Abnormalities in the T and NK lymphocyte phenotype in patients with Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by spontaneous chromosomal instability with predisposition to immunodeficiency and cancer. In order to assess the cellular basis of the compromised immune response of NBS patients, the distribution of functionally distinct lymphocyte subsets in peripheral blood was evaluated by means of double-colour flow cytometry. The study involved the 36 lymphopenic patients with a total lymphocyte count < or =1500 microl (group A) and seven patients (group B) having the absolute lymphocyte count comparable with the age-matched controls (> or =3000 microl). Regardless of the total lymphocyte count the NBS patients showed: (1) profound deficiency of CD4+ and CD3/CD8+ T cell subsets and up to fourfold increase in natural killer (NK) cells, almost lack of naive CD4+ T cells expressing CD45RA isoform, unchanged percentage of naive CD8+ cell subset (CD8/CD45RA+) but bearing the CD8 receptor of low density (CD8low); (2) normal expression of CD45RA isoform in the CD56+ lymphocyte subset, profound decrease in alpha beta but up to threefold increase in gamma delta-T cell-receptor (TCR)-positive T cells; (3) shift towards the memory phenotype in both CD4+ and CD8+ lymphocyte subpopulations expressing CD45RO isoform (over-expression of CD45RO in terms of both the fluorescence intensity for CD45RO isoform and the number of positive cells); and (4) an increase in fluorescence intensity for the CD45RA isoform in NK cells population. These results indicate either a failure in T cell regeneration in the thymic pathway (deficiency of naive CD4+ cells) and/or more dominant contribution of non-thymic pathways in lymphocyte renewal reflected by an increase in the population of CD4+ and CD8+ memory cells, gamma delta-TCR positive T as well as NK cell subsets.

Heterogeneity of humoral immune abnormalities in children with Nijmegen breakage syndrome: an 8-year follow-up study in a single centre.

During an 8-year period of observation, defects of immune responses were characterized and monitored in 40 of 50 Polish children with Nijmegen breakage syndrome referred to the Children's Memorial Health Institute in Warsaw. The following parameters were determined at diagnosis: (1) concentrations of serum IgM, IgG, IgA; (2) concentrations of IgG subclasses; and (3) lymphocyte subpopulations. In addition, naturally acquired specific antibodies against Streptococcus pneumoniae were determined in 20 patients with a history of recurrent respiratory infections. During follow-up, total serum immunoglobulins and IgG subclasses were monitored systematically in 17 patients who did not receive immunomodulatory therapy. Moreover, anti-HBs antibody response was measured after vaccination of 20 children against HBV. We found that the immune deficiency in NBS is profound, highly variable, with a tendency to progress over time. Systematic monitoring of the humoral response, despite good clinical condition, is essential for early medical intervention.

Immunological abnormalities in children with biliary atresia.

Since orthotopic liver transplantation is the treatment of choice for bilary atresia, the role of nutritional support preceding this procedure is significant. The aim of this study was to assess the selected parameters of both humoral and cellular immunity before and after nutritional support. Eight children aged 1.08-7 years. with biliary atresia, qualified to LTx, received high-calorie standard diet supplemented with MCT oil. The distribution of functionally different lymphocyte subpopulations in the peripheral blood was evaluated using double color flow cytometry (EPICS-MCL, Coulter). The concentrations of total serum immunoglobulins were measured by nephelometry (Beckman Array 360) and concentrations of IgG subclasses by ELISA. Abnormalities in the expression of lymphocyte surface markers as well as in immunoglobulin synthesis were as follows: 1) decrease in the percentage of total CD3+ (4/8), CD4+ (5/8), CD8+ (3/8) cells and markedly elevated percentage of CD19+ B cells (4/8); 2) reduction of the proportion of 'naive' CD4+ and CD8+ lymphocytes but normal percentage of 'memory' CD4+ and CD8+ cell subsets; 3) hypergammaglobulinemia with especially high levels of IgG (16.0-3.05 g/l) and IgA (2.6-6.66 g/l) was found in 6 out of 8 children. Treatment with hypercaloric diet did not improve the immunological parameters. We conclude that lymphopenia and possibly also hypergammaglobulinemia observed in BA children resulted mainly from the deficiency of the so-called 'naive', suppressor-inducer CD4+ T cell subset (CD4+/CD45RA+) that is known to maintain the proper level of immunoglobulin synthesis by inhibition of B cell differentiation into plasma cells.

IgG subclass distribution of hepatitis B surface antigen antibodies induced in children with chronic hepatitis B infection after interferon-alpha therapy.

The IgG subclass distribution of antibody to hepatitis B surface antigen (anti-HBs) was investigated in 19 children with chronic active hepatitis B infection who showed a complete serological seroconversion after interferon-alpha therapy. Determinations were done 6 and 12 months after treatment. Our results showed no selectivity in anti-HBs synthesis among IgG subclasses. All 4 IgG isotypes were involved in the response, with similar percentage contributions, on average, of IgG1 (35%), IgG3 (27%), and IgG4 (28%), followed by IgG2 (10%). IgG4 became the second most dominant isotype at the end of observation. These results are in contrast to those found after natural seroconversion, in which anti-HBs was highly restricted to neutralizing IgG1 and IgG3, with only a minor contribution from IgG2 and IgG4. It is postulated that analysis of the specific profiles of IgG subclasses may be of value for the estimation of the therapeutic efficacy of recombinant interferon-alpha used and may be helpful in choosing more-effective treatment.

The IgG subclass profile of anti-HBs response in vaccinated children and children seroconverted after natural infection.

The IgG subclass profiles of anti-HBs antibodies were investigated in 30 children who had recovered from acute hepatitis B and 40 children vaccinated against hepatitis B virus (HBV) with Engerix B. After natural seroconversion the mean geometric value of anti-HBs titres was ca 41-fold lower than at the peak of response in vaccinees, and specific antibodies were highly restricted to IgG1 subclass followed by IgG3 with only a minor contribution of IgG2 and IgG4. Conversely, in children immunized with recombinant HBsAg, IgG1 and IgG3 dominated after two doses of vaccine and 1 month after the third injection but the response was less selective and more variable. One year after vaccination IgG4 anti-HBs antibodies became the second dominating isotype. Significant statistical differences in the profiles of IgG anti-HBs were observed when the age and maturity of humoral response were considered. While children vaccinated below 5 years of age responded mainly with IgG1 and IgG3 subclasses, older children (> 5 years) showed a high individual variability in the specific profiles with a high contribution of IgG4. We concluded that vaccination at a younger age leads to the production of antibody subclasses which are more effective for virus neutralization.

[Angioedema is caused by a defect in C1-inhibitor synthesis]. / Obrzek naczynioruchowy zalezny od defektu syntezy C1-inhibitora.

Deficit of the first component of complement inhibitor (C1-inhibitor, C1-inh) may clinically be manifested as angioedema. The disease is characterized by episodic swellings of mucosa and subcutaneous tissue at different locations of the body. Laryngeal swelling can be life-threatening. The major mediators of edema are discussed to be bradykinin and C2b derived peptides. These mediators increase capillary permeability. Antifibrinolytic agents (aminocaproic acid, tranexamic acid) and attenuated androgens (danazol or stanazolol) are used for prophylaxis. Prolonged use both of them might result in more or less severe side effects. In experiments in vitro it has been shown that IFN-gamma, IL-1, IL-6 have a stimulatory effect on C1-inh synthesis. We want to verify the practical use of probiotics as natural inductors of IFN-gamma synthesis for elevating C1-inh level.

Further delineation of the classical Smith-Lemli-Opitz syndrome phenotype at different patient ages: clinical and biochemical studies.

We present here extensive clinical and biochemical data on thirteen SLOS (type I) patients with proven defect in cholesterol biosynthesis for further delineation of the classical SLOS phenotype at different patient ages.

Serum and salivary antigliadin antibodies and serum IgA anti-endomysium antibodies as a screening test for coeliac disease.

Serum and salivary IgA and IgG antigliadin antibodies were determined by an enzyme-linked immunosorbent assay in 18 children with villous atrophy and 30 children on a gluten-free diet for coeliac disease in whom normal intestinal mucosa was found. Serum IgA anti-endomysium antibodies were also determined by an immunofluorescence method in these children. Serum IgG antigliadin and IgA anti-endomysium antibodies had the highest sensitivity (100 and 94.4%, respectively), followed by serum IgA antibodies to gliadin (72.2%), salivary IgA antigliadin (61.2%) and IgG antigliadin (50%) antibodies. The highest specificity was found for serum IgA anti-endomysium (100%) and IgA antigliadin (96.6%) antibodies and salivary IgA and IgG antigliadin antibodies (93.3%), while serum IgG antigliadin antibodies were found to be least specific (63.3%).

[Regulation of Ige synthesis and cytokines in selected allergic diseases]. / Regulacja syntezy IgE oraz cytokin w wybranych zespolach alergicznych.

Some of CD4+ helper T cells-mediated mechanisms of IgE synthesis were discussed. The present division of helper T cells population into Th1 and Th2 subsets reflects their role in the induction as well as in the control of immune response. The Th1 cells are involved mainly in the cellular immune responses, and Th2 cells in the humoral immune responses, including IgE synthesis. Development of investigations on cytokines and evaluation of their influence on regulation of IgE synthesis may have a role in the treatment of allergic diseases in the future.

Eleven Polish patients with microcephaly, immunodeficiency, and chromosomal instability: the Nijmegen breakage syndrome.

We report on 11 patients from 8 independent families (3 pairs of sibs) with a complex clinical pattern including microcephaly, peculiar "bird-like" face, growth retardation, and, in some cases, mild-to-moderate mental deficiency. Most of the patients have recurring respiratory tract infections. One girl has developed B-cell lymphoma. A detailed anthropometric study of 15 physical parameters, including 3 cephalic traits, was performed. It was possible to study the chromosomes of PHA-stimulated lymphocytes in all of the patients. We found structural aberrations with multiple rearrangements, preferentially involving chromosomes 7 and 14 in a proportion of metaphases in all individuals. Profound humoral and cellular immune defects were observed. Serum AFP levels were within normal range. Radioresistant DNA synthesis was strongly increased in all 8 patients who were hitherto studied in this respect. Our patients fulfill the criteria of the Nijmegen breakage syndrome, which belongs to the growing category of ataxia telangiectasia-related genetic disorders. In light of the increased predisposition to malignancy in this syndrome, an accurate diagnosis is important for the patient.

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.

C1-INH defect as an example of deficiency disease.

Primary defect of C1-inhibitor (C1-INH), the regulatory protein of the initial classical pathway of complement, is the cause of hereditary angioedema. Clinical symptoms involve potentially fatal obstruction of the upper respiratory tract, abdominal pains, and subcutaneous edemas. Since the description of functional tests for C1-INH two types of hereditary defect have been known: type I and type II. Sixteen patients with the type I of hereditary angioedema were diagnosed and treated in our hospital. The onset of the disease occurred between 1.5-12 y. of age. Clinical symptoms were observed in skin, gastrointestinal and respiratory tracts. Mean concentration of C1-INH in sera of 16 patients was 3.25 mg/dl, below 8.75 mg/dl that is the critical for well-functioning C1-INH. Inhibitory activity of C1-INH for C1 esterase in plasma was zero in most of the patients, while it was 0.94 +/- 0.20 U/ml in plasma samples of 91 healthy blood donors. Three modalities of treatment are available: substitution with C1-INH concentrate in acute attacks and antifibrinolytic and/or anabolic drugs for prophylaxis. We have obtained good therapeutic results with epsilon-aminocaproic acid (antifibrinolytic), 2g daily during 3 months, with 6 months intervals.

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.

Concentration of C1 inhibitor in sera of healthy blood donors as studied by immunoenzymatic assay.

One hundred and forty sera from healthy blood donors (age 19-30 years) were studied for the concentration of C1 inhibitor. The determinations were performed by enzyme-linked immunosorbent assay with the use of specific anti-C1 inhibitor antibodies fixed to the wells of a microplate and peroxidase labeled as the second layer. The geometric mean of the C1 inhibitor value was 0.25 g/l with a standard deviation of 0.09 and a standard error of 0.0077 g/l while the median and mode values were equal to 0.27 g/l. The technique is relatively simple and can be used for the screening of hereditary or acquired angioedema. The technique can be applied also for the study of population differences in the C1 inhibitor concentration and for the study of its synthesis in in vitro systems.

Influence of immune complexes containing HBsAg and HBeAg on IL-2 dependent human lymphocyte proliferation.

Studies were undertaken to evaluate the effect of hepatitis B virus (HBV) immune complexes (HBV-IC) on IL-2 dependent human lymphocyte proliferation. The following parameters were studied: 1) Effect of HBV-IC (HBsAg-IgG or HBeAg-IgG) on PHA-mediated lymphocyte proliferation; 2) Influence of HBV-IC on the ability of PHA-stimulated peripheral blood lymphocytes (PBL) for IL-2 production and IL-2 receptor expression. HBV-IC induced a dose dependent and antigenic dependent suppression of PHA stimulated lymphocytes. The suppressor effect exerted by HBsAg-IgG was irreversible. In contrast, the suppression mediated by HBeAg-IgG was reversible: lymphocytes preincubated with this preparation washed and activated with PHA responded well to mitogen. The presence of HBV-IC in the cultures of PHA-activated PBL decreased their ability to produce IL-2: HBeAg-IgG exerted a stronger suppressor effect. This effect was partially reversible: removal of HBV-IC from the culture by washing and subsequent stimulation of PBL with PHA increased the capacity of lymphocytes to produce IL-2. This was particularly evident with HBeAg-IgG. Decreased activity of IL-2 observed in the cultures, was also partially dependent on the ability of HBV-IC to bind IL-2 present in the culture medium. Experiments performed using ultracentrifugation indicated that HBV-IC, especially HBsAg-IgG, may bind to IL-2 and inactivate it. HBV-IC had also an effect on IL-2 receptor expression: 1) their presence in the cultures of PHA-stimulated PBL decreased the number of Tac positive cells; 2) the response of HTCL to exogenous IL-2 was decreased by HBV-IC present in the culture medium. This was especially observed in the case of HBsAg-IgG. We suggest that the observed inhibition of PHA-induced lymphocyte proliferation exerted by immune complexes containing HBsAg-IgG or HBeAg-IgG may be caused mainly by their influence on IL-2 dependent mechanism of lymphoproliferation.

Effect of HBV antigens and their immune complexes on the PHA induced lymphocyte proliferation. Modulatory influence on interleukin-2 activity.

Studies were undertaken to evaluate immunomodulating properties of Hepatitis B virus (HBV) preparations: HBsAg, HBeAg and their complexes: HBsAg-IgG and HBeAg-IgG, on PHA-induced lymphocyte proliferation. Cells were obtained from blood of healthy individuals, serologically negative for HBV markers. HBV preparations were purified from sera of children with HBV-mediated glomerulonephritis. Suppression of lymphocyte proliferation observed in the presence of HBsAg and HBsAg-IgG complexes was irreversible. However, the suppressive effect of HBeAg and HBeAg-IgG was abolished when these preparations were removed from the culture. Addition of exogenous interleukin-2/IL-2/reversed only the suppressive effect of HBeAg-IgG which was constantly present in the culture. The inhibition of lymphocyte proliferation correlated well with the decreased level of IL-2 activity in cultures with HBV-preparations. Experiments performed using ultracentrifugation indicated that HBV preparations, especially HBsAg and HBsAg-IgG, may bind to IL-2 and inactivate it in supernatants. The experiments indicate that HBV antigens, as well as other viral products, can inhibit lymphocyte proliferative response to the mitogen. Furthermore, we suggest that this inhibition may occur via suppression of IL-2 synthesis.

Primary immunodeficiency diseases in children treated in the Children's Memorial Hospital, Poland.

One hundred and three cases of primary immunodeficiency diseases were diagnosed among children suffering mainly from chronic and severe infections in the period 1980-1987. Predominantly antibody defects were recognized in 48 patients (46.6%), combined immunodeficiencies in 36 patients (35%), phagocytic disorders in 12 patients (11.6%), complement defects in 6 patients (5.8%), and cell-mediated disease (Di George syndrome) in 1 patient. Allergic complications were observed in 25 patients (24.2%) and malignancy-in 3 patients (2.9%). More detailed immunological studies were performed in children with X-linked agammaglobulinemia in the course of intravenous immunoglobulin therapy and in children with ataxia telangiectasia.