Effect of intravenous immunoglobulin administration on erythrocyte and leucocyte parameters.

BACKGROUND: Intravenous immunoglobulins (IVIG) are an attractive therapeutic tool for therapy of toxic epidermal necrolysis and severe forms of certain autoimmune diseases, including dermatomyositis, autoimmune blistering diseases, systemic vasculitis and lupus erythematodes. OBJECTIVES: Prompted by a case of IVIG-associated haemolytic anaemia, the effects of IVIG administrations on haematological parameters in patients with dermatological conditions were investigated. METHODS: Erythrocyte and leucocyte parameters were retrospectively analysed in 16 patients who had received IVIG at doses from 1 to 3 g/kg bodyweight (n = 35 cycles). The influence of IVIG on leucocyte survival was determined in vitro. RESULTS: Decreased absolute erythrocyte numbers, haemoglobin and haematocrit levels and a case of haemolytic anaemia were linked to transfusion of high-, but not low-dose IVIG. In contrast, leucopenia post-IVIG occurred in the vast majority of the recipients, unrelated to the administered IVIG amounts. In vitro investigations revealed a dose-dependent impairment of cell survival by IVIG in the neutrophil and monocyte, but not in the lymphocyte subpopulations. In several IVIG preparations, substantial amounts of blood group anti-A/anti-B antibodies were detected which could have accounted for the observed changes in the haematological parameters in our study cohort. CONCLUSIONS: IVIG products should be administered strictly according to indications. Commercially available IVIG products can contain blood group-specific antibodies that may induce haemolysis in some recipients. Monitoring of blood counts during applied IVIG therapy, especially when high doses are administered, is recommended.

Molecular characterization of major histocompatibility complex class II gene expression and demonstration of antigen-specific T cell response indicate a new phenotype in class II-deficient patients.

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency. The disease is known as bare lymphocyte syndrome (BLS). BLS is characterized by a lack of constitutive MHC class II expression on macrophages and B cells as well as a lack of induced MHC class II expression on cells other than professional antigen-presenting cells (APCs) due to the absence of mRNA and protein of the human leukocyte antigen (HLA) class II molecules, designated HLA-DR, -DQ, and -DP. The defect in gene expression is located at the transcriptional level and affects all class II genes simultaneously. Here we have analyzed transcription and protein expression of class II antigens in Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines and mononuclear cells (MNCs) of twin brothers. Whereas flow cytometric analysis failed to detect class II antigens on the cell surface of the patients' EBV-B cells and MNCs, examination of the genes coding for HLA-DR, -DQ, -DP, and the invariant chain (Ii) by reverse transcriptase-polymerase chain reaction amplification resulted in an unusual mRNA pattern in the B cell lines of the patients (HLA-DR alpha +, -DR beta, -DQ alpha +, -DQ beta -, -DP alpha -; -DP beta +, Ii+). In accordance with these findings no HLA-DR beta-specific protein was detected by immunoblotting, whereas low levels of HLA-DR alpha and normal levels of Ii were present. In contrast to EBV-B cells, the MNCs of both patients displayed a residual HLA-DR beta, -DQ beta, and -DP alpha mRNA signal. Furthermore, HLA-DR beta-specific protein was found in addition to HLA-DR alpha by immunoblotting of cell lysates, even though it was clearly decreased as compared with controls. Our results indicate that the defect in class II antigen expression is not necessarily present to the same extent in B cells and cells of other lineages. mRNA levels of HLA-DR beta were found to be enriched in adherent cells within the MNC fraction. Further investigations indicated that the MHC class II expressed is functional in antigen presentation, as the two boys' CD4+ T cells became activated and expressed interleukin-2R after stimulation of peripheral blood mononuclear cell cultures with recall antigen (tetanus toxoid). Furthermore, T cells tested in one of the two patients responded to both MHC class I and II allostimulation, and this response was inhibited by monoclonal antibodies of the respective specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Defective TCR surface expression associated with impaired TCR beta-chain assembly in a patient with cutaneous T-cell lymphoma.

We report on a patient with cutaneous T-cell lymphoma (CTCL) of long-standing duration. Phenotypic analysis of his peripheral blood mononuclear cells revealed an increased CD4+ T-helper subset and a decreased CD8+ cytotoxic T-cell population. Eighty-three to ninety-three percent of the patient's CD4+ T cells in the peripheral blood and 70% of the CD4+ T cells in the lesional skin lacked surface expression of the TCR/CD3 complex and showed a clonal rearrangement pattern of the TCR gamma-chain gene (V11-J1/J2). The lack in TCR surface expression correlated with defective assembly of the TCR beta-chain. Although mRNA for the TCR constant region beta 1 was found in the patient's purified CD4+ TCR-CD3- T cells, no intracytoplasmic TCR beta protein was detectable. In contrast, the patient's purified CD4+ TCR-CD3- T cells not only expressed mRNA specific for the TCR alpha-chain and for all CD3 chains, but intracytoplasmic TCR alpha and CD3 epsilon proteins could also be found. The lack of TCR beta protein clearly explains the defective surface expression of the TCR/CD3 complex in the patient's malignant T cells.

Nitrocellulose particles adsorbed to immunoglobulins are a new and effective approach to induce cell activation dependent on receptor aggregation.

Nitrocellulose (NC) has proved to be a versatile tool for the isolation and characterization of various biomolecules. In this report we extend its scope by using antibody-coated NC particles to cross-link molecules on the surface of living cells. Ligation of receptors in Jurkat cells with NC-bound specific antibodies induced protein tyrosine phosphorylation patterns of cellular proteins comparable to conventional antibody cross-linking. In addition, the present study shows that application of NC particles coated with human IgA significantly activated monocytic cells via the Fc alpha receptor (Fc alphaR), whereas cross-linking of receptor-ligand complexes with isotype-specific antibody was less efficient. Subsequent immunoprecipitation and immunoblot analysis of aggregated Fc receptors (FcRs) complexed to Ig-adsorbed particles permits fast identification of molecules involved in the transmission of signals. Therefore, ligand-coated NC particles can be used to examine receptor-mediated cell activation events dependent upon extensive receptor aggregation.

Immunization of chimpanzees with the HIV-1 glycoprotein gp160 induces long-lasting T-cell memory.

The goal of the present study was to investigate the antigen-specific T-cell response to the recombinant HIV envelope glycoprotein (gp160) and to test the effect of various adjuvant formulations on the efficiency of T-cell priming as well as on magnitude and longevity of the gp160-specific T-cell response. Our studies revealed that, in combination with an appropriate adjuvant (lipid-based adjuvant or mineral carrier complex), immunization with recombinant gp160 led to the appearance of gp160-primed T cells. The T-cell response obtained was substantial (proliferative response of greater than 100,000 delta dpm after one primary and two booster immunizations), gp160-specific (proliferation only in response to gp160, no proliferation after addition of a mock gp160 preparation), and long-lasting (T cell responses of greater than 50,000 delta dpm were observed more than one year after the last booster). The results presented here differ from those of previous studies in that they show the presence of substantial and long-lasting T-cell memory toward the immunogen gp160. Therefore further investigations on the use of these preparations as HIV candidate vaccines appear to be justified.

Transient increase in mitogen-induced lymphoproliferative responses in patients with testicular cancer after BEP chemotherapy.

OBJECTIVES: To investigate the impact of polychemotherapy on cellular immunity in patients with testicular cancer. METHODS: Lymphocyte subpopulations, lymphoproliferative responses to mitogenic stimulation, and mitogen-induced release of soluble interleukin-2 receptor from peripheral blood mononuclear cells were investigated in 15 patients with testicular germ cell tumors a median of 61 months (range 7 to 73) after polychemotherapy with bleomycin, etoposide, and cisplatin (BEP). RESULTS: The numbers of peripheral blood T cells (CD3+), CD4+ and CD8+ subsets, and lymphoproliferative responses to pokeweed mitogen, phytohemagglutinin, and concanavalin A in patients were comparable to those of healthy control subjects. When two groups of patients were formed according to elapsed time from BEP polychemotherapy and study onset (group A, 12 months and group B, 69 months after termination of BEP), a significant increase in lymphoproliferative response to concanavalin A (P <0.05) was found in group A 1 year after chemotherapy. CONCLUSIONS: BEP chemotherapy administered to patients with testicular cancer does not result in impairment of cellular immunity but rather leads to a significant increase in the capacity of patients' lymphocytes to respond to mitogenic stimulation up to 1 year after polychemotherapy. Moreover, the increased T-cell activity found after BEP therapy may contribute to the high rate of long-term complete remission.

Immunomodulatory effect of immunoglobulins.

IVIG is clearly indicated as the treatment of choice on the basis of large clinical trials in a number of inflammatory and autoimmune diseases, e.g. Kawasaki disease, ITP, Guillain-Barré syndrome, etc. According to in vitro studies various mechanisms have been identified whereby IgG could modify immunologically mediated and inflammatory diseases. Fc-receptor blockade as well as true down-modulation of Fc-receptors, acting as a sump for activated complement components, have been demonstrated at the cellular level and in experimental animals. The possibility of interfering with the idiotype network has been discussed in connection with autoimmune diseases. Down-regulation of inflammatory cytokines as well as an increase in the production and release of IL-1 receptor antagonist appears to be of importance in inflammatory processes. Clinical studies have proven the efficacy of IVIG. Basic research has demonstrated its possible mechanisms of action; however, the question of exactly which mechanisms are responsible for the clinical efficacy in certain diseases still awaits clarification.

Common variable immunodeficiency: clinical aspects and recent progress in identifying the immunological defect(s).

Common variable immunodeficiency (CVID) comprises a heterogeneous group of patients with as yet undefined genetic defects. Patients with CVID have in common a decrease in the levels of one or more serum immunoglobulin isotypes and a severe defect in the production of specific antibodies. Typically, the patients suffer from recurrent infections of the upper and lower respiratory tract or the gastrointestinal tract. In consequence of these infections patients may develop severe organ damage, such as chronic pulmonary disease with bronchiectases, leading to pulmonary failure. Early diagnosis of CVID is important, as antibody deficiency can efficiently be treated by regular intravenous IgG (IVIG) substitution therapy. IVIG therapy prevents the occurrence of further acute infectious episodes and the development of long-term complications. The basic immunological defect(s) in patients with CVID are still unknown. There is currently no convincing evidence for an intrinsic B-cell defect in patients with CVID. A defect in T-cell activation due to impaired signal transduction upon T-cell receptor triggering has been described in a large subgroup of patients with CVID. Defective T-cell activation may lead to an impairment in cognate T-B-cell interaction due to impaired expression of CD40 ligand and/or abnormalities in the production T-cell-derived cytokines required for fully functional B-cell activation, proliferation and/or differentiation which could indeed explain the impairment in antibody production present in CVID patients.

The anti-inflammatory effect of an oral immunoglobulin (IgA-IgG) preparation and its possible relevance for the prevention of necrotizing enterocolitis.

An exaggerated release of inflammatory mediators has been implicated in the pathogenesis of necrotizing enterocolitis (NEC). Oral administration of a human immunoglobulin preparation (serum IgA-IgG) has been demonstrated to be an effective prophylaxis for NEC. The aim of the present study was to examine the regulatory effect of a human IgA-IgG preparation on the release of inflammatory cytokines in human monocytes. Our results indicate that the immunoglobulin preparation inhibits TNF-alpha and IL-6 release in monocytes following stimulation with heat-inactivated Hib in a dose-dependent manner. This might have a biological relevance in infants receiving oral immunoglobulin prophylaxis for NEC, since modulation of the release of inflammatory mediators at the level of the gastrointestinal mucosa could interfere with the development of noxious sequelae of acute and/or chronic inflammation initiated by microbial pathogens or their toxins that finally lead to the pathologic changes associated with NEC.

Heterozygous promotor haplotype LXA/LYB in MBL-deficiency associated with myopathy and left ventricular hypertrabeculation/noncompaction.

AIM: To report the genetic background of mannose-binding lectin (MBL)-deficiency in a patient with recurrent infections, cardiac disease, and myopathy. METHOD: Case report. RESULTS: In a 47-year-old male with recurrent respiratory infections, MBL-deficiency was diagnosed. He additionally had developed left bundle-branch-block, ventricular runs, and dilative cardiomyopathy. Left ventricular (LV)-hypertrabeculation and intra-myocardial calcifications were detected earlier. At age 44 years, unclassified myopathy, manifesting as easy fatigability, myalgias, and ptosis was diagnosed. After death from a sepsis with Staphylococcus aureus, autopsy revealed endocardial fibrosis and calcification, located over the compacted as well as non-compacted segments. The patient carried the heterozygous haplotype LXA/LYB in the MBL gene. MBL-deficiency was considered responsible for recurrent pulmonary infections and sepsis. The association between MBL-deficiency, LV-hypertrabeculation, endocardial fibrosis, and calcification remains speculative. CONCLUSIONS: MBL-deficiency due to the LXA/LYB genotype may be associated with recurrent pulmonary infections and fatal sepsis. Endocardial fibrosis and calcification results rather from LV-hypertrabeculation than MBL-deficiency.

Prevention of necrotizing enterocolitis in low-birth-weight infants by IgA-IgG feeding.

In a randomized clinical trial, we evaluated the efficacy of an oral immunoglobulin preparation (73 percent IgA and 26 percent IgG) in reducing the incidence of necrotizing enterocolitis in infants of low birth weight for whom breast milk from their mothers was not available. A total of 434 infants weighing between 800 and 2000 g were eligible for entry in the study. Of these, 255 were withdrawn - 234 during the first week of the study because breast milk from their mothers became available (123 in the treatment group and 111 in the control group), and 21 because of violations of protocol or because breast milk became available after the first week. The duration of follow-up was 28 days. Among the infants for whom breast milk did not become available during the study, there were no cases of necrotizing enterocolitis among the 88 receiving oral IgA-IgG, as compared with six cases among the 91 control infants (P = 0.0143). Of the infants withdrawn from the study, two assigned to the control group had necrotizing enterocolitis. We conclude that the oral administration of IgA-IgG may prevent the development of necrotizing enterocolitis in low-birth-weight infants.

Prophylaxis of necrotizing enterocolitis by oral IgA-IgG: review of a clinical study in low birth weight infants and discussion of the pathogenic role of infection.

Necrotizing enterocolitis, a severe gastrointestinal disease in the neonatal period, affects primarily premature infants. Perinatal complications that predispose the neonate to systemic hypoxia are frequent in infants with necrotizing enterocolitis. Ischemia of the intestinal mucosa may facilitate the invasion of enteric microorganisms in stressed low birth weight infants. Geographical and temporal clustering of outbreaks of the disease and the termination of epidemics by standard infection control underline the importance of infectious agents in the development of this disease. Several studies have established the immunoprotective effect of orally administered antibodies against infection of the gastrointestinal mucosa in children and adults. Anecdotal evidence suggested that feeding of human immune globulin might have a positive effect on the incidence of necrotizing enterocolitis in premature infants. This paper reviews a prospective, randomized, controlled trial of the efficacy of an oral immune globulin preparation (published in detail in the New England Journal of Medicine, Vol. 319, pp 1-7, 1988) and discusses the pathogenic role of infection in necrotizing enterocolitis.

Physical and functional association of Fc alpha R with protein tyrosine kinase Lyn.

In this report, we show that the Src family nonreceptor protein tyrosine kinase (PTK) Lyn associates with aggregated IgA Fc receptor (Fc alpha R) in the monocytic cell line THP-1. Receptor aggregation and subsequent immunoprecipitation of receptor complexes with huIgA adsorbed to nitrocellulose particles shows that Lyn associates with Fc alpha R by a mechanism sensitive to short treatment with the Src family-selective inhibitor PP1. However, interaction of Lyn with IgG Fc receptor (Fc gamma R) in THP-1 cells was unaffected by short treatment with the PTK inhibitor. Cross-linking of Fc alpha R induced tyrosine phosphorylation of several cellular proteins, including p72Syk, which appears to be a major target of early PTK activity. Unexpectedly, in vitro kinase assays showed that Fc alpha R aggregation-induced tyrosine phosphorylation of Syk did not result in upregulation of Syk activity. Despite the lack of enhanced Syk kinase activity, downstream signaling after Fc alpha R cross-linking was functional and induced the release of significant amounts of interleukin-1 receptor antagonist and interleukin-8. The induction of cytokine release was completely blocked by PP1, thus confirming the biological significance of the association of Lyn with aggregated Fc alpha R. Our data show that early signal transduction after Fc alpha R cross-linking as well as Fc alpha R-mediated activation of cellular effector functions depends on Src family kinase activity. The Src-family PTK involved in Fc alpha R-mediated tyrosine phosphorylation appears to be Lyn, which coprecipitated with aggregated Fc alpha R complexes.

Method for the isolation of biologically active monomeric immunoglobulin A from a plasma fraction.

A purification method for immunoglobulin A (IgA) yielding monomeric IgA with a purity of over 97% has been developed. This procedure uses ethanol-precipitated plasma (Cohn fraction III precipitate) as the starting material and includes heparin-Sepharose adsorption, dextran sulfate and ammonium sulfate precipitation, hydroxyapatite chromatography, batch adsorption by an anion-exchange matrix and gel permeation. Additional protein G Sepharose treatment leads to an IgA preparation of greater than 99% purity. The isolated IgA presented with an IgA subclass distribution, equivalent to IgA in unfractionated plasma, and was biologically active, as was shown by its ability to down-modulate Haemophilus influenzae-b-induced IL-6 secretion of human monocytes.

Cell-mediated immune functions in a patient with MHC class II deficiency.

This report focuses on cell-mediated immune functions in a patient with MHC class II deficiency. The patient described presented with a case of "classical" MHC class II deficiency (T and B cells within the normal range, normal lymphocyte proliferation in response to stimulation with mitogens, gene encoding for MHC class II present, no expression of MHC class II). The absence of MHC class II expression resulted in an incapability of the patient's antigen-presenting cells to function as accessory cells in the presentation of soluble protein antigens, while accessory functions required for the induction of alloantigen-induced lymphocyte proliferation or for the generation of cytotoxic T cells in response to an allostimulus were normal. The patient's T cells responded normally to alloantigenic stimulation and also had the capacity to develop antigen-specific cytotoxic functions. However, the T cells were completely naive with respect to activation by soluble protein antigens, even after presentation by accessory cells derived from the patient's healthy histoidentical brother. In this context it was interesting to note that the patient's CD4-positive cells showed a normal pattern of expression of the 4B4 marker, a marker generally present on memory T cells. These data make it tempting to speculate that in the absence of MHC class II, other cell surface structures may at least partially take over immune functions normally under the control of the MHC class II complex.

Effect of polymeric IgG on human monocyte functions.

Fc and iC3b receptors are involved in various biological functions of phagocytic cells, such as immune adherence and phagocytosis of opsonized particles, degranulation and superoxide generation. In the present study we examined the expression of specific receptors for the Fc portion of IgG (FcR) and for iC3b (CR3), a cleavage product of the third complement component, on human monocytes following in vitro treatment with polymeric and monomeric IgG. Interaction of polymeric IgG (fluid phase) with the monocyte membrane led to a concomitant modulation of both Fc and iC3b receptors. Monomeric IgG, however, down modulated Fc receptor expression only if surface bound. Under these conditions, no concomitant modulation of the iC3b receptor could be observed. The down modulation of Fc and iC3b receptors induced by fluid-phase IgG polymers was also accompanied by a decrease in monocyte functions as expressed by reduced Fc receptor-mediated phagocytosis, decreased release of oxygen metabolites following stimulation by aggregated IgG and opsonized zymosan, as well as in impaired killing of bacteria. These data suggest that a down modulation of Fc and iC3b receptors might have important implications for host defense mechanisms, since interaction with these receptors is required for the proper elimination of many pathogens.

Phenotypes of peripheral blood lymphocytes during acute hepatitis A.

We investigated peripheral blood lymphocyte phenotypes of 74 patients at weekly intervals during the course of acute hepatitis A. In the second week after onset of jaundice, a significant elevation of total lymphocytes was observed (4,096 X 10(6) +/- 1,003 X 10(6)/l vs. controls 3,038 X 10(6) +/- 1,208 X 10(6)/l, p less than 0.005). However, no change in the relative percentage of B-cells (CD20+), T-cells (CD3+ or CD2+), or T-cell subpopulations (CD4+ helper cells and CD8+ suppressor cells) could be demonstrated during the course of the disease. Activated T-cells (CD3+ DR+) were elevated during the first week (204 X 10(6) +/- 134 X 10(6)l vs. normal 91 X 10(6) +/- 54 X 10(6)/l, p less than 0.005) and during the second week (202 X 10(6) +/- 82 X 10(6)/l, p less than 0.0005) after onset of disease and returned to normal values until the third week. Cells expressing phenotypes of lymphocytes capable of exerting non-MHC-restricted cellular cytotoxicity, i.e. Natural Killer cell activity (CD57+, CD16+, and CD56+) were significantly elevated in percentage in the first week of disease, as compared to controls (CD57: 14.5 +/- 7.0% vs. 9.3 +/- 5.8%, p less than 0.05; CD16: 13.4 +/- 7.3 vs. 9.5 +/- 5.1%, p less than 0.05; CD56: 10.5 +/- 3.5% vs. 8.0 +/- 1.5%, p less than 0.005). Also the absolute numbers of these lymphocyte subpopulations were found to be elevated during the first and second week. The increase in NK cells in the initial phase of acute hepatitis A suggests an important role of these cells in the first line of defence in this disease.