T cell immune response predicts survival in severely ill COVID-19 patients requiring venovenous extracorporeal membrane oxygenation support.

Introduction: There is a critical gap in understanding which SARS-CoV-2 patients would benefit most from venovenous extracorporeal membrane oxygenation (VV-ECMO) support. The potential role of a dysregulated immune response is still unclear in this patient population. Objectives: To assess the potential predictive value of SARS-CoV-2 specific cellular and humoral immune responses for survival in critically ill COVID-19 patients requiring VV-ECMO. Methods: We conducted a prospective single-center observational study of unvaccinated patients requiring VV-ECMO support treated at the intensive care unit of Semmelweis University Heart and Vascular Center between March and December 2021. Peripheral blood samples were collected to measure the humoral and cellular immune statuses of the patients at the VV-ECMO cannulation. Patients were followed until hospital discharge. Results: Overall, 35 COVID-19 patients (63% men, median age 37 years) on VV-ECMO support were included in our study. The time from COVID-19 verification to ECMO support was a median (IQR) of 10 (7-14) days. Of the patients, 9 (26%) were discharged alive and 26 (74%) died during their hospital stay. Immune tests confirmed ongoing SARS-CoV-2 infection in all the patients, showing an increased humoral immune response. SARS-CoV-2-specific cellular immune response was significantly higher among survivors compared to the deceased patients. A higher probability of survival was observed in patients with markers indicating a higher T cell response detected by both QuantiFeron (QF) and flow cytometry (Flow) assays. (Flow S1 CD8+ ≥ 0.15%, Flow S1 CD4+ ≥ 0.02%, QF CD4 ≥ 0.07, QF whole genome ≥ 0.59). In univariate Cox proportional hazard regression analysis BMI, right ventricular (RV) failure, QF whole genome T cell level, and Flow S1 CD8+ T cell level were associated with mortality, and we found that an increased T cell response showed a significant negative association with mortality, independent of BMI and RV failure. Conclusion: Evaluation of SARS-CoV-2 specific T cell response before the cannulation can aid the risk stratification and evaluation of seriously ill COVID-19 patients undergoing VV-ECMO support by predicting survival, potentially changing our clinical practice in the future.

[Reducing the risk of infections in hereditary and acquired complement deficiencies.] / Infekciók kockázatának csökkentése veleszületett és szerzett komplementdefektusokban.

Hereditary complement deficiencies are relatively rare worldwide, they account for about 1-10% of primary immunodeficiencies. Acquired complement deficiencies are more prevalent and with the more frequent use of complement inhibitor therapy, the incidence of patients with iatrogenic complement deficiency is increasing. Alike in the inherited forms, patients have a high risk of severe and life-threatening infections caused by encapsulated bacteria (sepsis, meningitis). The most frequent pathogens are Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. C5 and C3 complement inhibitor therapies are available in Hungary, which are mostly indicated in the treatment of paroxysmal nocturnal hemoglobinuria, myasthenia gravis, neuromyelitis optica and atypical haemolytic uremic syndrome. It is of utmost importance to prevent severe, potentially life-threatening bacterial infections in this group of patients. Nevertheless, there is no Hungarian guidance to decrease the risk of infections, preventive measures are incomplete and not standardized posing potential risk of infections for these patients, so far. In this review, we aim to summarize the international clinical practices and guidance on the infection prevention in complement deficient patients. This recommendation might be a source of an evidence-based Hungarian guideline regarding vaccination and antibiotic prophylaxis in this specifically vulnerable group of patients. Orv Hetil. 2023; 164(25): 971-980.

Clinical and epidemiological characteristics of pediatric COVID-19 patients in a community-based study / Az alapellátásban kezelt COVID-19-fertozött gyermekek tünettani és epidemiológiai jellemzoi.

Összefoglaló. Bevezetés: A COVID-19-pandémia kapcsán számos tanulmány vizsgálta a tünetek gyakoriságát és a járványterjedés jellemzoit gyermekkorban, kevés azonban az alapellátás adatait összefoglaló publikáció. Közleményünkben 12 házi gyermekorvosi praxis 545 SARS-CoV-2-fertozött betegének adatait elemeztük a 2. (n = 293) és a 3. (n = 252) járványhullámban. Célkituzés: A gyermekkori fertozések tünettanának és epidemiológiai jellemzoinek összehasonlítása korcsoportok és járványhullámok között. Módszer: Valamennyi alapellátó praxis egységes retrospektív adatgyujtést végzett ugyanazon paraméterek regisztrálásával. Eredmények: A 10 év alatti betegekben a láz, a nátha és a köhögés dominált (30-50%), míg a 10 év felettiekben magas arányban regisztráltunk általános tüneteket is (30-40% fejfájás, gyengeség, szaglászavar). A 2. hullámban a 11-18 évesek (68%), a 3. hullámban a 0-10 évesek (53%) voltak többségben. A 3. hullámban szignifikánsan emelkedett a légúti tünetek elofordulása, az általános tünetek gyakorisága jelentosen csökkent, és szignifikánsan nott a családon belüli expozíció aránya (36% vs. 58%) a 2. hullámmal összehasonlítva. A gyermekrol családtagra történo továbbterjedés 24% és 16% volt a két járványhullámban, és mértékét az életkor befolyásolta. Megbeszélés: A klinikai kép az életkorral és a feltételezett vírusvariánssal mutatott összefüggést: 10 év alatt a légúti tünetek domináltak, 10 év felett szignifikánsan több általános tünetet regisztráltunk a 0-10 évesekhez képest. A 3. járványhullámban az alfa-variáns terjedésével gyakoribbá váltak a légúti tünetek, az iskolabezárások következtében megváltozott az életkori megoszlás, és megemelkedett a családi expozíció okozta fertozések aránya. A fertozés továbbadása háztartáson belül mindkét hullámban alacsony maradt. Következtetés: A COVID-19 klinikai megjelenését és terjedési jellemzoit jelentosen befolyásolta az érintett gyermekpopuláció életkori összetétele, a cirkuláló vírusvariáns és az aktuális korlátozó intézkedések. Orv Hetil. 2021; 162(44): 1751-1760. INTRODUCTION: During the COVID-19 pandemic, a large number of publications examined the frequency of symptoms and the mode of transmission in childhood but only a few community-based studies have been published. In our paper, 545 pediatric COVID-19 patients' data were collected by 12 primary care pediatricians in the second (n = 293) and third (n = 252) waves of the pandemic. OBJECTIVE: To compare the frequency of symptoms and household transmission in different age groups and between the two waves. METHOD: Patients' data and disease characteristics were recorded retrospectively in the same manner by all participating pediatricians. RESULTS: In patients of <10 years of age, fever, rhinorrhea and cough were registered the most frequently (30-50%), in contrast to patients of >10 years, where high frequency of general symptoms was found (30-40% headache, weakness, anosmia). In the third wave, the ratio of the age group 11-18 years declined from 68% to 47%, the frequency of respiratory symptoms increased significantly, while the ratio of general symptoms decreased. Household exposition was more frequent in the third wave (36% vs. 58%), while the transmission rate from children to family members was 24% and 16%, respectively, and it varied with age. DISCUSSION: Clinical manifestation showed relation to age and virus variant: the older age associated with higher frequency of general symptoms and the spread of the alpha variant led to the predominance of respiratory symptoms over general complaints. Prolonged school closures affected the age distribution and increased the frequency of household exposition. Secondary household transmission remained low. CONCLUSION: Clinical and epidemiological characteristics of pediatric COVID-19 disease were highly influenced by age, dominant virus variant and mitigation measures. Orv Hetil. 2021; 162(44): 1751-1760.

Identification of the best-suited donor for generating virus-specific T cells.

BACKGROUND AND OBJECTIVES: Administration of virus-specific T cells (VSTs) is a viable antiviral treatment strategy after allogeneic HSCT, even if conventional therapies fail. Third-party donors are often chosen for the generation of the VST product. The eligibility of the donor has to be tested in a rigorous donor screening procedure, since the isolation technology only targets pre-existing VSTs. MATERIALS AND METHODS: In a period of 3 years, we performed 32 VST treatments for 28 patients. Targeting four different viruses, 284 healthy individuals underwent 417 donor screening procedures. VSTs were counted by flow cytometry detecting interferon-gamma (IFN-γ) producing T cells. Generation of the VSTs was performed from leukapheresis products in a fully automated and closed system using magnetic cell separation. RESULTS: The mean circulating VST frequencies ranged from 0·006% to 0·328%. The average yield of viable VSTs in the product was 1·83·106 cells, while the average VST dose calculated for the patient's body weight was 4·63·104 /kg. The mean purity - percentage of VSTs within the T cells - of all T-cell products was 62·9%. Correlation was identified between the frequency of the VSTs in the peripheral blood of the donor and the VST numbers of the end product; the strongest correlation was seen for CMV. CONCLUSION: This paper focuses on the T-cell donors, highlighting some key points on the donor selection process. Based on the findings in connection with the CMV therapies, peripheral VST seems to be the best predictor of the VST content of the final product administered to the patient.

Early Experience With CliniMACS Prodigy CCS (IFN-gamma) System in Selection of Virus-specific T Cells From Third-party Donors for Pediatric Patients With Severe Viral Infections After Hematopoietic Stem Cell Transplantation.

Viral reactivation is a frequent complication of allogeneic hematopoietic stem cell transplantation especially in children. For refractory cases, rapid virus-specific T-cell therapy would be ideally implemented within a few days. Over the course of a year in our pediatric cohort of 43 allogeneic transplantation, 9 patients fulfilled criteria for virus-specific T-cell therapy. Viral infections were due to cytomegalovirus (CMV) in 3, Epstein-Barr virus (EBV) in 2, and adenovirus (AdV) in 1 case, whereas >1 virus was detected in 3 cases. Viral diseases necessitating a T-cell therapy were CMV pneumonitis and colitis, AdV enteritis and cystitis, and EBV-induced posttransplantation lymphoproliferative disease. Cells were produced by the CliniMACS Prodigy CCS (IFN-gamma) System within 24 hours after mononuclear leukapheresis. Eight patients became completely asymptomatic, whereas 7 also cleared the virus. Six patients are alive without viral illness or sequelae demonstrating viral DNA clearance in peripheral blood with a median follow-up of 535 (350-786) days. One patient with CMV pneumonitis died of respiratory insufficiency. In 2 cases the viral illness improved or cleared, however, the patients died of invasive aspergillosis. No cases of graft-versus-host disease, rejection, organ toxicity, or recurrent infection were noticed. Virus-specific T-cell therapy implemented by the CliniMACS Prodigy CCS (IFN-gamma) System is an automated, fast, safe, and probably effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation.

[Allogeneic hematopoietic stem cell transplantation in Hungary]. / Allogén vérképzoossejt-átültetés Magyarországon.

INTRODUCTION AND AIM: The publication summarizes the 2548 stem cell transplantations performed in the period of 1993-2015 in Szent Laszló Hospital, Budapest and provides a detailed discussion of the 425 allogeneic transplantations during 2007-2013. METHOD: The analysis explains the major steps of the evolution of allogeneic stem cell transplantation and compares the results of the unique Hungarian allogeneic center. RESULTS: The significant shift in the transplantation indications from chronic myeloid leukemia to myelodysplastic syndromes and the rising age of the recipients are in line with world wide tendencies. The latter one is the consequence of the introduction and improvement of the concept of reduced intensity conditioning regimens, originally arising from the idea of Endre Kelemen. The most limiting factor, the donor availability seems to be resolved with the use of a new immunomodulating regimen, the application of posttransplantation cyclophosphamide, which allows the transplantation through HLA barriers with haploidentical family donors with comparable results to the HLA matched volunteer unrelated donors. The above mentioned tendencies result the wider use of allogeneic stem cell transplantation less dependent from recipient age, comorbidities and even donor availability. CONCLUSIONS: The publication highlights the need of expanding the stem cell transplantation budget and the involvement of new centers in Hungary in allogeneic of stem cell transplantation. Orv. Hetil., 2017, 158(8), 291-297.

Lipoprotein Lipase as a Prognostic Marker in Chronic Lymphocytic Leukemia.

The marked clinical heterogeneity of CLL makes early prognosis assessment important. Lipoprotein lipase (LPL) has been shown to confer adverse prognosis in CLL, recent data indicating it might also contribute to CLL cell survival and metabolism. We determined LPL mRNA expression in unselected peripheral blood of 84 CLL patients by RT PCR. Results were correlated with other prognostic markers and outcome. 30/84 (40 %) of cases were LPL positive based on the cutoff established by ROC analysis. In LPL positive patients significantly shorter median survival (136 vs 258 months, p < 0.0001) and time to first treatment intervals (36 vs 144 months, p < 0.002) were documented. LPL values correlated with male gender, higher stages, more treatment requirement, CD38 positivity and unmutated IgVH genes. Among cases with 13q deletion, LPL positivity identified a subcohort with poor outcome (median survival 108 months vs NR, p < 0.0001). In multivariate analysis, cytogenetic aberrations and LPL had significant impact on survival. Our results confirm that LPL is a strong predictor of outcome in CLL, able to improve prognostic accuracy in good risk cytogenetic subgroups. The relationship between its prognostic and functional role in CLL needs to be explored further.

Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia.

Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) are genetic alterations in acute myeloid leukemia (AML). The aim of our study was to investigate the frequency and prognostic effect of IDH1/2 mutations together followed by an individual analysis of each substitution in a Hungarian cohort consisting of 376 patients with AML. IDH1(mut) and IDH2(mut) were mutually exclusive, detected in 8.5% and 7.5% of cases, respectively. IDH1/2(mut) was associated with: older age (p = 0.001), higher average platelet count (p = 0.001), intermediate karyotype (p < 0.0001), NPM1(mut) (p = 0.022) and lower mRNA expression level of ABCG2 gene (p = 0.006). Overall survival (OS), remission and relapse rates were not different in IDH1(mut) or IDH2(mut) vs. IDH(neg). IDH1(mut) and IDH2(mut) were associated differently with NPM1(mut); co-occurrence was observed in 14.3% of IDH1 R132C vs. 70% of R132H carriers (p = 0.02) and in 47.4% of IDH2 R140Q vs. 0% of R172K carriers (p = 0.02). IDH1 R132H negatively influenced OS compared to IDH(neg) (p = 0.02) or R132C (p = 0.019). Particular amino acid changes affecting the same IDH1 codon influence the clinical characteristics and treatment outcome in AML.

Applying a "double-feature" promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery.

Human embryonic stem (HuES) cells represent a new potential tool for cell-therapy and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)-expressing clones using a transposon-based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1alpha, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This "double-feature" promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter-driven EGFP transcription and expression of cardiomyocyte-specific genes. Our experiments indicate an efficient applicability of transposon-based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods.

High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells.

Expression of multidrug resistance ABC transporters has been suggested as a functional marker and chemoprotective element in early human progenitor cell types. In this study we examined the expression and function of the key multidrug-ABC transporters, ABCB1, ABCC1 and ABCG2 in two human embryonic stem (HuES) cell lines. We detected a high level ABCG2 expression in the undifferentiated HuES cells, while the expression of this protein significantly decreased during early cell differentiation. ABCG2 in HuES cells provided protection against mitoxantrone toxicity, with a drug-stimulated overexpression of the transporter. No significant expression of ABCB1/ABCC1 was found either in the undifferentiated or partially differentiated HuES cells. Examination of the ABCG2 mRNA in HuES cells indicated the use of selected promoter sites and a truncated 3' untranslated region, suggesting a functionally distinct regulation of this transporter in undifferentiated stem cells. The selective expression of the ABCG2 multidrug transporter indicates that ABCG2 can be applied as a marker for undifferentiated HuES cells. Moreover, protection of embryonic stem cells against xenobiotics and endobiotics may depend on ABCG2 expression and regulation.

Differential regulation of umbilical cord blood and leukemic B cells by interferon-alpha (IFN-alpha): observations in cultured cells.

The exact mechanism of the beneficial therapeutic action of interferon-a (IFN-alpha) in B-cell-lineage malignancies has not been adequately explained. Here we report on the differential effect of IFN-alpha2b on non-malignant B cells of umbilical cord blood and leukemic B-cell lines JY, BL-41 and BCBL-1. Leukemic cell proliferation was characterized by colony assay, whereas apoptosis was investigated by flow cytometry of propidium iodide-stained cells. The degree of differentiation was evaluated by measuring the expression level of Fcgamma receptor-II (FcgammaRII) labeled with anti-CD32-FITC monoclonal antibody using flow cytometry. IFN-alpha protected umbilical cord blood CD19-positive B lymphocytes from apoptotic cell death in vitro. IFN-alpha significantly decreased colony formation of all three cell lines, and in contrast to normal cells, induced apoptosis in JY and BL-41 and excessive necrosis in HHV-8 infected BCBL-1 cells. FcgammaRII was upregulated both in normal and in leukemic B cells as indicated by an increase both in the proportion of CD32-positive cells and the mean fluorescence intensity. From our results it seems that antiproliferative, apoptotic and differentiative effects of IFN-alpha are interrelated but distinct cellular events, which are differentially regulated in normal, leukemic and virus-infected cells of the B-cell lineage.

"Pros and cons" on how to measure multidrug resistance in leukemias.

Drug resistance is one of the most significant challenges in the treatment of various types of malignancies, however most of the experimental and clinical data in multidrug resistance (MDR) has been obtained in leukemias. MDR is the term that describes innate or acquired resistance of tumor cells to a wide range of anticancer drugs. As its presence determines treatment outcome in several forms of leukemias, it is imperative that clinical laboratories provide the most useful data on its expression. Here, a brief review is provided on the pathomechanism and diagnostics of MDR. From the diagnostic point of view it is fortunate that MDR proteins display similar effluxing activity towards many dissimilar agents some of which can be used in fluorescent assays. These tests mimic the real clinical problem i.e. the extrusion activity of MDR proteins towards xenobiotics. Thus, we believe that functional assays when carried out in a standardized way and particularly combined with labeling for various surface markers can be recommended as a front-line test in MDR measurement.