Kazimierz Madaliński

Functional C1-inhibitor diagnostics in hereditary angioedema: assay evaluation and recommendations.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition to low C4 levels, the most important laboratory parameter for correct diagnosis of HAE or angioedema due to acquired C1-Inh deficiency is reduced C1-Inh function (fC1-Inh). No direct recommendations about the assays for fC1-Inh or sample handling conditions are available, although this would prove especially useful when a laboratory first starts to offer assays on fC1-Inh for HAE diagnosis. In the present study we evaluated the performance of fC1-Inh assays in the 15 different laboratories that are specialised in HAE diagnostics and assessed inter-laboratory variation with each laboratory using their own assays and standards. A double-blind survey was conducted using plasma/serum samples from healthy donors and HAE patients and the uniformity of HAE diagnosis was evaluated. It can be concluded that the diagnosis of fC1-Inh deficiency was made correctly in most cases in this survey. We can recommend the chromogenic assay for the determination of fC1-Inh, while the complex ELISA needs further investigation.

Antibody responses to preS components after immunization of children with low doses of BioHepB.

BioHepB is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing HBs as well as preS1 and preS2 antigens, in their glycosylated and non-glycosylated forms. The vaccine was administered intramuscularly to 18 children aged 5 months to 11 years at 0, 1 and 6 months. One hundred percent seroconversion and seroprotection rates were achieved after primary and secondary immunization with the 2.5 microg doses of BioHepB. Ten out of the 18 children (56%) responded with the appearance of anti-preS1 and/or anti-preS2 antibodies in circulation, when analyzed 1, 2, 6, 7 and 12 months after the initiation of vaccination. In comparison with the emergence of the anti-HBs response, early (month 2, after two injections) or late (month 7, after three injections) peak responses were noted for the kinetics of anti-preS1 and anti-preS2 production during the course of immunization, demonstrating that the anti-preS1 and anti-preS2 responses are differently regulated, compared with the anti-HBs response. At month 6, just prior to the final injection, BioHepB caused significantly higher anti-HBs responses (GMT) in preS1-reactive children than in children without preS1 antibodies (P<0.005). Moreover, a significantly higher, anti-HBs response in GMT was also noted for anti-preS2-reactive children compared with anti-preS2-negative children (P<0.05). These findings demonstrated that recognition of the preS epitopes contained in the experimental preS1/preS2/S vaccine is accompanied by a more rapid onset and pronounced antibody response to the S-gene-derived protein in healthy children.

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.

PreS1 epitope recognition in newborns after vaccination with the third-generation Sci-B-Vac™ vaccine and their relation to the antibody response to hepatitis B surface antigen

BackgroundSci-B-Vac™ is a recombinant, hepatitis B vaccine derived from a mammalian cell line and containing hepatitis B surface antigen (HBsAg) as well as preS1 and preS2 antigens. Few studies have been performed on the antibody responses to preS1 in relation to the antibody to hepatitis B surface antigen (anti-HBs) response during immunisation of healthy children with preS-containing vaccines.ResultsIn this study 28 healthy newborns were randomly selected to receive either 2.5 ug or 5.0 ug of the Sci-B-Vac vaccine. Children received three doses of vaccine according to a 0-, 1-, 6-month scheme. Antibodies against the S-protein and three synthetic peptides mimicking three B-cell preS1 epitopes, (21–32 amino acid epitope), (32–47 amino acid epitope) and the C-terminal (amino acid epitope 94–117) were determined at 6 and 9 months. Fourteen (50%) of the 28 newborns had detectable levels of anti-preS1 (21–32) antibodies; 15 (54%) were anti-preS1 (32–47) reactive and 12 (43%) were anti-preS1 (94–117) reactive at 6 or 9 months after initiation of the vaccination. Significantly higher levels of anti-HBs were observed in the sera of patients with detectable anti-preS1 (32–47) reactivity (24 550 ± 7375 IU/L, mean ± SEM) as compared with the non-reactive sera (5991 ± 1530 IU/L, p < 0.05). The anti-HBs levels were significantly lower if none (p < 0.05) or one (p < 0.025) of the preS1 (21–32, 32–47, 94–117) peptides were recognised compared with the anti-HBs levels if two or three peptides were recognised.ConclusionRecognition of several preS1 epitopes, and in particular, the epitope contained within the second half of the hepatocyte binding site localised in the hepatitis B surface protein of the third-generation hepatitis B vaccine is accompanied by a more pronounced antibody response to the S-gene-derived protein in healthy newborns.

Possible disease-modifying factors: the mannan-binding lectin pathway and infections in hereditary angioedema of children and adults

IntroductionHereditary angioedema (HAE) is caused by mutations in the C1inh gene, leading to dysfunction of the C1-esterase inhibitor (C1-INH). C1-INH interacts with MASP-1 and MASP-2 proteases, participating in the mannan-binding lectin (MBL) pathway of complement activation. The aim of the study was to investigate the contribution of possible changes in MBL/MASP-2 complex activity and Helicobacter pylori, hepatitis B virus (HBV), and hepatitis C virus (HCV) infections to the severity and frequency of clinical symptoms of HAE.Materials and MethodsThe study was performed in 65 patients with HAE and 113 healthy persons. The parameters measured were C1-INH, C4, MBL concentration and MBL/MASP-2 complex activity, and serological markers of H. pylori, HBV, and HCV infection. Scores for the frequency and severity of HAE symptoms were determined.ResultsHAE scores were significantly higher in patients whose C1-INH activity did not exceed 10% than in patients with activity of 10-52% (p=0.016). No significant differences were found in the median levels of MBL concentration and MBL/MASP-2 complex activity between patients and the control group. There was a slight association between contact with H. pylori in patients and HAE symptom score (p=0.052, not significant). Adult patients showed a 2.6-times higher frequency of anti-HBc than the general population. HBV DNA was negative in anti-HBc(+) patients.ConclusionsThese results suggest that the MBL complement activation pathway itself does not contribute to the frequency of angioedema attacks. Infections with H. pylori and HBV may slightly influence the disease score (not significant).

IgG Subclass Distribution of Hepatitis B Surface Antigen Antibodies Induced in Children with Chronic Hepatitis B Infection after Interferon-α 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.

Hepatitis B virus genotypes in children with chronic hepatitis B in Poland.

Objectives To analyse the distribution of HBV genotypes in Polish children with chronic hepatitis B, and to assess the relation between the viral genotype and the severity of liver damage. Methods Serum samples from children with chronic hepatitis B were used for biochemical and serological testing, and for determination of HBV genotypes by a nested-multiplex-polymerase chain reaction. Liver biopsies were obtained for histological assessment, which was performed according to the Batts and Ludwig scoring system of chronic hepatitis. Results Of 78 children with chronic hepatitis B, 74 had an identifiable HBV genotype: 86.5% were infected with genotype A, and 13.5% were carriers of genotype D. The frequency of HBeAg clearance and the levels of alanine aminotransferase and serum aspartate transaminase were comparable in both genotype groups. There was no correlation between the HBV genotype and either activity of liver inflammation or liver fibrosis. Conclusions This study shows that the distribution of HBV genotypes in Polish children with chronic HBV infection reflects the general prevalence of HBV genotypes in Europe. The course of chronic hepatitis B in children is not significantly influenced by viral genotypes A or D.

Hepatitis C virus epidemiology and prevention in Polish and Swiss population – similar and contrasting experiences

OBJECTIVE The aim of the study was to review available data on HCV in Poland and Switzerland, in order to compare the two European countries with respect to epidemiological situation and efficiency of the response systems. MATERIALS AND METHOD A search of registries, published and grey literature was performed to assemble data on prevalence, rate of detection of new cases, identified risk factors for transmission, mortality due to HCV, prevalence of HCC and the consequent liver transplantations, as well as data on treatment in Poland and Switzerland. RESULTS Overall, the prevalence of anti-HCV antibodies was similar, not exceeding 1%. However, the major transmission routes of HCV infections were different: medical procedures in Poland and drug injections in Switzerland. By combining the available information it was also possible to demonstrate important differences in efficiency of the response systems. There was approximately 1 new diagnosis per 100 estimated undiagnosed cases in the population in Poland per year, compared to 6 in Switzerland, and the treatment rate per 100 estimated active infections was 2 and 4, respectively. CONCLUSIONS Scaling up of the diagnosis and treatment is necessary in both countries; however, the means to achieve this might differ, taking into account the higher concentration of the infections in risk groups in Switzerland than in Poland.

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.

Factors associated with hepatitis C prevalence differ by the stage of liver fibrosis: A cross-sectional study in the general population in Poland, 2012-2016

Background & aims There is a considerable burden of hepatitis C in Europe related to the lack of prompt diagnosis. We aimed to estimate the prevalence and related risk factors of HCV infections by the stages of liver fibrosis, using non-invasive methods, to understand testing needs in Poland. Methods A cross-sectional study was conducted in 2012–2016 adopting a stratified random sampling of primary health care units followed by systematic sampling of patients within each unit. Study participants filled a questionnaire and donated blood for laboratory HCV testing. Additionally, the results of liver function tests and platelet count were collected to calculate APRI and FIB-4 scores. Cases were classified according to the level of fibrosis: ‘significant fibrosis’ (APRI≥0.7 or FIB4≥1.45) and ‘no significant fibrosis’ (APRI<0.7 and FIB4<1.45). Results Of 21 875 study participants, 102 were HCV-RNA positive. Prevalence of HCV infections and significant fibrosis was estimated at 0.47% (95% CI 0.38% - 0.57%) and 0.12% (0.08% - 0.17%), respectively. Cases with significant fibrosis accounted for 51.6% (33.4%-69.9%) in men and 34.4% (17.3%-51.4%) in women. There was no correlation between the HCV prevalence and age. Blood transfusion prior to 1992 strongly predicted significant fibrosis as did the history of injecting drug use (IDU) and ever having an HCV-infected sexual partner in men and caesarean sections in women. Factors associated with HCV infection without significant fibrosis were tattooing in men and younger age in women. We acknowledge limited possibility to study the associations between IDU and ever having HCV-infected sexual partner, given small sample sizes for these exposures. Conclusions As no clear birth cohort affected by HCV could be identified, risk factor-based screening in the general population should be considered, taking into account the association between the increased risk of liver fibrosis and the history of transfusion prior to 1992 and caesarean sections.