Andrea Sümegi

High prevalence of IgA class anti-neutrophil cytoplasmic antibodies (ANCA) is associated with increased risk of bacterial infection in patients with cirrhosis

BACKGROUND & AIMS Anti-neutrophil cytoplasmic antibodies (ANCA) are a non-uniform family of antibodies recognizing diverse components of neutrophil granulocytes. ANCA formation might be induced by protracted bacterial infections or probably reflect an abnormal immune response to commensal microorganisms. Bacterial infections are common complications in cirrhosis with high incidence of episodes caused by enteric organisms, therefore, we sought to study the presence and clinical importance of ANCA in cirrhosis. METHODS Sera of 385 patients with cirrhosis of different etiologies were assayed for ANCA of IgG, IgA, IgA1, IgA2, and secretory IgA subtypes by indirect immunofluorescence and ELISAs. The control group comprised 202 patients with chronic liver diseases without cirrhosis and 100 healthy subjects. In cirrhosis, a 2-year follow-up, observational study was conducted to assess a possible association between the presence of ANCA and clinically significant bacterial infections. RESULTS Prevalence of ANCA IgA was significantly higher in cirrhosis (52.2%) compared to chronic liver diseases (18.6%) or healthy controls (0%, p<0.001 for both). ANCA IgA subtyping assays revealed marked increase in the proportion of IgA2 subtype (46% of total ANCA IgA) and presence of the secretory component concurrently. Presence of ANCA IgA was associated with disease-specific clinical characteristics (Child-Pugh stage and presence of ascites, p<0.001). During a 2-year follow-up period, risk of infections was higher among patients with ANCA IgA compared to those without (41.8% vs. 23.4%, p<0.001). ANCA IgA positivity was associated with a shorter time to the first infectious complication (pLogRank <0.001) in Kaplan-Meier analysis and was identified as an independent predictor in multivariate Cox-regression analysis (HR:1.74, 95% CI: 1.18-2.56, p=0.006). CONCLUSIONS Presence of IgA type ANCA is common in cirrhosis. Involvement of gut mucosal immune system is in center of their formation and probably reflects sustained exposure to bacterial constituents.

Lissencephaly and band heterotopia: LIS1, TUBA1A, and DCX mutations in Hungary

The spectrum of lissencephaly ranges from absent (agyria) or decreased (pachygyria) convolutions to less severe malformation known as subcortical band heterotopia. Mutations involving LIS1 and TUBA1A result in the classic form of lissencephaly, whereas mutations of the DCX gene cause lissencephaly in males and subcortical band heterotopia in females. This report describes the clinical manifestations and imaging and genetic findings in 2 boys with lissencephaly and a girl with subcortical band heterotopia. An ovel mutation (c.83_84delAT, p.Tyr28Phefs*31) was identified in LIS1 in 1 of the boys with lissencephaly and another novel mutation (c.200delG, p.Ile68Leufs*87) was found in DCX in the girl with subcortical band heterotopia. The mutations appeared in the first half of the genes and are predicted to result in truncated proteins. A mutation was found in the TUBA1A gene (c.1205G>A, p.Arg402His) in the other boy. This mutation affects the folding of tubulin heterodimers, changing the interactions with proteins that bind microtubules.

Analysis of Components of the CD14/TLR System on Leukocytes of Patients with Atopic Dermatitis

Background: Atopic dermatitis (AD) is frequently associated with skin infections that may be a consequence of an impaired function of the innate immune response. Conversely, the frequent bacterial colonization may also influence the systemic immune reactions, including the Toll-like receptor (TLR) system, through the translocation of bacterial components into the circulation. Therefore, we characterized phenotypic and functional properties of the TLR system in patients with extrinsic and intrinsic AD. Methods: The absolute number of surface CD14, TLR2, TLR4 and CD180 and the CD14-mediated uptake of bodipy-labeled endotoxin and bacteria by whole blood leukocytes was studied by flow cytometry. We measured the serum soluble CD14 concentration by an inhibitory flow cytometric method. Results: We observed a significant overexpression of TLR2 and TLR4 on monocytes, TLR2 and CD14 on granulocytes and CD180 on lymphocytes of intrinsic AD patients compared to healthy controls. The serum soluble CD14 was not different in the intrinsic AD patients, while it was diminished in the extrinsic AD group compared to the controls. The endotoxin and bacterium uptake showed no differences. Conclusions: The observed upregulation of CD14, TLR2, TLR4 and CD180 on peripheral leukocytes seems to be rather a consequence than the cause of the repeated bacterial infections in AD.

Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods

Abstract Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods – PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) – was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%–2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes. Conclusions: This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.

Meiotic segregation study of a novel t(3;6)(q21;q23) in an infertile man using fluorescence in situ hybridization (FISH)

Male carriers with balanced reciprocal translocations can produce a variable proportion of unbalanced gametes resulting in reproductive failures. The presence of a structural rearrangement may induce an interchromosomal effect. This is characterized by abnormal bivalents not involved in the reorganization thereby yielding non-disjunction, which would present as aneuploid spermatozoa for these chromosomes. In the present case report segregation analysis of the sperm and investigation of interchromosomal effect were carried out using cytogenetic and fluorescence in situ hybridization (FISH) analysis on blood lymphocytes. The karyotype of the patient was 46,XY,t(3;6)(q21;q23). During sperm segregation analysis a total of 2,002 sperms were evaluated, of which 46.8% showed normal/balanced (alternate segregation mode) and 53.2% of sperm showed an abnormal signal pattern. A significant difference in the frequency of the estimated number of chromosome anomalies was observed in the translocation carrier when compared to the normozoospermic group (P < 0.0001) and the oligozoospermic group (P < 0.0001). Meiotic segregation analysis of sperm together with aneuploidy assessment for X, Y, and 17 chromosomes using FISH allows for the determination of a reproductive prognosis in male balanced translocation carriers and can be used for appropriate genetic counseling.

Presepsin teardown - Pitfalls of biomarkers in the diagnosis and prognosis of bacterial infection in cirrhosis

AIM To evaluate the diagnostic and prognostic value of presepsin in cirrhosis-associated bacterial infections. METHODS Two hundred and sixteen patients with cirrhosis were enrolled. At admission, the presence of bacterial infections and level of plasma presepsin, serum C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Patients were followed for three months to assess the possible association between presepsin level and short-term mortality. RESULTS Present 34.7 of patients had bacterial infection. Presepsin levels were significantly higher in patients with infection than without (median, 1002 pg/mL vs 477 pg/mL, P < 0.001), increasing with the severity of infection [organ failure (OF): Yes vs No, 2358 pg/mL vs 710 pg/mL, P < 0.001]. Diagnostic accuracy of presepsin for severe infections was similar to PCT and superior to CRP (AUC-ROC: 0.85, 0.85 and 0.66, respectively, P = NS for presepsin vs PCT and P < 0.01 for presepsin vs CRP). At the optimal cut-off value of presepsin > 1206 pg/mL sensitivity, specificity, positive predictive values and negative predictive values were as follows: 87.5%, 74.5%, 61.8% and 92.7%. The accuracy of presepsin, however, decreased in advanced stage of the disease or in the presence of renal failure, most probably because of the significantly elevated presepsin levels in non-infected patients. 28-d mortality rate was higher among patients with > 1277 pg/mL compared to those with ≤ 1277 pg/mL (46.9% vs 11.6%, P < 0.001). In a binary logistic regression analysis, however, only PCT (OR = 1.81, 95%CI: 1.09-3.01, P = 0.022) but neither presepsin nor CRP were independent risk factor for 28-d mortality after adjusting with MELD score and leukocyte count. CONCLUSION Presepsin is a valuable new biomarker for defining severe infections in cirrhosis, proving same efficacy as PCT. However, it is not a useful marker of short-term mortality.

Fusion of the Fc part of human IgG1 to CD14 enhances its binding to Gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils

Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 - a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes. Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes.

Functional polymorphisms of innate immunity receptors are not risk factors for the non-SBP type bacterial infections in cirrhosis

Pattern recognition receptors (PRRs) have a key role in the innate host defense. Functional polymorphisms of various PRRs have been established to contribute to an increased susceptibility to spontaneous bacterial peritonitis (SBP). Their role in the development of cirrhosis‐associated bacterial infections (BI), beyond SBP or progressive disease course related to pathological bacterial translocation (BT) remains unknown.