Diminished response to interleukin-10 and reduced antibody-dependent cellular cytotoxicity of cord blood monocyte-derived macrophages

Monocyte-derived macrophage (MPhi) subsets are generated by antagonistic induction pathways. A helper MPhi-type (Mh-MPhi) is induced by interferon gamma (IFN-gamma), whereas a cytotoxic MPhi-type (Mc-MPhi), induced by interleukin-10 (IL-10), is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Compared with MPhi from healthy adults [peripheral blood monocyte-derived macrophages (PBMPhi)], cord blood MPhi (CBMPhi) were found less capable of generating Mh-MPhi. Here we tested the hypothesis that their generation of Mc-MPhi via IL-10 is also impaired. MPhi surface markers were phenotyped. IL-10 protein and mRNA production were detected after stimulation [alphaCD3 monoclonal antibody (mAb)]. CBMPhi or PBMPhi were co-cultured with MPhi-depleted mononuclear cells of adults and CD4-targeting antibodies as models for ADCC were added. In cord blood, we found diminished alphaCD3-induced IL-10 protein and mRNA production (p < 0.05 versus adults). Basal CD16 and HLA-DR expressions on CBMPhi of preterm and full-term neonates were lower (p < 0.05 versus PBMPhi). IL-10 had reduced effects on CD16 up- and HLA-DR down-modulation on CBMPhi (p < 0.05 versus PBMPhi). CD4-directed receptor modulation and deletion were reduced in the presence of CBMPhi (p < 0.05 versus PBMPhi). IL-10 failed to enhance their ADCC capacity, which was in contrast to PBMPhi (p < 0.05). These data suggest that CBMPhi have an impaired cytotoxic capacity via lower sensitivity toward IL-10.     

Fc and complement receptor (CR1 and CR3) expression on neonatal human polymorphonuclear leukocytes

Fc gamma receptor III (Fc gamma RIII) and complement receptors (CR1 and CR3) were examined on polymorphonuclear leukocytes (PMN) from neonatal cord blood and adult blood using monoclonal antibodies directed against these receptors. Receptor expression was determined by flow cytometry. Fc gamma RIII, CR1 and CR3 expression was examined in whole blood at 4 degrees C, at 37 degrees C with or without stimulation with the chemotactic peptide f-met-leu-phe, and on PMN isolated by Ficoll-Hypaque centrifugation and dextran sedimentation. There was no significant difference between adult and cord PMN in the percent of cells which expressed Fc gamma RIII, CR1 and CR3 when examined in whole blood at 4 or 37 degrees C, or following stimulation with f-met-leu-phe. The percentage of PMN expressing CR1 and CR3 was lower on cord PMN compared to adult PMN when these cells were examined following Ficoll-Hypaque centrifugation and dextran sedimentation. The mean peak fluorescence of PMN which stained positively for CR1 and CR3 increased following f-met-leu-phe treatment of whole blood from adults and neonates. Since neonatal cord PMN were capable of upregulating complement receptors in response to chemotactic factors these results do not explain the increased susceptibility to infection exhibited by neonates.     

Cytokine inhibitors for sepsis and septic shock

Lipopolysaccharide and other bacterial products stimulate the immune cells of the host to elicit secondary inflammatory mediators, including cytokines. The 2 primary proinflammatory cytokines elicited in septic shock are tumor necrosis factor (TNF) and interleukin-1 (IL-1). Because of the importance of these cytokines in the biologic response to sepsis, they stand out as potential targets for adjunctive therapies of sepsis. This review focuses on therapies designed to inhibit the responses of TNF and IL-1, including studies in animals and humans evaluating TNF monoclonal antibodies, TNF receptor fusion proteins, IL-1 receptor antagonists, IL-1 receptor antibodies, and phosphodiesterase inhibitors. In general, the human trials have been disappointing in comparison with corresponding animal studies. As of yet, no cytokine inhibitor therapy that significantly decreases mortality in patients with severe sepsis or septic shock has been found. Copyright © 2001 by W.B. Saunders Company     

B cell-dependent T cell development

T and B cells are thought to develop independently. While it is widely recognized that T cells help B cells in the production of antibodies to protein antigens, less well understood is whether or how B cells contribute to T cell development and function. Defects in cell-mediated immunity in individuals with B cell deficiency and in B cell-deficient mice suggest that B cells contribute to T cell function. The question of whether T cell development is B cell dependent was revisited using two novel mouse strains: mice with monoclonal T cells (MT) and mice with monoclonal compartments of both B and T cells (MBT). It was found that T cell development and thymocyte selection is modified by the presence of B cells. These results suggest that B cells, or B cell products, contribute to thymocyte selection and T cell development.     

B cell-dependent T cell development

T and B cells are thought to develop independently. While it is widely recognized that T cells help B cells in the production of antibodies to protein antigens, less well understood is whether or how B cells contribute to T cell development and function. Defects in cell-mediated immunity in individuals with B cell deficiency and in B cell-deficient mice suggest that B cells contribute to T cell function. The question of whether T cell development is B cell dependent was revisited using two novel mouse strains: mice with monoclonal T cells (MT) and mice with monoclonal compartments of both B and T cells (MBT). It was found that T cell development and thymocyte selection is modified by the presence of B cells. These results suggest that B cells, or B cell products, contribute to thymocyte selection and T cell development.     

Study of antibody-dependent cell mediated cytotoxicity and thyroid growth blocking antibodies in congenital hypothyroidism

Antibody-dependent cell mediated cytotoxicity (ADCC) and thyroid growth immunoglobulin blocking (TGI block) which have been found in autoimmune thyroiditis in adults, as well as TSH receptors binding inhibitory antibodies (TBI ab) and antimicrosomal (Mc ab) and antithyroglobulin (Tg ab) antibodies were search in 42 mothers-infants pairs called at hospital after a positive screening for congenital hypothyroidism. The etiologic diagnoses were: 12 athyreosis, 12 ectopies, 7 anatomically normal glands and 11 transients. Tg ab and Mc ab were measured by commercial hemagglutination tests, TBI ab were determined using a radio ligand assay. ADCC in a 51Cr release assay by human thyroid cells in culture and TGI block by incorporation of 3H-thymidine using the same cells. Results were 38% for TBI ab in infants mainly in patients with dysgenesis without any concordance between mothers and infants. ADCC were found in 24% and TGI block in 24% with respectively mothers-infants concordance of 90% and 84%. Five mothers had autoimmune diseases (2 thyroiditis, 2 Graves' diseases and 1 insulin-dependent diabetes). Beside these rare cases of maternal diseases, the significantly high number of antibodies without any expression in the mothers suggests that autoimmunity plays a role in the etiology of congenital hypothyroidism.     

Iso-immune neonatal neutropenia due to an anti-Fc receptor III (CD16) antibody

We report a case of transient neonatal neutropenia due to a maternal iso-immunization against a non polymorphic region of the glycosylphosphatidylinositollinked Fc receptor type III (CD16) on granulocytes. The mother's granulocytes were typed NA1-negative, NA2-negative and CD16-negative with human and monoclonal antibodies whereas her lymphocytes express the CD16 molecule. Expression of other markers were comparable to the controls. Flow cytometric analysis showed that maternal antibody recognized the granulocytes but not the lymphocytes from blood bank donors and that its binding was decreased on normal, phospholipase C-treated, granulocytes. The binding of commercial CD16 monoclonal antibodies was also dramatically decreased on normal granulocytes pre-incubated with maternal serum. The CD16 specificity of the antibody was confirmed by negative reactions with another CD16-deficient granulocytes. This observation leads us to conclude that celllineage specific differences of CD16 molecules are recognized by the patient's antibody. Moreover, we confirm that the absence of the FcRIII (CD16) on granulocytes is not associated with any pathology or susceptibility to infections and that, in the children, the blockade of this receptor by the maternal antibody only led to moderate neutropenia.     

IgE response and its regulation in allergic diseases

The distinguishing feature of the allergic person is his or her elevation of serum IgE. This propensity to develop a sustained IgE response is determined genetically. The biologic effects of IgE are mediated via Fc receptors (Fc epsilon R) present on mast cells and basophils (Fc epsilon R type 1) and subpopulations of monocytes, macrophages, eosinophils, and platelets (Fc epsilon R type 2). Interaction of allergen with IgE on these cells results in receptor "bridging" and the release of histamine and other inflammatory mediators. Fc epsilon R type 2 on lymphocytes and monocytes are upregulated in atopic disease and may play a role in the allergic inflammatory reaction. The activation of B cells to synthesize IgE requires several stages (see Fig. 2). T cells play an important role in the regulation of IgE synthesis. In vitro activation of resting B cells to synthesize IgE requires direct cellular interaction with T cells or the presence of IL4 for activation. The latter effect is inhibited by alpha-interferon. Preactivated B cells are influenced in an isotype-specific manner by T-cell-derived IgE binding factors (IgE-BF), which may act as IgE-potentiating or IgE-suppressive factors, depending on their degree of glycosylation. The regulation of IgE synthesis is an important area of investigation. It provides us with an understanding of the basis of the human allergic response and ultimately may provide the basis for novel strategies in the treatment of allergic diseases.     

The tumor microenvironment and immune targeting therapy in pediatric renal tumors

This review highlights the role of several immunomodulating elements contributing to the tumor microenvironment of various pediatric renal tumors including Wilms tumor. The roles of innate and adaptive immune cells in renal tumors are summarized as well as immunomodulatory cytokines and other proteins. The expression and the predictive role of checkpoint modulators like PD-L1 and immunomodulating proteins like glypican-3, B7-H3, COX-2 are highlighted with a translational view toward potential therapeutic innovations. We further discuss the current state of preclinical models in advancing this field of study. Finally, examples of clinical trials of immunomodulating strategies such as monoclonal antibodies and chimeric antigen receptor T (CAR-T) cells for relapsed/refractory/progressive pediatric renal tumors are described.     

Neutrophil adhesion abnormality with deficient surface membrane proteins (gp 110 and p 98): the effect of their antibodies on the function of normal neutrophils

A sister and brother previously described with neutrophil adhesion defects and a lack of two neutrophil membrane proteins, glycoprotein with a molecular weight of 110 K and 115 K, were further studied. Rabbit polyclonal IgG antibodies were raised against neutrophil membrane proteins of approximately 110 K from normal individuals, and absorbed with the siblings' neutrophil membrane proteins. The antibodies thus absorbed reacted with two normal neutrophil membrane proteins, a 110 K glycoprotein and a 98 K protein, which were totally deficient in the siblings' neutrophils. The polyclonal antibodies were also reactive with a 95 K membrane protein of normal lymphocytes. The protein was missing in the siblings' lymphocytes. Three membrane proteins, Mac-1, LFA-1, and p 150, 95 each consisting of a specific alpha-subunit and a beta-subunit common to them, have been reported to be deficient in neutrophils from several patients with a neutrophil adhesion disease. Using monoclonal antibodies to Mac-1 alpha, LFA-1 alpha, and to the beta-subunit, it was deduced that the siblings' neutrophils lacked the above three membrane proteins. The disease in the siblings is thus identical with that previously described. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of immunoprecipitates, formed by reacting labeled normal neutrophil membrane proteins with our polyclonal antibodies, revealed three bands, almost identical with that of immunoprecipitates formed with the anti-beta-subunit monoclonal antibody. This finding indicates that the polyclonal antibodies reacted mainly with the beta-subunit of the membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initial characterization of the apoptosis-inducing receptor for natural human anti-neuroblastoma IgM

BACKGROUND: Human neuroblastoma (NB) cells contain a 260 kDa surface antigen (NB-p260), which serves as receptor for natural human IgM antibodies (anti-NB IgM). Upon binding to NB-p260, these antibodies induce apoptosis in human NB cells. PROCEDURE AND RESULTS: In this study, we purified NB-p260 to homogeneity from human LA-N-1 NB cells by sequential ion exchange chromatography followed by preparative SDS gel electrophoresis. Purified NB-p260 exhibited rapid autodegradation despite the presence of various protease inhibitors. The autodegradation process precluded extensive N-terminal sequencing. However, from repeat N-terminal sequence analysis, a consensus sequence of seven amino acid residues emerged that exhibited significant homology to the subunit c of the human mitochondrial ATP synthase, a hydrophobic membrane protein of 7.6 kDa. Western blot analyses demonstrated that purified NB-p260 was recognized by polyclonal antibodies raised against both subunit c-containing storage bodies and a synthetic peptide consisting of amino acid residues 32-45 of subunit c. In addition to peptide sequences related to subunit c, NB-p260 also contained epitopes related to the human heat shock protein HSP90. In Western blots, a monoclonal anti-HSP90 antibody reacted with purified NB-p260 as well as with a predominant protein fragment of approximately 90 kDa that appeared during the process of NB-p260 autodegradation. The anti-HSP90 antibody was also capable of binding to the surface of LA-N-1 cells and inhibiting the binding of human anti-NB IgM in a dose-dependent manner. CONCLUSIONS: Collectively, our data suggest that NB-p260, the apoptosis-mediating receptor for natural human anti-NB IgM, represents a novel surface protein of human NB cells containing polypeptide sequences related to the subunit c of the mitochondrial ATP synthase and the heat shock protein HSP90.     

IgE receptor-bearing lymphocytes in allergic and nonallergic children

Using a monoclonal anti-human IgE receptor (Fc epsilon R) antibody, the percentage of Fc epsilon R(+) cells among peripheral blood lymphocytes in children with or without allergic disorders was determined. The percentage of Fc epsilon R(+) cells in 63 nonallergic children was 4.3 +/- 1.5%, which did not vary with age and was equal to that of adults (4.2 +/- 1.2%). Allergic younger children (0-2 yr) showed a significantly higher percentage of Fc epsilon R(+) cells (7.7 +/- 3.0%) than nonallergic younger children (0-2 yr) (4.0 +/- 1.3%, p less than 0.001). Similarly, in allergic younger children, serum IgE levels (geometric mean = 58.9 IU/ml) were also significantly higher than those of nonallergic younger children (geometric mean = 2.0 IU/ml) (p less than 0.01). A positive correlation between the percentages of Fc epsilon R(+) cells and serum IgE levels was observed (Spearman rank = 0.88, p less than 0.01] in eight allergic younger children (0-2 yr) with serum IgE levels higher than 100 IU/ml. The increase in the percentage of Fc epsilon R(+) cells in allergic younger children (0-2 yr) was not a secondary phenomenon caused by serum IgE because serum IgE levels in these children were much lower than the concentration at which IgE enhance Fc epsilon R expression on lymphocytes. In conclusion, Fc epsilon R(+) lymphocytes may play a regulatory role in IgE synthesis in allergic younger children (0-2 yr).     

Developmental biology of the dendritic cell system

Aim : To determine whether an imbalance of dendritic cell subsets might contribute to diminished adaptive host responses observed in newborn infants. It was hypothesized that the proportion of lymphoid dendritic cells would be greater than that of myeloid dendritic cells in cord blood. Methods : To investigate this, dendritic cell subsets were evaluated in whole cord blood by flow cytometry. Circulating dendritic cells were also isolated from cord blood based on CD1c and BDCA-2 expression. Myeloid dendritic cells were also obtained by culturing cord and adult blood monocytes. Surface phenotypes of these cells were determined by flow cytometry using monoclonal antibodies directed against lineage, major histocompatibility, adhesion, co-stimulation and cytokine receptor molecules. Antigen-presenting functions of dendritic cell subsets were determined by mixed leukocyte reactions. Results : Circulating myeloid dendritic cells were higher in cord blood than previously reported in adult blood, whereas lymphoid dendritic cell numbers were similar between cord and adult blood. Expression of CD11c, CD45RA and CD45RO did not accurately differentiate between dendritic cell subsets circulating in cord blood. Fresh and culture-derived cord blood myeloid dendritic cells stimulated adult allogeneic leukocyte proliferation, while lymphoid dendritic cells were less effective inducers of an adult allogeneic leukocyte response. Culture-derived dendritic cells induced modest autologous cord blood leukocyte proliferation, but freshly isolated myeloid and lymphoid dendritic cells did not stimulated autologous leukocytes.
Conclusion : Contrary to the hypothesis, an imbalance in the ratio of circulating myeloid to lymphoid dendritic cell subsets does not exist and, therefore, does not contribute to diminished adaptive immune responses in newborn infants.     

Developmental biology of the dendritic cell system

AIM: To determine whether an imbalance of dendritic cell subsets might contribute to diminished adaptive host responses observed in newborn infants. It was hypothesized that the proportion of lymphoid dendritic cells would be greater than that of myeloid dendritic cells in cord blood. METHODS: To investigate this, dendritic cell subsets were evaluated in whole cord blood by flow cytometry. Circulating dendritic cells were also isolated from cord blood based on CD1c and BDCA-2 expression. Myeloid dendritic cells were also obtained by culturing cord and adult blood monocytes. Surface phenotypes of these cells were determined by flow cytometry using monoclonal antibodies directed against lineage, major histocompatibility, adhesion, co-stimulation and cytokine receptor molecules. Antigen-presenting functions of dendritic cell subsets were determined by mixed leukocyte reactions. RESULTS: Circulating myeloid dendritic cells were higher in cord blood than previously reported in adult blood, whereas lymphoid dendritic cell numbers were similar between cord and adult blood. Expression of CD11c, CD45RA and CD45RO did not accurately differentiate between dendritic cell subsets circulating in cord blood. Fresh and culture-derived cord blood myeloid dendritic cells stimulated adult allogeneic leukocyte proliferation, while lymphoid dendritic cells were less effective inducers of an adult allogeneic leukocyte response. Culture-derived dendritic cells induced modest autologous cord blood leukocyte proliferation, but freshly isolated myeloid and lymphoid dendritic cells did not stimulated autologous leukocytes. CONCLUSION: Contrary to the hypothesis, an imbalance in the ratio of circulating myeloid to lymphoid dendritic cell subsets does not exist and, therefore, does not contribute to diminished adaptive immune responses in newborn infants.     

Immunodiagnosis and immunotherapy of childhood malignancies

Recent advances in immunologic techniques have allowed the generation of monoclonal antibodies against antigens on tumor cells and their normal counterparts. Monoclonal antibodies useful for diagnosing and defining subtypes of acute leukemias and neuroblastoma have been prepared, although the prognostic significance of the subtypes defined by such antibodies remains to be determined. The usefulness of these reagents for therapeutic purposes either ex vivo, in association with autologous bone marrow transplantation, or in vivo, as carriers of cytotoxic agents, is currently under investigation.     

Central role for CD40/CD40 ligand (CD154) interactions in transplant rejection

Major advances have been made in understanding the expression and function of CD40 and its ligand CD154. It is now clear that CD40/CD154 interactions are critical in many aspects of the immune response, including T cell activation, T cell-dependent macrophage activation, T cell-B cell interactions and endothelial activation. Moreover, increasing evidence supports a central role for CD40/CD154 interactions in the immune processes of allograft rejection. Functional studies using blocking monoclonal antibodies have revealed beneficial effects of interupting CD40/CD154 co-stimulation in animal models of transplantation, particularly in association with interuption of the CD28/B7 pathway. A next step is to develop new therapeutic approaches to interrupting this pathway in humans, either through the development of receptor antagonists or through the understanding of intracellular signaling pathways utilized by these molecules.     

Update on therapeutic monoclonal antibodies

Monoclonal antibodies are among the most important class of drugs introduced into the therapeutic armamentarium since the introduction of antimicrobials in the 1930s. The first therapeutic monoclonal antibody, the anti T-cell monoclonal antibody OKT4, was licensed in 1986. Since then, 18 additional antibodies have been licensed in the US, with many more in the pipeline. Before 1986, many monoclonal antibodies were available for laboratory studies, notably to identify specific cells in the blood and tissues. This is best illustrated by the cluster designation (CD) system for antigens present on hematopoietic cells, now numbering over 200.     

Presence of endopeptidase 24.11 during the ontogeny of the small intestine in different species

The presence of endopeptidase 24.11 has been demonstrated at different periods of development in the small intestine of rat, rabbit and human, by immunoblotting using monoclonal antibodies raised against the rabbit kidney enzyme. The kinetic characteristics of the rabbit intestinal enzyme were determined after its purification by immunoaffinity column using the same antibodies. The endopeptidase 24.11 appears in the suckling rabbit as a low-affinity, high-capacity form, suggesting a role of this enzyme in the extracellular digestion of proteins in the young animal.     

Immunological abnormalities in a patient with lysinuric protein intolerance

We report an 8-year-old girl with lysimuric protein intolerance and immunological abnormalities including impaired function of lymphocytes, the presence of LE cells, antinuclear antibodies, and hypergammaglobulinaemia. These abnormalities have not been reported before and may be due to an amino acid imbalance or protein malnutrition in cells or tissues. The coincidence of a pre-stage of systemic lupus erythematosus (SLE) and LPI is not excluded.Abbreviations LPI lysinuric protein intolerance - SLE systemic lupus erythematosus - ANA antinuclear antibodies - PHA phytohaemagglutinin - Con A concanavalin A - ADCC antibody-dependent cell-mediated cytotoxicity     

Expression of C-kit in Ewing Family of Tumors: A Comparison of Different Immunohistochemical Protocols

Ewing sarcoma is a small round blue cell tumor with a high incidence of metastasis and poor survival. The tyrosine kinase receptor, c-kit, is a growth factor receptor that is expressed in a variety of tumors including Ewing sarcoma. Blockade of c-kit by imatinib mesylate (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, NJ) has been successfully used in the treatment of chronic myelogenous leukemia and gastrointestinal tumors. Detection of c-kit expression in Ewing sarcoma indicates a possible role of c-kit in tumor progression and a potential use of anti-c-kit therapy in Ewing sarcoma. Ki-67 is a proliferation marker found at all stages of the cell cycle. Expression of c-kit and Ki-67 was studied in 17 patients with Ewing sarcoma. Sections from paraffin-embedded tumor samples were immunostained, using standard immunohistochemical protocols, with c-kit and Ki-67 monoclonal antibodies, polyclonal c-kit antibody without antigen retrieval, and c-kit polyclonal antibody with antigen retrieval. Eleven out of 17 cases (65%) stained with c-kit monoclonal antibody; the staining was diffuse in 6/17 (35%) cases. C-kit expression did not correlate with Ki-67 proliferation rates. Using the polyclonal c-kit-antibody without antigen retrieval methods, c-kit expression was demonstrated in 1/11 (9%) cases. Incorporating antigen retrieval methods, c-kit expression increased to 53%. Concordance between monoclonal antibodies in detecting c-kit expression was observed in 12/17 cases (71%). We conclude that c-kit is variably expressed in Ewing sarcoma, using either monoclonal or polyclonal antibodies. Detection of c-kit expression in Ewing sarcoma improves with the use of antigen retrieval methods.