Serum Blocking Factors Research Articles

Interpretation of blocking activity in maternal serum depends on the equation used for calculation of mixed lymphocyte culture results

Immunosuppressive blocking factors in maternal serum are usually determined by inhibition of mixed lymphocyte cultures (MLC), but reports on the importance of these factors for successful pregnancy are conflicting. Here we measured serum blocking activity in men, non-pregnant nulliparous women, non-pregnant multiparous women, women with normal pregnancies, and in women who had had recurrent spontaneous abortions and were treated with leucocyte immunizations. Three different equations were used for calculation of blocking activity: blocking effect index (BEI); stimulation index (SI); and blocking index (BI). By all three methods of calculation, significantly lower levels of blocking activity were noted for men and women compared with pregnant women and multiparae. In the patients with a history of recurrent spontaneous abortions blocking activity as determined by BEI and BI increased into the positive range after treatment with infusions of third-party donor leucocytes in a statistically significant number of women (P less than 0.05). However, blocking activity as determined by BEI had a higher predictive value for successful pregnancy than did that determined by BI or SI. Our data suggest that the equation used for calculating BEI is superior to other methods for the determination of blocking activity when monitoring the response to leucocyte immunization in women with recurrent spontaneous abortion. However, these results also cast doubt on the importance of blocking antibodies in histories of recurrent abortion, since pregnancies occurred in the absence and spontaneous abortions occurred in the presence of blocking activity.

Interferon gamma production in whole peripheral blood culture in acute hepatitis B

Interferon gamma (IFNg) production in whole peripheral blood (WPB) and mononuclear (MN) cell culture in acute hepatitis B (AHB) was compared. IFNg production was induced by phytogem agglutinin and measured in the cell supernatants of 14 AHB patients in the course of the disease. There were some up-regulating factors of IFNg production that probably operated in WPB culture: the presence of autoerythrocytes as well as the low content of monocytes. Autoserum regulated IFNg production in a stage-dependent way: it decreased IFNg activity at the bilirubin peak in hepatitis B infection, but not in convalescence. In contrast, we did not find a serum blocking effect in the corresponding stage of acute hepatitis A. The nature of this serum blocking factor in AHB is unclear.

Leukocyte Adherence Inhibition Test: Cell-Mediated Reactivity to Tumor Antigens in Colorectal and Lung Tumor Long Survivors

Cell-mediated immunity versus tumor antigens (cytosols) of the same histotype and site was evaluated by means of the leukocyte adherence inhibition (LAI) test in 44 colorectal adenocarcinoma and 17 lung squamous-cell carcinoma patients 3-10 years after surgical resection. LAI-positivity was observed in 17/44 (38.6%) and 13/17 (76.5%) patients, respectively, together with serum blocking factors in 7/17 (41.2%) compared to 0/13. These results indicate that a high percentage of long survivors retain an immunologic memory of the tumor antigen to which they have been exposed. The prognostic significance of serum blocking factors is less clear.

Identification of tumor necrosis blocking factor in serum and tumor tissue from patients with recurrent glioblastomas: Robert S. Yamamoto, Gene Ioli, Skip Jacques, Yancy Beamer, Edward Jeffes, and Gale A. Granger. University of California Irvine, Memorial Cancer Institute, Long Beach Memorial Medical Center, Healthcare Medical Center Tustin and Good Samaritan Hospital of Los Angeles

Identification of tumor necrosis blocking factor in serum and tumor tissue from patients with recurrent glioblastomas: Robert S. Yamamoto, Gene Ioli, Skip Jacques, Yancy Beamer, Edward Jeffes, and Gale A. Granger. University of California Irvine, Memorial Cancer Institute, Long Beach Memorial Medical Center, Healthcare Medical Center Tustin and Good Samaritan Hospital of Los Angeles

Inhibition of lymphokine-activated killer cell generation by blocking factors in sera of patients with head and neck cancer.

Cytolytic activation of peripheral blood lymphocytes by recombinant interleukin-2 (rIL-2) in patients with squamous cell carcinoma (SCC) of the head and neck may be inhibited by serum blocking factors, and this could influence therapeutic efficacy. Peripheral blood lymphocytes from 21 patients with this disease and 17 controls were incubated with 10-1000 U rIL-2 for 6 days in supplemented complete medium (containing 10% fetal calf serum) or the same medium plus 10% autologous serum. After washing the effector cells, we determined their cytotoxicity against K562 and MDA1386, a lymphokine-activated-killer(LAK)-sensitive SCC cell line, using a 51Cr-release assay. Patient sera inhibited LAK-generated lysis of both MDA1386 and K562, while control sera from healthy persons inhibited LAK-generated lysis of MDA1386. The blocking activity of patient sera tended to be greater than that of control sera. The sera of patients with untreated or recurrent disease and those who were free of disease had equivalent inhibitory capacity. The serum blocking factor acted in a dose-dependent manner, and inhibition was overcome by increasing the dose of rIL-2. Levels of circulating immune complexes (measured by the C1q binding method) did not correlate significantly with inhibition. A clinical protocol of repeated plasma exchange in patients with advanced and recurrent squamous cell carcinoma of the head and neck allowed sequential study of one patients's serum before, during, and after treatments. Plasmapheresis removed serum inhibitory factors, albeit temporarily. The activity of serum blocking factors in patients with this disease can be modulated by increasing doses of rIL-2 and by plasma exchange. This modulation may be important to improving clinical response rates for patients undergoing immunotherapy.

ELISA test for the detection of an immunological blocking factor in human pregnancy serum

We have previously shown that progesterone-treated lymphocytes of healthy pregnant women can produce a 34 kDa protein, progesterone-induced blocking factor (PIBF) capable of blocking lymphocyte function in vitro. Lymphocytes of women with idiopathic threatened pre-term delivery failed to produce this factor. On the assumption that the PIBF appears in the serum of healthy pregnant women but not in that of women at risk for immunologically based abortion of pre-term delivery, an ELISA has been designed for detecting the blocking factor in serum samples. Sera obtained at the time of delivery, as well as those from women with pre-term deliveries or miscarriages, contained significantly less PIBF than sera of 209 healthy pregnant women. Data obtained by testing sera of women at 16 weeks gestation whose pregnancies ended in spontaneous abortion showed that the predictive value of the test depended on the time interval between blood sampling and the onset of abortion. In 11 of 13 women with uterine contractions the outcome of pregnancy was predictable by normal or lower than normal levels of PIBF in the sera taken at the time the symptoms were presented. The data suggest that determination of PIBF in pregnancy serum might contribute to the diagnosis of immunologically bases pre-term disruption of pregnancy.

Effector lymphocyte response to homologous tumor antigens in various stages of malignant disease as monitored by leukocyte adherence inhibition — cell mediated immunity (LAI-CMI)

Leukocyte adherence inhibition-cell mediated immunity (LAI-CMI) studies were performed on leukocytes obtained from patients with various stages of breast cancer, colon carcinoma and lung cancer in order to monitor cell mediated immunity during tumor progression. In the presence of autologous serum, all patients with localized tumors showed positive LAI-CMI indexes (> 20%), while significant reduction of homologous tumor antigen recognition as measured by the LAI-CMI responses was observed in nearly all patients with Stage IV breast cancer, Duke C colon cancer and Stage III lung cancer. On substituting autologous serum with normal AB serum, leukocytes from patients with large tumor burdens responded to homologous tumor antigens. These results indicate the existence of organ-specific serum factor(s) which may mask the receptor sites on effector cells for tumor recognition. Patients with such serum blocking factor(s) showed significant increase of IgG immune complexes IgM, IgA and alpha-2-macroglobulins. Application of a protein A affinity column purification resulted in a major reduction of IgG and other immune globulins but not of alpha-2-macroglobulin. The blocking effects of autologous serum, however, were not completely abrogated by fitration on the protein A column, thus suggesting that SBF may be heterogeneous in nature and may occur in other serum protein fractions beside the immune globulins.

What is known about blocking factors in renal allograft recipients.

A variety of substances present in serum or plasma, either at the time of renal transplant or during stable graft function in long-term recipients, may interfere with cell-mediated immune functions. In several cases the presence of serum blocking factors has been correlated with decreased graft vulnerability to acute rejection. The question is: are serum blocking factors important for the induction or maintenance of allograft tolerance or, alternatively, are they merely by-products of the tolerant state? Perhaps the most compelling case for an essential role of serum blocking factors can be made in instances where vigorous cell-mediated immune responses can be demonstrated in vitro, but anergy (for example, absence of DTH response) is seen in vivo. However, in renal transplant recipients the majority of studies that show the presence of receptor blocking antibodies or other immunoregulatory serum factors also found a decreased cell-mediated immune response in vitro. Thus, allograft tolerance would appear to involve multiple mechanisms as suggested elsewhere. Due to the recent discovery of the molecular structure of the T cell receptor 33,34, it is now possible to identify determinants associated with the binding of T cell receptor blocking antibodies in renal transplant recipients; for example, one should be able to determine if families of V beta genes are involved. Alternatively, it may be possible to identify polymorphic structures on T cells other than the alpha, beta receptor complex as ligands for blocking antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunodetection and radioimmunotherapy of cutaneous T cell lymphomas using an 131I-labeled monoclonal antibody: an Illinois Cancer Council Study.

A radiolabeled murine monoclonal antibody (T101) was used for imaging and therapy of six patients with cutaneous T cell lymphoma (CTCL). Radioimmunodetection was performed with a 5.6 to 13.1 mCi 131I-T101 preparation (9.6 to 10.5 mg). A therapeutic dose of 100.5 to 150.1 mCi 131I on 9.9 to 16.9 mg of antibody was administered to five patients, with subsequent retreatment following plasmapheresis in three patients at the time of disease progression. All patients responded to their initial therapy and two patients responded to retreatment. Regression of skin lesions and peripheral adenopathy was witnessed. All patients reported resolution of their chronic pruritus. The duration of response ranged from 3 weeks to 3 months. Acute toxicity included fevers, pruritus, and mild dyspnea in two instances. Myelosuppression was seen in patients receiving the 144.7 mCi, 145.0 mCi, and 150.1 mCi 131I-T101 doses. Radioimmunodiagnostic and therapy studies included gamma scintigraphy, plasma, urinary, and wholebody antibody clearances, and biodistribution determined from skin, bone marrow, and liver biopsies. Immunologic studies included immunoperoxidase staining of target tissues, immunofluorescent flow cytometric analysis on peripheral blood and bone marrow, assays for serum blocking factors, determination of a human antimouse antibody (HAMA) response, and quantitation of circulating T101 levels. These preliminary data suggest that 131I-T101 has therapeutic potential in CTCL and that myelosuppression will be the limiting toxicity.

Inhibition of histamine release in vitro by a blocking factor from human serum: comparison with the iron binding proteins transferrin and lactoferrin

Recently, we described the presence of a blocking factor (BF) in rat serum, which inhibited the histamine release from rat mast cells in vivo and in vitro. The blocking activity was demonstrated in human serum as well. Qualitative analysis of the purified preparations demonstrated a major component with an apparent molecular weight of 70,000 daltons. In human serum the blocking factor was identified as transferrin (TF) by serological and biochemical methods. BF (DEAE-peak 1) and the iron binding proteins transferrin and lactoferrin (LF) are shown to inhibit the histamine release in vitro. The dose response curves reveal that inhibition by these proteins is dependent on their degree of iron saturation. Furthermore, unlike lactoferrin, the effects of transferrin and BF (DEAE-peak 1) follow the same pattern. Their mechanism of action remains to be elucidated.

Inhibition of histamine release from rat peritoneal mast cells by a factor from human serum — identification as transferrin

Recently, we described the presence of a blocking factor (BF) in rat serum which inhibited the histamine release from rat mast cells in vivo and in vitro. The blocking activity was demonstrated in human serum as well. Purification of the human-BF was carried out in a similar way as previously described for the rodent molecule. Both protein fractions produced a marked suppression of histamine release from rat mast cells and human basophils in a dose-dependent fashion. Qualitative analysis of the purified preparations demonstrated a major component with a molecular weight of 70,000 daltons. In human serum the blocking factor was identified as transferrin by serological and biochemical methods. It is suggested that this molecule may play an important role in regulating histamine release during allergic and inflammatory reactions.

Efficacy of large-volume plasma exchange in patients with chemotherapy-resistant metastatic colorectal cancer.

23 patients with advanced metastatic colorectal adenocarcinoma and measurable metastases were treated with chemotherapy until resistance to chemotherapy was evident. Chemotherapy was then not discontinued but administered for at least a further cycle combined with large-volume plasma exchange (PE). 15 of 23 patients responded again for 4-45 weeks, an average of 13. This effect was thought to be due to dilution or elimination of serum-blocking factors, which could be measured by mixed lymphocyte culture (MLC) assay. 16 of 20 patients showed a positive correlation between the clinical course and MLC activity, if the basic MLC reactivity was compared with the MLC levels at the start of each subsequent PE in each patient. It is postulated that in some tumors there is a resistance to chemotherapy mediated by plasma-blocking factors.

Detection of serum blocking factors and antibodies to the albumin receptor on HBsAG particles in healthy persons and patients with liver diseases.

An enzyme-linked immunosorbent assay (ELISA) for the detection of serum blocking factors (BF), or antibodies to the albumin receptor on HBsAg particles, was developed, and its clinical usefulness was examined in healthy persons and patients with liver diseases. Thirteen of 80 anti-HBs-positive female (16.3%) had BF, but all 25 male anti-HBs-positive, 41 female and 32 male anti-HBs-negative subjects were negative for BF. The activity of BF in BF-positive cases was not associated with the positive reciprocal hemagglutination titer of anti-HBs. For a neutralization test of BF, the BFs from 5 cases were absorbed with IgG-immunobeads. It was determined that these IgG-BFs were antibodies to the albumin receptors on HBsAg particles. No significance between positive-BF and abnormal S-GPT levels was recognized. These results suggest that the present test for the detection of BF, or anti-albumin receptor antibody, different from anti-HBs, might be useful for diagnosis of hepatitis B and as a marker for HB virus.

The Efficacy of Large Volume Plasma Exchange in Chemotherapy Resistant Malignancies

Chemotherapy resistance in cancer patients may be due to serum blocking factors, which can be diminished or eliminated by large volume plasma exchange (PE). This procedure was performed with the IBM blood cell separator in 69 patients resistant to chemotherapy. Immediately after PE the chemotherapy was given but it was reinstituted, if clinical evaluation revealed partial remission, minor response or no change. 37 out of 69 patients (53.6%) responded again, 32 (46.4%) did not. Response duration ranged from 2 to 45 weeks. Best clinical results were obtained in patients with colorectal cancer, 15 out of 23 showed improvement between 4 to 45 weeks. Serum blocking activity was measured using a modified mixed lymphocyte culture assay (MLC). There was a 80% positive correlation between clinical course of patients and MLC levels, if basic activity before the first PE was compared to MLC inhibition before the following PE's.

Active enhancement of rat cardiac allografts induced by donor specific semisoluble antigens.

Active enhancement was induced in inbred rats with cardiac allografts using semisoluble antigens. The optimal time of antigen pretreatment and optimal dose of semisoluble antigens were examined. The presence of serum blocking factors in the sera of rats having had allografts for a long time was examined with a macrophage migration inhibition test and lymphocyte microcytotoxicity assay. Since the blocking factors of macrophage migration inhibition were increasing on the 7th day, that day was determined to be the optimal time of antigen pretreatment. The mean survival time (MST) of cardiac allografts in untreated rats was 17.2 +/- 7.5 days. Semisoluble antigens were administered at 2 mg/kg body weight 7 days before the graft, 4 mg/kg 7 days before the graft and 2 mg/kg divided over three days, 15, 8 and 1 day before the graft, and the MSTs of cardiac allografts of rats receiving these treatments were 71.2 +/- 39.9, 62.6 +/- 42.2 and 79.3 +/- 31.0 days, respectively. The MST in each group of the treated rats was significantly longer than that of the control group (p less than 0.01). Rejection of the allograft, however, was accelerated in a group treated with 8 mg/kg 7 days before the graft (MST: 8.4 +/- 3.2 days). Serum blocking factors were detected in the sera of approximately half of the rats having cardiac allografts which survived a long time.

Cell-mediated immunity of experimental brain tumors

A brain tumor model was produced by intracerebral inoculation of 104 syngeneic malignant glioma cells induced with 20-methylcholanthrene into the right basal ganglia of C57BL/6 mice. Mean survival days of this model was 22.8 ± 3.1 days. Brain tumors were visible on Day, 12 after tumor inoculation and comprized 37 percent of the brain weight by Day 22. The results were as follows. (1) Body weight decreased gradually from Day 15 after tumor inoculation. Spleen and thymus weight, and the number of white blood cells and lymphocytes were reduced from Day 17. Mild splenomegaly was observed on Day 12. (2) Lymphocyte proliferative responses to phytohemagglutinin (PHA) of spleen cells from tumor-bearing mice were impaired from Day 12 and suppressed remarkably on Day 22. When serum from tumor-bearing mice was added to lymphocyte culture medium, blastogenesis of normal spleen cells was also impaired from Day 12 and remarkably suppressed on Day 22. (3) Natural cytotoxic activities of spleen cells and brain mononuclear cells from normal mice against malignant glioma cells in a 15 hr 51Cr release assay were recognized. Cytotoxic activities of spleen cells and brain mononuclear cells from tumor-bearing mice in the same assay were enhanced mildly on Day 12 and recognized by Day 22. (4) Natural cytotoxic cells were observed in the adherent cell fraction of brain mononuclear cells, and in both the adherent and nonadherent cell fractions of spleen cells. It is suggested that changes of cell-mediated immunity appeared at a relatively early stage in tumorbearing mice, although weight loss of organs, leukopenia, and lymphopenia were observed at the terminal stage. Nonspecific activity of lymphocytes was apparently suppressed with the growth of the tumor, as demonstrated by the appearance of serum blocking factor and lymphopenia. Natural cytotoxicity mediated in part by macrophages was maintained even in the terminal stage.

Serum blocking factors in tumor bearing hosts: the value of extended plasma exchange on in vitro immune parameters of patients with advanced cancer.

Experimental and clinical studies (reviewed in Ref. 1) indicate that serum blocking factors may alter immunological and inflammatory responses promoting tumor growth. In recent years, many plasma components including as yet undefined soluble factors especially glyco-proteins have been described as inhibitory in in vitro and in vivo bio-assays. It is still controversial if these factors are tumor products or if they are consequent to a tumor induced host response. In the present study, we investigated the possibility of reducing or eliminating such factors by large volume plasma-exchange with the aim of modulating the host tumor response.KeywordsPlasma ExchangeBlocking ActivityTumor BearingSerum BlockingTritiated Thymidine UptakeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Enhanced detection with an enzyme-linked immunosorbent assay of candida mannan in antibody-containing serum after heat extraction.

A double-antibody, solid-phase enzyme-linked immunosorbent assay was developed for detection of candida antigen(s) in serum and body fluids. The assay detected mannan purified from Candida albicans cells with a sensitivity of 1 ng/ml in buffer, but it was 1,000-fold less sensitive in pooled human serum. Because antibody appeared to be the major blocking factor in serum, a rapid and efficient heat-extraction method for recovering mannan from antibody-containing serum was devised. The assay detected antigenemia within 24 hr in all of 30 lethally infected rats, and antigenemia persisted until their deaths. Eight of 15 patients with documented or suspected candidiasis had antigenemia at levels ranging from 5 to greater than 1,000 ng/ml.

Abnormal autologous mixed lymphocyte reaction in autoimmune thrombocytopenic purpura

The autologous mixed lymphocyte reaction (auto-MLR) measures the ability of non-T cells to stimulate autologous T cells to proliferate in tissue culture. The auto-MLR was studied in 11 patients with autoimmune thrombocytopenic purpura (ATP). Seven patients had decreased auto-MLR, which averaged 4440 +/- 3364 cpm (SEM) compared to 15,360 +/- 6905 cpm for simultaneously studied controls. The average of the ratios of cpm incorporated by patients/cpm incorporated by control subjects was 0.20 +/- 0.06 (p less than 0.01). Serum from all 7 patients with low auto-MLR decreased the auto-MLR of normal subjects by an average of 56% +/- 8.5 (p less than 0.001). Preliminary results indicate that the inhibitory effect was mediated by a component of the IgG immunoglobulin fraction of serum. Sera from normal persons and from ATP patients with normal or high auto-MLR did not affect the auto-MLR of normal subjects. It was further shown that non-T cells from 3 of 5 patients with decreased auto-MLR failed to stimulate allogeneic T cells normally. It is concluded that many patients with ATP have decreased auto-MLR apparently due to the presence of a serum blocking factor and, perhaps, a defective stimulatory capacity of non-T-cells.

Abnormal Autologous Mixed Lymphocyte Reaction in Autoimmune Thrombocytopenic Purpura

The autologous mixed lymphocyte reaction (auto-MLR) measures the ability of non-T cells to stimulate autologous T cells to proliferate in tissue culture. The auto-MLR was studied in 11 patients with autoimmune thrombocytopenic purpura (ATP). Seven patients had decreased auto-MLR, which averaged 4440 +/- 3364 cpm (SEM) compared to 15,360 +/- 6905 cpm for simultaneously studied controls. The average of the ratios of cpm incorporated by patients/cpm incorporated by control subjects was 0.20 +/- 0.06 (p less than 0.01). Serum from all 7 patients with low auto-MLR decreased the auto-MLR of normal subjects by an average of 56% +/- 8.5 (p less than 0.001). Preliminary results indicate that the inhibitory effect was mediated by a component of the IgG immunoglobulin fraction of serum. Sera from normal persons and from ATP patients with normal or high auto-MLR did not affect the auto-MLR of normal subjects. It was further shown that non-T cells from 3 of 5 patients with decreased auto-MLR failed to stimulate allogeneic T cells normally. It is concluded that many patients with ATP have decreased auto-MLR apparently due to the presence of a serum blocking factor and, perhaps, a defective stimulatory capacity of non-T-cells.