Deficiency of the expression of CD45RA isoform of CD45 common leukocyte antigen in CD4+ T lymphocytes in children with infantile cholestasis.

The immunological background of the pathological changes that appear in infantile cholestasis (infections, inflammatory process in the liver) is largely unknown. With the use of double color flow cytometry, we assessed the distribution of functionally different lymphocyte subpopulations in the peripheral blood of 29 infants with extra and intra-hepatic cholestasis (12 and 17 patients, respectively), aged from 1 to 8.6 months. Control group consisted of 15 age-matched, healthy infants. We examined: (1) the expression of CD3, CD4, CD8, CD19 lymphocyte surface receptors; and (2) the distribution of lymphocyte subsets with distinctive surface Ag characteristics of 'naive' (CD45RA+) and 'memory' (CD45RO+) cells in both CD4+ and CD8+ cell populations. The surface markers expression was evaluated in terms of percentage of positive cells and receptor density. The following changes in the expression of lymphocyte surface markers are described: (1) a decrease in the percentage of total CD3+, CD4+ cells but normal percentage of CD8+ cells and elevated proportion of CD19+ B cells; (2) a reduction of the proportion of 'naive' CD4+ lymphocytes but normal percentage of 'naive' CD8+ as well as 'memory' CD4+ and CD8+ cell subsets; (3) a decrease in density of CD3, CD4+, CD8 receptors, and D45RA isoform in a subset of 'naive' CD4+ cells. We conclude that deficiency of 'naive' CD4+ T cell subset which possess important effector and immunoregulatory functions, and low expression of certain lymphocyte receptors known to be engaged in T cell activation, possibly reflect a defect of cell mediated immunity that may account for viral and bacterial infections, often observed in infants with cholestasis.

Effect of Pseudomonas aeruginosa exotoxin A on CD3-induced human T-cell activation.

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.

Effect of Pseudomonas aeruginosa exotoxin A on IFN-gamma synthesis: expression of costimulatory molecules on monocytes and activity of NK cells.

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis. Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis. Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma. P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.

Primary resistance to clarithromycin in clinical strains of Helicobacter pylori isolated from children in Poland.

Helicobacter pylori resistance to clarithromycin is an important factor in the failure of eradication therapy. The resistance results from point mutations in the 23S rRNA gene of H. pylori. The prevalence of primary resistance of H. pylori to clarithromycin in children and mutations associated with resistance were studied and it was found that 23.5% (23/98) of H. pylori strains isolated in our hospital during 1998-2000 were resistant to clarithromycin. The primary resistance was mainly caused by an A2143G mutation, but the isolates with an A2142G mutation had higher MICs for clarithromycin compared with those with an A2143G mutation: median MIC 256 versus 16 mg/l. Comparison of our data with previous results showed that the prevalence of H. pylori resistance to clarithromycin in children has increased in Poland over the last three years, however the difference was not significant (23.5 vs. 17%, P=0.22).

Reduction of CD45RA Isoform Expression and Decrease in CD4 and CD8 Receptor Density in Lymphocytes of Patients with Primary Biliary Cirrhosis.

BACKGROUND: The immunological background of primary biliary cirrhosis (PBC) remains largely obscure. METHODS: Using double colour flow cytometry, we estimated the distribution of functionally different lymphocyte subpopulations in the peripheral blood of 25 PBC patients and 18 controls. We examined: 1) the expression of CD3, CD4, CD8, CD 19 and CD56 surface receptors, 2) the distribution of lymphocyte subsets bearing 'naive' (CD45RA+) and 'memory' (CD45RO+) phenotypes in both CD4+ and CD8+ cell populations, 3) the expression of an early activation marker (CD69), 4) the distribution of C1.7 mAb binding cytotoxic effectors in CD3+, CD8+ and CD56+ cells. The surface marker expression was evaluated in terms of percentage of positive cells and receptor density. RESULTS: We found: 1) a decrease in the percentage of total CD3+ and CD4+ cells, an unchanged proportion of CD8+ cells but elevated proportion of CD 19+ cells and NK lymphocytes; 2) a reduction in the percentage of 'naive' CD4+ but normal proportion of 'naive' CD8+ as well as CD4+ and CD8+ 'memory' cell subsets; 3) a decrease in the density of CD4 and CD8 receptors in the subsets of 'naive' and 'memory' T cells, 4) an increase in the percentage of CD69 receptor bearing T cells but unchanged proportion of C1.7 mAb. CONCLUSIONS: It is concluded that the reduction in number of 'suppressor-inducer-like 'naive' CD4+ T-cell subsets in association with the decrease in fluorescence intensity for CD4 and CD8 may significantly contribute to the mechanisms that could account for a development of PBC.

Detection and quantitation of immune complexes by polyethylene glycol-laser nephelometer assay. I. Immunological study in chronic serum sickness in rabbits.

Polyethylene glycol (PEG)-laser nephelometer assay for the detection of circulating immune complexes (i.c.) has been developed. The method proved to be useful in the detection of i.c. in sera of rabbits with chronic serum sickness. I.c. were detected in 28 animal experimental sera and in samples with preformed immune complexes, but not in sera of 10 healthy rabbits and 5 rabbits prior to immunization. Laser nephelometer assay is a rapid, simple, quantitative screening test suited for clinical use as a first step in i.c. detection.

Effectiveness of treatment of children with hypogammaglobulinemia: comparison of fresh plasma and intravenous immunoglobulin preparation.

Eight children aged 2 to 16 years who were diagnosed as having primary humoral immunodeficiency were treated with fresh plasma substitution, during the first 2 years. Subsequently these children and 5 other children with primary humoral immunodeficiency (ID) were treated during the next 10 months with intravenous preparation of IgG. Eight children received doses of 500 mg/kg body weight (group A), while five children received doses of 150 mg/kg (group B) at monthly intervals. After five months of this therapy the average increase of IgG concentration was in group A--395 mg/dl, while in group B--100 mg/dl, the difference being statistically significant. Substitution therapy with intravenous IgG was more effective than therapy with fresh plasma and caused appreciable clinical improvement. Only in one out of 130 cases of intravenous injections mild post-transfusion reaction was observed.

Immune complexes in sera of children with HBV-mediated glomerulonephritis.

Multiple serum samples from 27 children with hepatitis B virus (HBV)--mediated glomerulonephritis (GN) were screened for the presence of immune complexes by an antigen-specific method. For this purpose an immunoenzymatic test was set up, applying a solid-phase Clq and the enzyme-conjugated antibodies: anti-HBs and anti-HBe. Complexes of HBsAg were found in sera of 15 children (55.6%), while complexes of HBeAg--in sera of 12 children (44.4%). Molecular weight of complexes was measured in sera of three patients, disclosing the values of 2.5-3.3 X 10(6) daltons for HBsAg complexes and 2.5-3.2 X 10(5) daltons for HBeAg complexes. Immune deposits consisting of hepatitis B virus antigen (HBeAg), IgG and C3 were detected in the glomeruli by immunoperoxidase and immunofluorescent assays respectively, in 3 out of 4 patients with membranous glomerulonephritis (MGN). No genetic defect of the complement system was found by the measurement of total haemolytic activity of complement and concentration of early complement components. From the analysis of clinical and laboratory data it was concluded that the appearance of HBeAg complexes correlated with more severe course of the disease.

Isolation and partial characterization of circulating immune complexes in sera of children with HBV-mediated glomerulonephritis.

Circulating immune complexes (CIC) containing HBsAg and HBeAg were identified in sera of 5 out of 6 children with hepatitis B mediated membranous glomerulonephritis. CIC were precipitated from sera by 3.5% PEG, washed and subsequently analysed after acid dissociation and trypsin digestion. HBsAg, anti-HBs and albumin; HBeAg, anti-HBe and anti-HBc were recovered from the isolated complexes and these findings are discussed. Analysis of 3.5% PEG mediated precipitate of human serum proteins showed the relatively high content of IgG classical pathway complement components: C1q, C4 and C3.

Immunopathology of hepatitis B mediated membranous glomerulonephritis.

The study was undertaken to establish the clinical and immunological aspects of HBV-mediated membranous glomerulonephritis in children. Out of 54 children with membranous GN treated in the Children's Memorial Hospital 51 children (94.4%) had the disease related to HBV infection. The inhibitory effect of immune complexes: HBsAg-IgG and HBeAg-IgG, isolated from sera of these children on lymphocyte proliferation of blood donors was observed. This may partly explain the status of immune tolerance towards HBV in the infected children.

Inherited defect of alpha-1-antitrypsin associated with chronic liver and respiratory tract diseases in children.

The frequency of occurrence of alpha-1-antitrypsin (A1AT) deficiency among total of 3228 Polish children with chronic liver diseases and chronic disease of respiratory tract was determined. It was observed that among children with chronic liver diseases which disclosed more frequent defect (concentration of A1AT below 150 mg/dl was found in 10.3% of children), the highest occurrence of deficiency was in children with neonatal hepatitis (23.1%). The deficiency was connected with the presence of ZZ and MZ phenotypes of A1AT.

Familial occurrence of C1-inhibitor deficiency and the effect of treatment.

Seventy one persons (67 children and 4 of their mothers) who came to the out-patient department of our hospital because of recurrent edemas of subcutaneous tissue and mucous membranes were the subjects of study. Serum haemolytic activity of complement and concentration of C1-esterase inhibitor, C1q, C4 and C3 were measured. Twenty six persons were related: altogether 12 families were studied, mostly sisters and brothers. In six patients (k 8.5% of the group studied) inherited type I defect of C1-inhibitor was detected and the diagnosis of angioneurotic edema confirmed. The results of complement determination in these patients (5 girls and 1 mother) were compared to the rest of the group and to healthy blood donors and showed clear differences in concentrations of C1-inhibitor, C1q, C4 and in haemolytic activity. Treatment consisted of fibrinolysis inhibitor, epsilon-aminocaproic acid and of antihistamine drugs. The value of this kind of therapy in the treatment of a very well responding child is discussed.

Immunoglobulin synthesis by lymphocytes of eight immuno-deficient children.

The group of patients consisted of 4 pairs of male siblings with the family history suggesting X-linked immunodeficiency. The concentration of serum immunoglobulins (Ig) and the titer of anti-E. coli and anti-Candida albicans antibodies was extremely low in comparison to the control group. Only 2 siblings showed normal number of lymphocytes with surface Ig. Four out of 8 children had the increased number of Fc-IgG bearing T cells. Blast transformation of lymphocytes revealed the decreased response to PHA and Con A in 2 children. Cellular immunoglobulin (cIg) synthesis of in vitro stimulated peripheral blood lymphocytes (PBL) was decreased in all 8 children. Prednisolone, when added to the PBL cultures, increased cIg synthesis in 2 children. Thymosin (TFX) caused an enhancement of cIg production by lymphocytes of 4 children. Cowan I--elicited stimulation of PBL was low in all but 2 children. Our data further support the view on the heterogeneity of etiopathogenesis of hypogammaglobulinemia.

Evaluation of cell-mediated and humoral immunity in children suffering from spinal muscular atrophy.

A study of certain parameters of cellular and humoral immunity of 56 children with spinal muscular atrophy (SMA), treated in Children's Memorial Hospital, was undertaken to explain the observed frequent and serious respiratory tract infections. The main differences between a group of patients and the control group were found in the serum IgA and IgM concentrations, a number of peripheral B cells, and in the response to skin tests. Among all the methods applied, the skin tests seem to provide the best information concerning the child immunity system. The observed abnormalities were almost parallel to the severity of the SMA course.

Effect of Pseudomonas aeruginosa exotoxin a on PHA-induced lymphocyte proliferation.

P. aeruginosa Exotoxin A induced a dose dependent suppression of PHA-stimulated human lymphocyte proliferation. The suppressor effect was not reversed by interleukin 2 (IL-2) added to the culture medium. Expression of IL-2 receptors (IL-2R) on PHA-blasts a well as their ability for IL-2 binding were decreased by Exotoxin A. Kinetic studies of IL-2 production showed that IL-2 activity was still present in cultures of lymphocytes activated for 72 hours with PHA and Exotoxin A. In contrast, supernatants of lymphocyte cultures activated with PHA and Exotoxin A for 18 hours had low activity of IL-2. Hence, Exotoxin A probably could decrease the utilization of IL-2 due to either inhibition of IL-2R expression or suppression of their ability for IL-2 binding.

Complement and circulating immune complexes in children with mumps meningitis.

Haemolytic activity of complement (CH50) and occurrence of circulating immune, complexes (CIC) were studied in serum samples from children with mumps meningitis taken at regular intervals. The decrease of CH50 was observed in 46 out of 77 samples (60%) in the Ist examination, between 1-6th day from the appearance of symptoms, in 43/67 samples (64%) in the IInd examination, between 12--16th day of disease, and in 7/22 samples (32%) in the IIIrd examination, between 12--14th month of observation since the appearance of symptoms. Typical changes of complement level during the disease were of such profile that the most pronounced decrease was in the Ist and IInd examination with the normalization in the IIIrd examination. The presence of CIC was found in 21%, 15% and 14% of samples in the Ist, IInd, and IIIrd examination respectively. Serum sample form the child on 5th day of disease, with high concentration of CIC (482 micrograms/ml), was subjected to sepharose 6B gel filtration on the calibrated column. The molecular weight of thus analyzed immune complexes was as follows: 2.5 X 10(6) and 5.0 X 10(5) daltons. CIC contained anti-mumps antibodies, determined by Elisa, in IgG, IgA and IgM classes.

The influence of D-Ala1-peptide T amide, an analogue of HIV glycoprotein 120 fragment, on the CD4--anti CD4 lymphocyte interaction.

D-Ala1-peptide T amide (DAPTA), synthetic analogue of the HIV glycoprotein 120 sequence, was used to study its binding to specific cellular receptor of HIV, CD4. The analogue contains eight aminoacid residues including 4 threonine residues; its molecular weight being 992. We have shown the temperature- and dose-dependent inhibitory effect of peptide T on the CD4 - anti CD4 binding. The strongest effect was noted at 37 degrees C with the peptide dose of 150 nM: 62% inhibition of binding. Other means of possible blocking of HIV attachment to the host cellular receptor are discussed.

Development of IgG subclasses in healthy Polish children.

To determine the ontogeny of IgG subclasses in healthy Polish children IgG1, IgG2, IgG3, IgG4 concentrations were measured by enzyme-linked immunosorbent assay in 30 cord blood sera and sera from 152 healthy children aged 4 months to 16 years. The geometric mean of concentrations all of IgG subclasses in age-related groups of children were calculated. In cord sera, four IgG subclasses were found with the IgG1 level reaching about 90%, IgG2--52%, IgG3--80% and IgG4--33% of the mean adult values. In the group of youngest children (4-6 months of age) the concentration of all IgG subclasses were significantly decreased in comparison to initial values. Then, IgG1 and IgG3 levels increased rapidly reaching ca. 50% of the mean adult values by 1 year of age. We also demonstrated much slower maturation of IgG2 and IgG4 which lagged behind IgG1 and IgG3 development, especially in younger children.

Immune complexes in children with different forms of glomerulonephritis.

The occurrence of circulating immune complexes was tested in 43 children with different forms of glomerulonephritis (GN). The presence of immune complexes was demonstrated in sera of 6 out of 10 patients with GN of hepatitis B etiology, in 6 out of 11 patients with GN of bacterial etiology, and in 3 out of 10 patients with GN of unknown etiology. Immune complexes were not found in sera of 3 patients with Schönlein-Henoch disease, in 6 patients with lipoid nephrosis, and in 3 patients with GN and hepatitis B as a coexisting infection. In a patient with membrane-capillary GN which appeared after infection with hepatitis B virus we demonstrated immune complexes of M.W. 4.0 X 10(6) and 7.5 X 10(5).

Deficiency of short-lived suppressor cells controlling T-lymphocyte colony formation in acute lymphoblastic leukemia.

Short-lived suppressor cell (SLSC) activity controlling T-lymphocyte colony formation in vitro was evaluated in patients with acute lymphoblastic leukemia (ALL). No SLSC activity was found in patients in acute phase of the disease. On the other hand, patients in remission frequently had higher SLSC activity than controls. No correlation between colony count and thymidine incorporation as well as T-cell numbers was found in ALL patients.