorionic gonadotrophin vaccine in India. Several sperm antigens have been identified as potential contraceptive immunogens and zona pellucida antigens have been reported that reversibly control fertility.

PIP: Birth control vaccines, once proven to be safe, effective, and reversible, may be superior to available methods in terms of the reduced number of doses required, the lack of administration of pharmacological agents, and the absence of risk due to improper use. The reproductive process can be interrupted by immune effectors at several points, making several potential birth control vaccines possible. For example, pregnancy can be blocked by immune effectors which can either inactivate an hormone which is indispensable to reproduction or counteract a gamete antigen crucial for gamete development and/or fertilization. First evidence of a birth control vaccine in humans has emerged from Phase II trials on the human chorionic gonadotrophin vaccine in India. Several sperm antigens have been identified as potential contraceptive immunogens and zona pellucida antigens have been reported which reversibly control fertility. Several problems still have to be resolved before fertility control vaccines become available for routine use, but research is nonetheless yielding encouraging results. Sections discuss human chorionic gonadotrophin vaccines, gonadotropin releasing hormone vaccine, carrier-induced suppression, sperm antigen vaccines, egg antigens, other candidate immunogens, and future perspectives.

A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens.

This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens. This CELISA has the advantages of convenience and rapidity and is therefore ideally suited for the screening of a large number of hybridoma culture supernatants. The basic procedure involves the direct drying of cell suspensions onto the wells of enzyme immunoassay (EIA) plates and a subsequent EIA with appropriate blocking reagents. In order to overcome high background binding of primary antibodies to Fc receptors and of secondary antibodies to surface Ig (sIg), this method involves a blocking step consisting of unlabelled secondary antibodies. Once CELISA plates are prepared, they can be stored for a period of at least 6 months and hence this assay does not rely on the availability of fresh, viable cells for each assay. This assay is simple, reproducible and sensitive. The results can be assessed in an objective manner and can also be adapted for the detection of cellular antigens. This paper describes a CELISA for the detection of antibodies to blood group antigens and human leukocyte (HLA) antigens.

Fate of antipaternal H-2 antibodies bound to the placenta in vivo.

We have previously demonstrated that the placental spongiotrophoblast and yolk sac venous plexus express paternally derived H-2K and D (class I) antigens in a manner accessible to maternal circulation, and that the placenta serves to prevent antipaternal antibodies from reaching the fetus. Kinetic studies indicated that the placenta is capable of reexpressing its H-2 antigens after having been bound by anti-H-2 antibodies, suggesting that the placental cells somehow have the ability to eliminate the bound antibodies. In order to investigate the fate of the antibodies, we have followed the uptake and fate of radiolabeled anti-H-2Kk monoclonal antibody in the placentas of target allogeneic and control syngeneic pregnancies. Chromatographic analyses indicate that most of the intercellular radiolabel is associated with fragments smaller than IgG, and similar degradation was not observed with syngeneic control placentas. We conclude that the placenta is capable of binding, ingesting, and then digesting antipaternal H-2 antibodies, further substantiating the immunoabsorbent barrier hypothesis of allogeneic fetal survival.

Characterization and cross-reactivity of human and mouse oncofetal antigens. Use of a new solid phase assay for detection of cell surface antigens.

Oncofetal (OF) antigens have been isolated from mouse F9 teratocarcinoma cells, mouse testicular cells, and human molar tissue by detergent extraction followed by dialysis. The soluble antigens have been used in solid phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent (ELISA) assay. Specific antibodies have been raised to these antigens in mice. By using these antisera, extensive cross-reactivity was found between mouse and human OF antigens. A human trophoblastic tumor cell line BEWO absorbed out mouse anti-F9 reactivity. Patients with tumors of germinal origin were found to have antibodies which cross-react with mouse and human OF antigens. This new assay is a rapid and sensitive method for the screening of monoclonal antibodies against these antigens as well as for detecting antibodies to tumors bearing these antigens in patients.

Influence of the genetic background and carrier protein on the antibody response to GnRH.

A vaccine against the gonadotropin releasing hormone (GnRH) is being developed as an immunological method for the treatment of prostatic enlargement. The work described here was aimed at investigating the influence of the genetic background on immune responses to GnRH conjugated to diphtheria toxoid (DT). Mice of different strains were injected with the conjugate and the antibody levels against GnRH and DT quantitated in order to examine the effect of genetic background on immune responses to the hapten and the carrier. All immunized animals produced antibodies to DT. Anti-GnRH antibodies were generated by all strains of mice except 129. The low anti-GnRH response in the 129 strain mice did not appear to be MHC-linked, as C57BL/6 mice, which bear the same MHC haplotype as 129 mice, were able to generate a strong anti-GnRH response. The non-responsiveness to the hapten (GnRH) in 129 strain mice was overcome by the use of an 'alternate carrier' approach.

Hyporesponsiveness to a GnRH vaccine in a non-responder mouse strain is T-cell mediated.

Immunization of rats and monkeys with the decapeptide gonadotropin releasing hormone (GnRH) linked to carriers such as diphtheria toxoid (DT) or tetanus toxoid (TT) results in a marked atrophy of the prostate. This vaccine is now being explored for its potential in the "immunosurgery" of prostatic hypertrophy in men and is currently undergoing Phase I/II clinical trials. We have been investigating immunogenetic aspects of immune responses to this hapten-carrier conjugate, and in a recent communication we described the responses of different strains of mice to GnRH conjugated to DT (GnRH-DT). Mice of the 129 (H-2b) strain were found to be non-responders to GnRH. However, further immunization of GnRH-DT-immunized 129 mice with GnRH linked to an alternate carrier, TT, resulted in the production of high levels of anti-GnRH antibodies. This showed that 129 mice are not deficient in GnRH-specific B cells and that the lack of response to GnRH in 129 mice is possibly due to (i) the lack of appropriate helper T-cells or (ii) the presence of suppressor cells. In this report we present evidence to support the existence of suppressor cells in GnRH-DT-immunized 129 mice.

Cytokine patterns in maternal blood after premature rupture of membranes.

OBJECTIVE: To compare two types of cytokines, type 1, which activate cell-mediated reactions and are important in cytotoxic and delayed-type hypersensitivity reactions, and type 2, which encourage vigorous antibody production and are commonly found in association with humoral immune responses, in blood of women with premature rupture of membranes (PROM). METHODS: Forty-four women with histories of at least three successful pregnancies and who currently delivered normally served as controls. The PROM group consisted of 30 women with spontaneous rupture of fetal membranes at term. Peripheral blood mononuclear cells were stimulated separately with a mitogen, placental cells, and a trophoblast antigen extract, and the supernatants examined for type 1 and type 2 cytokines. RESULTS: Mitogen-stimulated blood cells produced significantly higher levels of type 1 cytokines in PROM women than in normal controls. Higher levels of the type 1 cytokine interferon-gamma were produced by PROM samples stimulated with autologous placental cells and with trophoblast antigens. Ratios of type 1 to type 2 cytokines were higher in PROM compared with normal pregnancy, and in some cases as much as 25-fold higher. CONCLUSION: Women in the PROM group had a stronger type 1 reactivity whereas normal women were more predisposed to type 2 immunity; thus, PROM appears to be associated with a maternal type 1 bias.

Quality of paper boards from arecanut leaf sheath.

A study was carried on utilizing arecanut leaf sheath for making paper boards. Paper boards were made with various combinations of arecanut leaf sheath with waste paper, 1:1, 1:2, 1:3, 3:1, 2:1, control (100% areca leaf sheath) and the qualities of these paper boards were tested as per the Bureau of Indian Standards (IS: 1060 (part-I)-1966). The paper boards made with more arecanut sheath materials had more resistance to water absorption. The addition of paper increased the substance weight of the paper boards. The 2:1 and 3:1 combinations of arecanut leaf sheath and waste paper had best tear strength, tensile strength, bursting strength and water resistance with minimum substance weight.

A single human monoclonal antibody that confers total protection from tetanus.

Protective human monoclonal antibodies (HuMAbs) are superior to hyperimmune sera and murine monoclonal antibodies as far as human immunotherapy is concerned. In this report, we describe the successful generation of triomas secreting HuMAbs to tetanus toxin (tt). Lymphoblastoid cell lines secreting anti-tt antibodies were stabilized by back-fusion with a mouse x human heterohybrid myeloma partner, SBC-H20. One of the antibodies so produced, confers total protection of mice from tetanus, unlike a few recent reports where only partial protection (delay in the onset of tetanus) was achieved with single HuMAbs. Experiments to localize the neutralizing epitope(s) of the toxin using the protective monoclonal antibodies revealed that the antibody recognizes a conformational determinant that is destroyed on SDS-treatment. Preliminary studies show that Fab preparations of the protective antibody are capable of neutralizing tetanus toxin, suggesting that it might be possible to clone and express the Fab in a stable vector for large scale production.

Efficacy and safety of infliximab in active SLE: a pilot study.

Tumour necrosis factor-alpha (TNF-alpha) plays a major role in propagating the inflammatory processes responsible for tissue damage in systemic lupus erythematosus (SLE) and is overexpressed both systemically and locally in this disease. Hence, this pilot study was carried out to assess the safety and efficacy of TNF blockade in patients with active SLE. A total of 46 individuals (27 patients with active SLE and 19 healthy control volunteers) were the subjects of this study. Nine patients with SLE were allocated to treatment arm and 18 were allocated to control arm. In addition to conventional treatment, treatment arm received infliximab infusions 3 mg/kg body weight at 0, 2, 6 weeks and then q 8 weeks for a total of 24 weeks, that is, a total of five doses. Patients were closely monitored for infection. Clinical, laboratory and treatment data were entered into a pre-designed proforma. Health status (SF-36), patient global assessment (PGA) of disease activity, disease activity scores by SLEDAI and organ damage by SLICC/ACR-DI (American College Rheumatology) were measured at baseline and end of the study. Relevant immunological studies included serum levels of TNF-alpha and soluble TNF receptors-1 (p55 srTNF-alpha) and -2 (p75 srTNF-alpha), C3 and C4 complement levels, anti-dsDNA antibody titres (IgM, IgG and IgA isotypes), anti-cardiolipin titres (IgM, IgG and IgA isotypes) and anti-beta2GPI (Glycoprotein I) antibody titres (IgM, IgG and IgA isotypes). Four patients from treatment arm dropped out due to infliximab infusion reaction and 12 patients dropped out from the control arm. The treatment group showed significantly greater improvement in Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Improvements in several SF-36 subscales, PGA and VAS-Fatigue (Visual Analogue Scale) were also greater in the treatment group but did not achieve statistical significance. The mean levels of TNF-alpha, soluble TNF receptors-1 (p55 srTNF-alpha) and -2 (p75 srTNF-alpha) were higher in the SLE group compared with the healthy controls but did not change significantly over the study period. We did not face any safety issues with infliximab in this study. In view of improvement in several SLE parameters and good safety profile of infliximab, anti-TNF-alpha therapy is an interesting candidate approach for treating SLE.

Progesterone-induced blocking factor (PIBF) modulates cytokine production by lymphocytes from women with recurrent miscarriage or preterm delivery.

Spontaneous miscarriage and preterm delivery are common complications of pregnancy. Pro-inflammatory cytokines have been shown to be associated with recurrent spontaneous miscarriage (RSM) and preterm delivery (PTD) and these have led to exploration of ways to downregulate pro-inflammatory cytokines and/or to upregulate anti-inflammatory cytokines. Progesterone-induced blocking factor (PIBF) is a molecule with inhibitory effects on cell-mediated immune reactions. We have ascertained the effects of PIBF on secretion of selected type 1 and type 2 cytokines by peripheral blood mononuclear cells from healthy non-pregnant women, women undergoing normal pregnancy, women with unexplained RSM and women with PTD. Peripheral blood mononuclear cells from 30 women with a history of unexplained RSM, 18 women undergoing PTD, 11 women with normal pregnancy and 13 non-pregnant healthy women were stimulated with a mitogen in the absence and presence of PIBF after which the levels of cytokines released into culture supernatants were determined by ELISA. Production of the type 2 cytokines IL-4, IL-6 and IL-10 by lymphocytes from the RSM and PTD groups and of IL-4 and IL-10 by lymphocytes from healthy pregnant women was significantly increased upon exposure to PIBF, while the levels of type 1 cytokines were not affected. Ratios of type 1:type 2 cytokines were decreased, suggesting a shift towards Th2 bias. PIBF did not affect cytokine production by lymphocytes from non-pregnant women. Thus, PIBF acts on lymphocytes in pregnancy to induce a type 1 to type 2 cytokine shift by upregulating the production of type 2 cytokines.