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.     

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.     

DiGeorge syndrome with hypogammaglobulinaemia: a patient with excess suppressor T cell activity treated with fetal thymus transplantation

A male infant with DiGeorge syndrome had hypogammaglobulinaemia with a normal number of B cells. CD3(+) T cells were reduced and the CD4(+)/CD8(+) ratio was reversed. Proliferative responses of T cells to mitogens and to allogeneic cells were low. The pokeweed mitogen (PWM)-induced B cell differentiation assay revealed a higher than normal suppressor T cell activity. This suggests that some T cells had differentiated into functionally mature cells resulting in an imbalance of regulatory T cell functions and that excess suppressor activity might play a role in hypogammaglobulinaemia. Fetal thymus transplantation improved both cellular and humoral immunity. The patient's susceptibility to viral and bacterial infections, proliferative response of T cells and serum Ig concentration returned to normal. The excess suppressor activity seen before transplantation disappeared. Hypocalcaemia did not improve. These results show that fetal thymus transplantation was effective not only in reconstituting cellular immunity but also in normalizing the imbalance of regulatory T cell functions in this patient with DiGeorge syndrome.Abbreviations MNC mononuclear cells - ConA concanavalin A - PHA phytohaemagglutinin P - PWM pokeweed mitogen - ³H-TdR tritiated thymidine - PBL peripheral blood lymphocytes     

Deficient lymphokine production of newborn lymphocytes

Lymphokine production by newborn lymphocytes was assessed by measuring migration inhibition factor (MIF) and leukocyte inhibition factor (LIF) of isolated mononuclear cells from cord blood, 1-7-days-old newborns, and adult controls. Ficoll-Hypaque separated mononuclear cells were stimulated with phytohemagglutinin (PHA) or allogeneic lymphocytes in a mixed leukocyte culture (MLC), and the supernatants were harvested at optimal times for lymphokine assays. Thymidine incorporation into DNA was also assayed to calculate a proliferative index. MIF was assessed by the inhibition of adult mononuclear phagocyte cell migration under agarose; LIF was assessed by polymorphonuclear cell migration under agarose. Although the proliferative responses of cord and newborn cells are equivalent or greater than those of adult controls, the PHA-induced MIF production in cord blood and newborn lymphocytes was only 46% and 12.5% respectively of mean adult levels; MLC-induced MIF production was 44% and 7%, respectively of mean adult levels. PHA-induced LIF production in cord blood was 27% of adult levels. These differences are only appreciated if dilutions of the supernatants are assayed. Simultaneous assay of MIF and LIF production in dilution of supernatants from adult lymphocytes showed higher LIF activity, whereas in cord lymphocytes MIF activity was greater than LIF activity. This further emphasizes the non-identity of MIF and LIF. These results indicate another abnormality of T cellular immunity in newborns not detected by T-cell enumeration or proliferative responses and parallels other defects in specialized T cell function such as cytotoxicity and immune interferon production.     

Immune response of young children using ATP‐based Cylex® assay: A brief report

Jayaram A, Zeevi A, Bentlejewski C, Lin Y, Michaels MG. Immune response of young children using ATP‐based Cylex^® assay: A brief report. Pediatr Transplantation 2010: 14:664–666. © 2010 John Wiley & Sons A/S. Abstract: Data on immune responses of young children using ATP release–based Cylex assay are insufficient. This study measured the immune response of healthy children less than three years of age to mitogens, PHA and Con‐A. Blood was obtained from children attending routine health care visits. The Cylex^® assay was used to measure ATP production by CD4+ and CD3+ cells in response to PHA and Con‐A, respectively. Samples from 20 children less than three years (range 10–27 months) were evaluated. The mean ATP production by CD4+ lymphocytes following PHA stimulation was 376 ng/mL (95% CI 17.1–735), which was similar to the response of older children in Hooper et al.’s (Clin Transplant 2005;19:834) study (p‐value 0.28). The mean and median ATP production by CD3+ cells following Con‐A stimulation were 114 ng/mL and 93.3 ng/mL, respectively (95% CI for median 45.2,148.6). The data suggest that although the immune system of young infants and toddlers is evolving, they are still able to respond to mitogen stimulation similar to older children.     

Lack of suppression by concanavalin A-activated neonatal mononuclear cells

We studied the mixed leukocyte culture suppression generated by cord and newborn mononuclear cells stimulated by concanavalin A (Con A) compared to normal adult cells and cells from patients with systemic lupus erythematosus. Also we studied the effect of supernatants from cord or adult cells stimulated with Con A linked to sepharose (Con A sepharose). Peripheral blood mononuclear cells from 15 adults, 10 normal newborns, 23 cord blood samples, and 11 patients with systemic lupus erythematosus were preincubated with Con A for 48 hr, irradiated, and added to a one-way mixed leukocyte culture. Adult Con A-activated cells suppressed the mixed leukocyte culture by 30 +/- 6.5%. By contrast cord cells and newborn cells had no suppressive activity; these cells resulted in stimulation of 18 +/- 12.1 and 18 +/- 7%, respectively. This lack of suppression also was present in systemic lupus erythematosus cells (11.2 +/- 13.3). The supernatants of both Con A sepharose-stimulated cord and adult cells showed significant suppressive activity and there was some suppressive activity of sepharose-stimulated cells alone. These results suggest that the mixed leukocyte cultures suppressive activity observed previously by newborn cells is radiosensitive and dependent on ongoing cell division for its expression. It also is independent of prior mitogenic stimulation.     

Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults

To evaluate the development of the neonatal immune system, we measured T lymphocyte response to Con A, intracellular IL-2, IL-4, IFN-gamma and IL-10 production, and natural killer cell (NKC) activity in 12 very preterm, 12 preterm and 20 term neonates, 10 children and 10 adults. Immunoproliferation to Con A was significantly lower in cord blood than in children or adults. The percentage of CD4+ lymphocytes was significantly higher in newborns while CD8+ cells were higher at older ages, with a resulting gradual decline of the CD4+/CD8+ ratio. The percentage of IL-2-producing CD4+ and CD8+ cells was higher in all newborn groups than in children and adults, while the percentage of IL-4-producing cells was higher for CD8+ and lower for CD4+ cells in cord blood than in children and adults. Neonates had substantially lower percentages of CD4+ and CD8+ IFN-gamma-producing cells. A significant negative correlation was observed between gestational age and IFN-gamma-CD4+-, IL-2-CD8+-, and IL-10- CD4+-producing cells. In addition, a positive correlation was found between gestational age and IL-10-CD8+-producing cells. Percentages of CD4+/CD45RA+ cells were higher and CD4+/CD45RO+ percentages were lower in newborns than in children and adults. NKC activity in infants was significantly correlated with gestational age and significantly impaired compared to children and adults. On the whole, these results suggest a gradual development of immunity during gestation and show significant immaturity of cellular immune response at birth. The reduction of NKC activity, the lower proliferative response of T cells, the reduced cytotoxic response and a dysregulated cytokine production may contribute to the neonatal increased risk of infection and to the low incidence of graft-versus-host disease after cord blood transplantation.     

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.     

In vivo and in vitro effects of theophylline on the peripheral blood mononuclear cells from preterm infants

The in vivo effect of aminophylline (theophylline ethylenediamine) on cAMP levels and the mitogenic response of peripheral blood mononuclear cells (PBMC) from preterm infants were investigated. Increased cAMP levels and reduced response of these cells to concanavalin A (Con A) were found in preterm newborns who received intravenous aminophylline. No inhibitory effect of the drug on phytohemagglutinin (PHA)-induced proliferation was observed. cAMP levels of PBMC derived from preterm infants were significantly lower than those of term newborns. The in vitro effect of theophylline on the mitogenic response of PBMC from adults, term and preterm infants was also compared. It was found that PBMC from adults were significantly more sensitive to the inhibitory effect of theophylline on Con A-induced proliferation than were PBMC from either term or preterm infants, whereas the effect of the drug on the mitogenic response to PHA was similar in the three groups. These data suggest differences either in the sensitivity to increased cAMP concentration between PBMC from adults and newborns or in their lymphocyte subpopulations.     

Human placental cells that regulate lymphocyte function

We examined normal human placenta for an immunologic function by measuring the release of soluble inhibitory factor (SIF). SIF is a product of normal T lymphocytes and of the JEG-3 choriocarcinoma cell line, and blocks proliferative and antibody-producing responses of mononuclear cells. SIF can be further characterized by a noncovalently linked subcomponent, lipid suppressor substance. The villous surfaces of six normal human placentae were digested with collagenase to obtain a population of predominantly multinucleated giant cells. These cells were maintained in standard culture for 5 days after which the cell-free conditioned culture medium was assayed for SIF content by measuring suppression of [3H]thymidine incorporation into lymphocytes stimulated by low-dose phytohemagglutinin. Undiluted placental SIF induced 88% inhibition of this response (p less than 0.001). The placental SIF was found to contain lipid suppressor substance, as does SIF from mononuclear cells. We determined this by thin-layer chromatography where a peak of suppressive activity occurred at Rf 0.32 [( 3H]thymidine incorporation reduced from 21810 +/- 308 to 4121 +/- 214 cpm); this is the position on thin-layer chromatography to which mononuclear cell lipid suppressor substance migrates. Ion exchange chromatography comparing the elution patterns of lymphocyte-SIF and placental-SIF indicated that both eluted in the fraction of 40-50 mM PO4 means buffer, further suggesting identity between these two substances. SIF from placental and lymphocyte sources functioned by inducing the presence of suppressor cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro studies of bone marrow stem-cell function in uraemic children

Anaemia is a frequent symptom in children with chronic uraemia. There is only limited information about committed haemopoietic stem-cell function in renal insufficiency. The ability of bone-marrow cells to form erythropoietic and granulopoietic cell colonies in vitro was therefore tested in 13 children with chronic renal insufficiency.Following separation of mononuclear cells from bone-marrow aspirates by Ficoll-Isopaque gradient centrifugation, erythropoietic precursor cells were stimulated in plasma clots by erythropoietin. Granulopoietic precursor cells were stimulated in soft-agar gel using feeder layers of normal human leukocytes. The colonies were identified by staining and counted.The number and proliferative capacity of erythropoietic precursor cells (CFU-E) did not seem to be significantly suppressed. Under in vitro conditions, the responsiveness of CFU-E to erythropoietin seemed to be normal.Addition of autologous serum resulted in different degrees of inhibition of erythroid colony formation. The inhibitory effect of the sera was also obvious when uraemic sera were added to bone-marrow cells from subjects without renal disease. Preliminary data indicate that haemodialysis is effective in reducing this inhibitory activity which may be an important factor in the pathogenesis of anaemia in chronic renal insufficiency. The numbers and proliferative capacity of granulopoietic precursor cells (CFU-C) were normal in all the children tested.     

Transient immunodeficiency during asymptomatic Epstein-Barr virus infection

In vivo and in vitro humoral and cellular immune responses were studied in a 2 1/2-year-old girl immediately before, during, and after an asymptomatic infection with Epstein-Barr virus. During the acute EBV infection, the patient's peripheral blood mononuclear cells were deficient in immunoglobulin synthesis and suppressed the in vitro immunoglobulin synthesis of normal allogeneic cells. In vitro mitogen transformation of lymphocytes was reduced. In vivo antibody responses to the T cell-dependent antigens bacteriophage phi X 174 and Keyhole limpet hemocyanin were markedly depressed. These studies suggest that suppressor cells induced during acute EBV infection not only suppress immunoglobulin synthesis in vitro, but also interfere with in vivo antibody synthesis.     

Inhibition of lymphocyte DNA synthesis by plasma from patients with Kawasaki disease

Plasma obtained from patients with Kawasaki disease during the acute phase markedly inhibited DNA synthesis in autologous peripheral blood lymphocytes (PBLs) stimulated by phytohaemagglutinin-P (PHA-P) or concanavalin A (Con A). The inhibition became less marked with the progression of the disease and there was no effect on DNA synthesis in PBLs stimulated by pokeweed mitogen (PWM). The plasma also inhibited DNA synthesis in PBLs obtained from healthy adults. The postulated suppressors markedly inhibited DNA synthesis in PBLs from healthy adults stimulated by PHA-P, Con A, purified protein derivative (PPD) or mixed lymphocyte culture reaction (MLR) but they had little effect on the DNA synthesis stimulated by PWM or protein A. With respect to the mechanism, the suppression was found to be potentiated by an increase in the concentration of the patients' plasma, and not to be associated with cytotoxicity nor with a deficiency of factor(s) indispensable for PBL proliferation. It was also evident that the suppression was not related to the concentration of the stimulant, to the lengths of the culturing period nor to the presence of prostaglandins.     

Restoration of suppressor T-cell functions in children with AIDS following intravenous gamma globulin treatment

Suppressor T-cell function was analyzed in seven children with acquired autoimmunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). Four of the patients had markedly increased serum IgG levels. All patients had elevated percentages and absolute numbers of peripheral blood T8 cells. In vitro concanavalin A generation of suppressor cells for T-cell mitogenic responses and suppression of pokeweed mitogen-driven immunoglobulin secretion were diminished in all patients. After intravenous treatment with gamma globulin, four patients regained in vitro suppression of pokeweed mitogen-driven gamma globulin secretion. Treatment with intravenous gamma globulin also modified in vitro suppressor T-cell functions in children with AIDS or ARC.     

In vitro analysis of lymphocyte functions in common variable immunodeficiency: heterogeneity in B-cell defects

Ten patients with common variable immunodeficiency were classified into three groups according to the number of circulating B-cells, i.e. B-cells being absent (three patients), very low (three patients) or within the normal range (four patients). The four patients in the last group showed significant proliferative responses to the T-independent B-cell mitogen, formalin-fixed Staphylococcus aureus, Cowan I. Further study of these patients by co-cultures with allogeneic T or B-cells in various combinations with pokeweed mitogen showed that two patients had an intrinsic B-cell defect without T-cell defect. The third patient had a T-cell dysfunction (i.e. his T-cell could only help the B-cells of some individuals) resulting in a defect in Ig production. The T-cells of the fourth patient showed poor helper function towards all controls. All six patients with absent or very low numbers of B-cells in group I and II had normal T-cell helper function. This study demonstrates that the immunological defect in common variable immunodeficiency is most often a B-cell defect at different stages of their differentiation with sometimes an additional T-cell dysfunction.Abbreviations Con A concanavalin A - CVID common variable immunodeficiency - E erythrocyte - EAC erythrocyte-antibody-complement - FCS fetal calf serum - HEPES N-2-Hydroxyethylpiperazine-N-Ethanesulphonic Acid - Ig immunoglobulin - PBL peripheral blood lymphocyte - PHA phytohaemagglutinin - PWM pokeweed mitogen - RFC rosette-forming cells - RPMI Roswell Park Memorial Institute - Staph A formalin-fixed Staphylococcus aureus, Cowan I     

CD8+ T cell-mediated suppression of human immunodeficiency virus replication in older children with acquired immunodeficiency syndrome

BACKGROUND: Suppression of HIV replication by CD8+ T cells and/or their products correlated with the survival of infants. We sought to elucidate the role of CD8+ T cell-mediated suppression in seven older children with AIDS. METHODS: After separation of each child's CD4+ and CD8+ T cells, three different HIV culture assays were performed: (1) patient CD4+ T cells and phytohemagglutinin (PHA)-stimulated donor peripheral blood mononuclear cells (PBMC); (2) patient CD8+ T cells added to the CD4+ T cells and the PHA-stimulated donor PBMC (to test for CD8-mediated T cell suppression of HIV); (3) patient CD8+ cells added across a semipermeable membrane to the CD4+ T cells and the PHA-stimulated donor PBMC [to determine whether the CD8 cells secreted a soluble factor(s) that suppressed HIV]. RESULTS: Cultures from four of seven children showed greater HIV replication with CD4 cells alone than with CD4 and CD8 cells together, demonstrating CD8 suppression; evidence of soluble suppression was also seen. Cultures from two of the seven children showed HIV replication and no evidence of CD8 cell suppression. Cultures from one of the seven children had no appreciable replication of HIV even after removal of CD8 cells. CONCLUSIONS: CD8-mediated suppression is present in at least some children with AIDS. Additional mechanisms may be operating to slow the progression of the disease.     

Immunoregulation with levamisole in children with frequently relapsing steroid responsive nephrotic syndrome

The immunological and clinical effects of levamisole were studied in 10 children with frequently relapsing steroid responsive nephrotic syndrome (SRNS). The efficacy of the drug was tested during remission of the disease with all patients on alternate day steroid therapy. The lymphocyte proliferative response to phytohemagglutinin (PHA), concanavalin-A (Con-A) and pokeweed mitogen (PWM) were normal. The Con-A induced suppressor T-lymphocyte activity of 7 patients was low before treatment with levamisole 8 +/- 3.7% and increased to normal values during therapy 34 +/- 6%; p less than 0.001 (control 32 +/- 5%). In these 7 children prednisolone dosage could be decreased significantly or discontinued altogether (44.1 +/- 5.3%). Patients without immunoregulatory abnormalities did not respond to levamisole. In 3 out of 4 children tested the percentage of OKT8+ cells rose during levamisole therapy from 19.7 +/- 2.1 to 37 +/- 2.3 (p less than 0.001), thus correcting the elevated pre-treatment OKT4+/OKT8+ ratio from 3.1 +/- 0.2 to 1.5 +/- 0.2; p less than 0.001 (control 1.47 +/- 0.2). These data support the hypothesis that abnormal immunoregulation may play a role in the pathogenesis of SRNS. Treatment with levamisole can be useful in some patients with the frequently relapsing form of the disease.     

Granulopoiesis in Shwachman's syndrome (pancreatic insufficiency and bone marrow dysfunction).

Granulopoiesis was studied in 10 children with Shwachman's syndrome (chronic neutropenia and exocrine pancreatic insufficiency). Marrow proliferative activity assessed by determination of mitotic indices and tritiated thymidine uptake into granulocytic cells was normal. Assay of bone marrow granulocyte colony-forming cells (CFU-C) in a methylcellulose tissue culture system demonstrated normal CFU-C numbers in four patients and reduced numbers in five. The granulocyte colonies formed were indistinguishable from normal colonies morphologically. Production of colony-stimulating activity (CSA) from patients' peripheral blood leukocytes appeared normal when tested on control marrow. No serum inhibitors against CFU-C or CSA could be demonstrated using both control and autologous marrow, and co-culture of patients' peripheral blood lymphocytes with control marrow did not inhibit CFU-C growth. We conclude that in Shwachman's syndrome committed granulocytic stem cells are present, and the numbers detected in vitro vary widely as does the clinical neutropenia. The proliferative activity of recognizable granulocytic cells is normal and neither a deficiency of humoral stimulators nor the presence of serum or cellular inhibitors of granulopoiesis can be demonstrated.     

Suppressed lymphocyte mitogen-responsiveness in urinary tract infections of children and its correlation to pyelonephritis.

Cell-mediated immunity (CMI) was studied in a group of 48 children with urinary tract infections (UTI) using a whole blood micromethod for lymphocyte stimulation in vitro. The patients were subdivided into pyelonephritis group (27 cases) and lower urinary tract infection (LUTI) group (21 cases) on the basis of fever, erythrocyte sedimentation rate, C-reative protein and renal concentration capacity. At the acute stage of infection the lymphocyte responsiveness to leucoagglutinin (LA) and concanavalin A (Con A) was suppressed in both groups, but the suppression was much greater in those with pyelonephritis. By 6 weeks after infection the lymphocyte responses were normal in most but not all cases. We conclude that an acute pyelonephritis is associated with marked suppression of CMI and that the latter can be used as an additional criterion for establishing the level of infection. Patients with UTI did not generally appear to have any primary defect of CMI but when suppression of CMI was present, it seemed secondary to an ongoing infection.     

SUPPRESSED LYMPHOCYTE MITOGEN-RESPONSIVENESS IN URINARY TRACT INFECTIONS OF CHILDREN AND ITS CORRELATION TO PYELONEPHRITIS

Abstract. Cell-mediated immunity (CMI) was studied in a group of 48 children with urinary tract infections (UTI) using a whole blood micromethod for lymphocyte stimulation in vitro. The patients were subdivided into pyelonephritis group (27 cases) and lower urinary tract infection (LUTI) group (21 cases) on the basis of fever, erythrocyte sedimentation rate, C-reactive protein and renal concentration capacity. At the acute stage of infection the lymphocyte responsiveness to leucoagglutinin (LA) and concanavalin A (Con A) was suppressed in both groups, but the suppression was much greater in those with pyelonephritis. By 6 weeks after infection the lymphocyte responses were normal in most but not all cases. We conclude that an acute pyelonephritis is associated with marked suppression of CMI and that the latter can be used as an additional criterion for establishing the level of infection. Patients with UTI did not generally appear to have any primary defect of CMI but when suppression of CMI was present, it seemed secondary to an ongoing infection.