Functional Analyses of Cord IgM: Cytotoxic Antibody Against Common Killer T Cells

We have analysed cytotoxic antibody against T lymphocytes (TLFA) in cord IgM. TLFA IgM bound to T lymphocytes as determined by the radio immuno assay. TLFA IgM inhibited lymphocyte blastogenesis induced by Con A and PHA but did not inhibit that induced by PWM in the presence of complement. Based on these results, further studies were done to determine which function(S) of Con A- and PHA-induced lymphocytes could be inhibited by TLFA IgM. TLFA IgM inhibited Con A-induced T cell activity to suppress PWM-induced antibody formation and autologous mixed lymphocyte culture. In addition, TLFA IgM markedly inhibited PHA-mitogen factor induced killer T cell colony formation and their activity as determined by the colony assay and lymphocyte-mediated cytolysis using K562 and Daudi cell lines respectively. TLFA appears to play a role in protecting a fetus from immunologic rejection by the mother, by killing maternal killer cells which may trasfer across the placenta and harm the fetal tissues.     

Antifilarial and anti PPD IgM antibodies in cord blood

Tuberculosis and Filariasis are commonly encountered in South India, more so in the low socio-economic group. This study was undertaken to find out the fetal immune response to maternal filariasis and tuberculosis. The presence of anti-filarial and anti-PPD IgG and IgM antibodies in the mother and cord blood at the time of delivery was identified by ELISA. Six out of 29 cases showed IgM against PPD in the cord blood and 7 out of 29 cases showed IgM againstBrugia malayi antigen in the cord blood. In both instances, 6 out of 29 for PPD and 7 out of 29 for B.malayi, the maternal blood too showed the presence of IgM to these two antigens. Comparison with anti-Ascaris lumbricoides IgM antibodies in mother and cord blood in these (6 out of 29 against PPD and 7 out of 29 against S.malayi) cases did not show a similar distribution of IgM antibodies in mother and cord blood, indicating that there is a fetal response to maternal filariasis and tuberculosis.     

Suppression of B lymphocytes in mature newborn infants

The ability of cord blood lymphocytes to secrete immunoglobulins during in vitro culture was investigated by means of a reverse hemolytic plaque forming cell (PFC) assay. Mononuclear cord blood cells did not differentiate into immunoglobulin-secreting cells after stimulation with the polyclonal B lymphocyte activator pokeweed mitogen (PWM), contrary to the findings in normal adults. Mononuclear cord blood cells were then separated into T-enriched and T-depleted blood lymphocyte subsets. When these were co-cultured, the PWM-induced immunoglobulin secretion was still low; following irradiation of the T-enriched cells, the numbers of IgM-PFC but not of IgG- or IgA-PFC increased considerably. The effect of irradiation of the T-enriched cells on the PWM-induced IgM response was dose-dependent, with maximal effect at 2500 rad. It is concluded that the low PWM responses obtained using cord blood lymphocytes are in part due to suppression by radiosensitive T suppressor cells. Following removal of this suppression by means of irradiation, B lymphocytes can be induced to secrete IgM, but not IgG or IgA.     

SUPPRESSION OF B LYMPHOCYTES IN MATURE NEWBORN INFANTS

ABSTRACT. The ability of cord blood lymphocytes to secrete immunoglobulins during in vitro culture was investigated by means of a reverse hemolytic plaque forming cell (PFC) assay. Mononuclear cord blood cells did not differentiate into immunoglobulin-secreting cells after stimulation with the polyclonal B lymphocyte activator pokeweed mitogen (PWM), contrary to the findings in normal adults. Mononuclear cord blood cells were then separated into T-enriched and T-depleted blood lymphocyte subsets. When these were co-cultured, the PWM-induced immunoglobulin secretion was still low; following irradiation of the T-enriched cells, the numbers of IgM-PFC but not of IgG- or IgA-PFC increased considerably. The effect of irradiation of the T-enriched cells on the PWM-induced IgM response was dose-dependent, with maximal effect at 2500 rad. It is concluded that the low PWM responses obtained using cord blood lymphocytes are in part due to suppression by radiosensitive T suppressor cells. Following removal of this suppression by means of irradiation, B lymphocytes can be induced to secrete IgM, but not IgG or IgA.     

Fetal Response to Maternal Ascariasis as Evidenced by Anti Ascaris Lumbricoides IgM Antibodies in the Cord Blood

ABSTRACT. The immune response of the fetus to maternal infection with Ascaris lumbricoides was studied by estimating IgG and IgM antibodies, using ELISA, in 28 paired samples of mother and cord blood. A. lumbricoides adult male, cuticle and unembryonated egg antigen was used. Toxocara canis larva antigen was used as a specificity control. Presence of IgG in the cord blood does not signify fetal response, but the presence of IgM antibodies does. IgM antibodies to Ascaris adult male antigen were present in 27 out of 28 maternal sera and 1 out of 28 cord sera. All maternal sera and only 6 cord sera showed IgM antibodies against Ascaris cuticle and 20 out of 22 maternal sera and 8 out of 22 cord sera tested, showed IgM antibodies to Ascaris unembryonated egg antigen. Sixteen out of 22 maternal sera and none out of 22 cord sera showed IgM antibody against Toxocara canis antigen, ruling out cross reaction and transplacental leak. The results of our study suggest that the Ascaris infection can spread to the fetus and elicit immune response.     

Fetal response to maternal ascariasis as evidenced by anti ascaris lumbricoides IgM antibodies in the cord blood.

The immune response of the fetus to maternal infection with Ascaris lumbricoides was studied by estimating IgG and IgM antibodies, using ELISA, in 28 paired samples of mother and cord blood. A. lumbricoides adult male, cuticle and unembryonated egg antigen was used. Toxocara canis larva antigen was used as a specificity control. Presence of IgG in the cord blood does not signify fetal response, but the presence of IgM antibodies does. IgM antibodies to Ascaris adult male antigen were present in 27 out of 28 maternal sera and 1 out of 28 cord sera. All maternal sera and only 6 cord sera showed IgM antibodies against Ascaris cuticle and 20 out of 22 maternal sera and 8 out of 22 cord sera tested, showed IgM antibodies to Ascaris unembryonated egg antigen. Sixteen out of 22 maternal sera and none out of 22 cord sera showed IgM antibody against Toxocara canis antigen, ruling out cross reaction and transplacental leak. The results of our study suggest that the Ascaris infection can spread to the fetus and elicit immune response.     

T cell response to anti-CD3 antibody in Down's syndrome.

The non-specific mitogen phytohaemagglutinin (PHA) and an anti-CD3 (OKT3) monoclonal antibody were used to measure the lymphocyte proliferative response in blood samples from 15 subjects with Down''s syndrome. Blood from 15 healthy controls closely matched for age and sex was also assayed. The mean blastogenic value in PHA stimulated patient lymphocyte cultures was similar to that calculated in the controls. In contrast, the mitogenic response of lymphocytes from patients with Down''s syndrome to anti-CD3 stimulation was on average significantly reduced. Immunofluorescence studies and additional experiments carried out by using semiallogeneic (maternal) monocytes as a source of antigen presenting cells showed that the impaired anti-CD3 induced mitogenesis in Down''s syndrome could not be ascribed either to a lack of binding of the antibody to the trisomic cells, or to a defective monocyte-T cell interaction. These findings help to explain the cellular basis of the immune defect in Down''s syndrome.     

Cell mediated immunity in childhood malaria

Children with malarial infection, due to P.Vivax and P.falcifarum, were tested for cell mediated immunity (CMI) by lymphocyte proliferate response to mitogens PHA (phytohaemagglutinin)and PWM (poke weed mitogen) and antigen PPD (purified protein derivative). This was done during the period of parasitemia and after treatment, and compared to 19 normal matched controls. There was no significant difference between the patients and the control group with regard to PHA (patients 57.4 ±50.5; controls 61.3 ±54.9); PWM (patients 27.4 ±19.9, controls 29.9 ±24.5); PPD (patients 2.2 ±1.2, controls 1.9 ±1.4). There was also no significant difference in the lymphocyte responses during the period of parasitemia and after treatment. Hence, there does not seem to be any depression of CMI as shown by lymphocyte proliferative responses during childhood malaria.     

Immunological Profile in Children with Minimal Change Nephrotic Syndrome

ABSTRACT. Peripheral blood from patients with active stage of minimal change nephrotic syndrome (MCNS) was examined for concanavalin A (ConA)-inducible suppressor T cell activity, proliferative response to phytohemagglutinin (PHA) and in the autologous (AMLR) and allogeneic (MLR) mixed lymphocyte reaction, proportions of T cells with receptors for IgM (Tu) or IgG (Tγ) and the levels of serum immunoglobulin M, G and A. Six of 9 patients with MCNS studies showed deficiency of ConA-induced suppressor cell activity. In the AMLR, only one of 9 patients with MCNS demonstrated depressed proliferative response (p<0.05). In the allogeneic MLR, T cells from 5 of 9 patients with MCNS demonstrated poor proliferative response when stimulated with normal control non-T cells. Five of 9 patients with MCNS had depressed proliferative response to PHA. The proportion of total T cells, Tu cells and Ty cells in the patient group were comparable to healthy control group. Serum IgG was significantly decreased in 7 of 11 patients. This study demonstrates multiple immunological abnormalities in patients with MCNS that might play a role in its pathogenesis.     

Passive immunity acquisition of maternal anti-enterohemorrhagic Escherichia coli (EHEC) O157:H7 IgG antibodies by the newborn

Enterohaemorrhagic Escherichia coli (EHEC) strains are among the main causes of haemorrhagic colitis (HC) and haemolytic-uremic syndrome (HUS) in industrialised countries. In Brazil, EHEC have been detected in the faeces of patients with non-bloody diarrhoea, though an association between EHEC and HUS has been detected recently. These observations suggest that there is a pre-existing immunity triggered by the contact with EHEC and other categories of bacteria, such as EPEC, that share similar virulence factors and to which our population is highly exposed. Our aim was to evaluate the placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens. We evaluated 28 paired maternal and cord sera for the presence of IgG against EHEC O157:H7 protein antigens and IgG and IgM to O157 LPS employing ELISA and IB technique. Total IgG and IgM level analyses were also made. Anti-EHEC O157:H7 and anti-LPS O157 IgG antibody levels in cord sera were equivalent to those of their maternal sera. A good correlation between the mothers’ anti-LPS O157 IgM and total IgM levels was found. Anti-LPS O157 IgM levels were higher than anti-LPS O157 IgG levels in the same samples, and anti-LPS IgM antibodies were not detected in cord sera. Identical patterns of recognition of bacterial protein antigens by specific IgG were found in the paired samples and the recombinant purified variable region of γ intimin was specifically recognized by one paired maternal and cord sample. In conclusion, although the antibody profile varied among individuals, all paired cord and maternal serum samples showed an identical recognition pattern, indicating an efficient placental transfer of IgG antibodies reactive to EHEC O157:H7 antigens. Dr. Patricia Palmeira and Leonardo Y. Ito contributed equally to this work.     

Immunological profile in children with minimal change nephrotic syndrome

Peripheral blood from patients with active stage of minimal change nephrotic syndrome (MCNS) was examined for concanavalin A (ConA)-inducible suppressor T cell activity, proliferative response to phytohemagglutinin (PHA) and in the autologous (AMLR) and allogeneic (MLR) mixed lymphocyte reaction, proportions of T cells with receptors for IgM (Tu) or IgG (T gamma) and the levels of serum immunoglobulin M, G and A. Six of 9 patients with MCNS studies showed deficiency of ConA-induced suppressor cell activity. In the AMLR, only one of 9 patients with MCNS demonstrated depressed proliferative response (p less than 0.05). In the allogeneic MLR, T cells from 5 of 9 patients with MCNS demonstrated poor proliferative response when stimulated with normal control non-T cells. Five of 9 patients with MCNS had depressed proliferative response to PHA. The proportion of total T cells, Tu cells and T gamma cells in the patient group were comparable to healthy control group. Serum IgG was significantly decreased in 7 of 11 patients. This study demonstrates multiple immunological abnormalities in patients with MCNS that might play a role in its pathogenesis.     

Development of B lymphocyte function in childhood

The capacity of blood lymphocytes of children aged from birth to six years to produce immunoglobulins was studied in vitro at the cell level using a direct B lymphocyte activator (Epstein-Barr virus) or a T lymphocyte dependent B lymphocyte activator (pokeweed mitogen). Umbilical cord blood lymphocytes secreted IgM at adult levels after Epstein-Barr virus stimulation, while the ability to synthesize IgG and IgA increased up to the ages of 1 and 2 years, respectively, but not beyond this period. IgG3 production preceded that of the other IgG subclasses. The T lymphocyte dependent IgM synthesis was low at birth, but approached adult levels at two years of age. T cell dependent IgG and IgA secretion, however, remained reduced even up to 6 years of age.     

Development of B Lymphocyte Function in Childhood

ABSTRACT. The capacity of blood lymphocytes of children aged from birth to six years to produce immunoglobulins was studied in vitro at the cell level using a direct B lymphocyte activator (Epstein-Barr virus) or a T lymphocyte dependent B lymphocyte activator (pokeweed mitogen). Umbilical cord blood lymphocytes secreted IgM at adult levels after Epstein-Barr virus stimulation, while the ability to synthesize IgG and IgA increased up to the ages of 1 and 2 years, respectively, but not beyond this period. IgG3 production preceded that of the other IgG subclasses. The T lymphocyte dependent IgM synthesis was low at birth, but approached adult levels at two years of age. T cell dependent IgG and IgA secretion, however, remained reduced even up to 6 years of age.     

Augmentation of pokeweed mitogen-induced immunoglobulin production by cord T-cell supernatants

Adult mononuclear cells were stimulated with pokeweed mitogen (PWM) in the presence of supernatants from cord T cells which had been previously stimulated with PWM for 24 h. Increased IgM, IgG and IgA production, as measured by ELISA, was observed with the addition of increasing concentrations of cord T-cell supernatants. The most significant increase in immunoglobulin production was observed with IgM (p less than 0.001). Cord T lymphocytes appear to be capable of producing soluble helper factors which augment immunoglobulin production by adult lymphocytes.     

Absence of complement receptor type 3 and lymphocyte function antigen 1 causing deficient phagocyte and lymphocyte functions

We describe a patient with delayed umbilical cord detachment, recurrent bacterial infections, and inability to form pus, despite persistent leucocytosis. Immunofluorescence studies with specific monoclonal antibodies showed a severe deficiency in the expression of -chains of the receptor for the C3bi fragment of C3, complement receptor type 3, and the lymphocyte function antigen 1 molecule, found on neutrophil, monocyte and lymphocyte membranes. These membrane antigen defects were responsible for abnormalities in adhesive cell functions. Polymorphonuclear leucoytes demonstrated a markedly reduced chemiluminescence response as well as an impaired nitroblue tetrazolium test and superoxide generation to a particulate stimulus (zymosan), while the responses to a soluble stimulus (phorbol myristate acetate) were normal. In addition, random migration und chemotactic response to zymosan-activated serum were impaired. The lymphocytes demonstrated abolished natural killer cell cytotoxicity as well as abnormal humoral immunity and a lack of antibody response to pertussis and tetanus antigens.Abbreviations CL chemiluminescence - CR3 complement receptor type 3 - CTL cytotoxic T lymphocyte - LFA-1 lymphocyte function antigen 1 - Mab monoclonal antibody - MN monocytes - NBT nitroblue tetrazolium - NK cell natural killer cell - OPZ opsonized zymosan - PBMC peripheral blood mononuclear cells - PBL peripheral blood lymphocytes - PMN polymorphonuclear cells - PHA phytochaemagglutinin - PMA phorbol myristate acetate - ZAS zymosan-activated serum - CH50 total classical pathway complement - AP50 total alternative pathway complement     

Analysis of proliferating lymphocyte subpopulations in newborns and adults

Surface markers of peripheral lymphocyte subpopulations were determined before and after in vitro stimulation of neonatal and adult mononuclear cells with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). The type of cell responding to each mitogen was identified by a method combining autoradiography and the peroxidase-antiperoxidase (PAP) staining method. In comparison with adults, there is a lower proportion of OKT3-positive lymphocytes in neonates (43% vs. 72%). Analysis of lymphocyte subpopulations after stimulating mononuclear cells with PHA and PWM showed that in neonates nearly the same percentages of OKT4-and OKT8-positive proliferating cells (43% and 40%, respectively) could be observed, whereas in the proliferating cells from the adults, the OKT8 surface marker predominated (41% vs. 18%).Abbreviations PHA phytohaemagglutinin - PWM pokeweed mitogen - PAP peroxidase-antiperoxidase - MEM minimal essential medium - FCS fetal calf serum - MIF migration inhibition factor - LIF leukocyte migration inhibition factor - ConA concanavalin A     

Selective immunoglobulin M deficiency associated with disseminated molluscum contagiosum

A 16-year-old girl with disseminated molluscum contagiosum (MC) was found to have a very low level of serum IgM, elevated levels of IgG and IgA, and a high level of IgE. She had normal numbers of peripheral blood IgM⁺, IgG⁺ and IgA⁺ B-lymphocytes but their terminal differentiation into plasma cells could not be induced by pokeweed mitogen (PWM) in vitro. On the other hand, the patient's T-cells showed normal helper functions in the PWM system and normal interferon (IFN) production in vitro. However, the IgM⁺ B-cells can be induced to differentiate into IgM secreting cells by Epstein-Barr virus (EBV), suggesting that the genetic mechanism for synthesis of the component immunoglobulin proteins is present. T-cell functions were impaired, as shown by delayed type cutancous hypersensitivity (DTH) and mitogen response. The data suggest that the selective IgM deficiency of the patient is due mainly to defects in B-cells at the terminal differentiation stage, but immunological abnormalities are present in both B and T-cell systems. Neutrophil functions examined were normal. MC was treated by intravenous injection of IFN without any side effects; however, no clinical improvement was achieved.Abbreviations MC molluscum contagiosum - IFN interferon - DTH delayed type cutaneous hypersensitivity - MNC mononuclear cells - FCS fetal calf serum - PBL peripheral blood lymphocytes - sIg surface Ig - PWM pokeweed mitogen - PHA phytohaemaggulutinin - Con A Concanavalin A - DHEA-SO₂ dehydroepiandrosterone sulphate - E₂ Oestradiol-17 - E₃ Oestriol - TRH thyloid-releasing hormone - PRL prolactin - FITC fluorescein isothiocyanate - FL cells human fibroblasts - EBV Epstein-Barr virus     

Anti-insulin antibodies and birth weight in pregnancies complicated by diabetes

Free insulin cannot cross the placenta but insulin complexed to anti-insulin antibodies has been demonstrated in cord blood. We studied whether antibody-bound insulin in diabetic patients can evoke fetal macrosomia independently of maternal metabolic control. In 457 non insulin-treated controls and 173 insulin-treated diabetic patients we measured 1187 anti-insulin antibody levels and maternal blood glucose, maternal fructosamine, cord blood insulin, cord blood C-peptide, cord blood fructosamine and amniotic fluid insulin. Mean anti-insulin antibody levels in maternal blood and cord blood were significantly higher in insulin treated diabetic patients (4.6 and 5.4 U/ml) than in controls (1.8 and 1.7 U/ml) with maxima of 89.2 in maternal and 120.0 U/ml in cord blood, respectively. In insulin treated diabetic patients 16.6% (maternal blood) and 22% (cord blood) anti-insulin antibody levels were above the 97th percentile. There was a high significant correlation between maternal and cord blood anti-insulin antibodies (R = 0.987, P = < 0.0001), but no correlation of anti-insulin antibodies with maternal (glucose, fructosamine) or fetal (insulin, C-peptide, and fructosamine in cord blood, amniotic fluid insulin) metabolic parameters. While maternal and fetal metabolic parameters correlated with birth weight neither maternal nor cord blood anti-insulin antibody levels correlated with birth weight. These findings do not support the hypothesis that maternal anti-insulin antibodies independently influence fetal weight.     

Lymphocyte abnormalities in infants born to drug-abusing mothers

To evaluate the possible effects of maternal intravenous drug use on infant immunity, we measured the in vitro peripheral blood mononuclear cell proliferative responses to phytohemagglutinin (PHA) and pokeweed mitogen, T cell subset numbers, immunoglobulin levels, and titers of antibodies to cytomegalovirus (CMV) and human immunodeficiency virus (HIV) in a group of drug-abusing mothers and their infants. Infants of drug abusers had a lower proliferative response to mitogen, associated with altered kinetics of the maximum response to PHA. The OKT4/OKT8 ratio decreased with age in the drug-exposed infants compared with control infants (P less than 0.005). There was no evidence of CMV infection in either group. One mother and her infant had antibody to HIV. Our data demonstrate that infants of intravenous drug-using mothers have distinct immunologic differences at birth compared with non-drug-exposed infants and that these persist throughout the first year of life. The cause appears unrelated to intrauterine viral infection, suggesting a direct toxic effect of the drugs on fetal immunologic development.     

Immune function in growth hormone-deficient children treated with biosynthetic growth hormone

Conflicting data regarding the immune function in growth hormone (GH) -deficient children or changes in immune parameters during substitutive GH therapy have been reported. We have studied the immune function in 13 patients with GH deficiency before and during treatment with biosynthetic GH (12 IU/m2 body surface/week) after 6 and 12 months of therapy. We found that the absolute number of total T lymphocytes and T-cell subsets (using monoclonal Ab as markers), Natural Killer cell activity (target K562) and response of lymphocytes to polyclonal mitogens (PHA, ConA, PWM) were all in the normal range and remained so after 6 and 12 months of therapy. The absolute number of B lymphocytes was in the normal range before treatment and after 6 months of therapy but dropped significantly after 12 months of treatment. Serum immunoglobulins (IgG, IgA, IgM) did not show a parallel drop and remained normal throughout the whole study. Our GH-deficient patients did not show any undue susceptibility to infections and our data thus seem to confirm that the immune function is basically intact in these children and that it is not suppressed by GH treatment. Although a drop in B lymphocytes was observed, the normal level of immunoglobulins and the normal functional response to PWM seem to demonstrate the maintenance of a normal humoral immune response.