SARS-CoV-2 viral load assessment in lung transplantation.

In the era of COVID-19 pandemic, organ transplantation programs were facing serious challenges. The lung transplantation donor pool was extremely limited and SARS-CoV-2 viral load assessment has become a crucial part of selecting an optimal organ donor. Since COVID-19 is a respiratory disease, the viral load is thought to be more important in lung transplantations as compared to other solid organ transplantations. We present two challenging cases of potential lung donors with a questionable COVID-19 status. Based on these cases, we suggest that the cycle threshold (Ct) value should always be requested from the laboratory and the decision whether to proceed with transplantation should be made upon complex evaluation of diverse criteria, including the nasopharyngeal swab and bronchoalveolar lavage PCR results, the Ct value, imaging findings and the medical history. However, as the presence of viral RNA does not ensure infectivity, it is still to be clarified which Ct values are associated with the viral viability. Anti-SARS-CoV-2 IgA antibodies may support the diagnosis and moreover, novel methods, such as quantifying SARS-CoV-2 nucleocapsid antigen in serum may provide important answers in organ transplantations and donor selections.

Changes in phenotypic patterns of blood monocytes after kidney transplantation and during acute rejection.

Peripheral blood monocytes, which serve as precursors for tissue macrophages and dendritic cells (DC), play a key role in the immune response to kidney allograft, reparation processes and homeostasis regulation. In this prospective study, we used multicolor flow cytometry to monitor the phenotypic patterns of peripheral monocytes in subjects with uncomplicated outcomes and those with acute rejection. We found a reciprocal increase in the proportion of "classical monocytes" (CD14+CD16-) along with a decline in pro-inflammatory "intermediary" (CD14+CD16+) and "non-classical" (CD14lowCD16+) monocytes in subjects with normal outcomes. In subjects with acute rejection, we observed no reduction in "intermediary" monocytes and no increase in "classical" monocytes. Patients with uncomplicated outcomes exhibited downregulated HLA-DR in all three monocyte subpopulations. However, non-classical monocytes were unaffected in subjects with acute rejection. Expression of CD47 was downregulated after transplantation, while patients with antibody-mediated rejection and donor-specific antibodies showed higher pre-transplant values. In monocytes isolated at the time of biopsy, CD47 expression was higher in individuals with acute rejection compared to patients with normal outcomes one year post-transplant. Expression of CD209 (DC-SIGN) and the proportion of CD163+CD206+ subpopulations were upregulated during the first week after kidney transplantation. CD209 was also upregulated in samples taken on the day of biopsy confirming acute rejection. Our data demonstrate that kidney allograft transplantation is associated with phenotypic changes in peripheral blood monocytes during acute rejection.

Up-regulation of CD163 expression in subpopulations of blood monocytes after kidney allograft transplantation.

M2 macrophages expressing CD163 are known to suppress immune responses but have been also found in biopsies of patients with chronic kidney allograft injury associated with interstitial fibrosis. The aim of our study was to evaluate the expression of CD163 in blood monocytes, precursors of tissue macrophages, in kidney allograft recipients with uncomplicated outcome (n=94) compared with those developing acute rejection (n=44). Blood samples were collected before the transplantation and at 1 week, 1 month and 1 year. The expression of CD163 increased during the first week after the transplantation not only in classical (CD14+CD16-) but also in intermediate (CD14+CD16+) and nonclassical (CD14lowCD16+) monocytes in all patients regardless of their rejection status. In patients developing acute rejection, higher pre-transplant expression of CD163 on blood monocytes was found. In vitro experiments confirmed strong induction of membrane CD163 on monocytes together with CD206 (an alternative marker of M2 macrophages) in response to IL-10. We assume from our data that dramatic upregulation of CD163 by peripheral blood monocytes may have a pathophysiological role in early phases after kidney allograft transplantation and high pre-transplant expression of CD163 on blood monocytes might be involved in events leading to acute rejection.

Long-term peritoneal dialysis treatment provokes activation of genes related to adaptive immunity.

Permanent irritation of the peritoneum during peritoneal dialysis (PD) treatment leads to local chronic inflammation and subsequently activation of processes driving fibrogenesis in the long-term. The aim of the study was to compare the peritoneal effluent transcriptome of 20 patients treated less and 13 patients treated more than 2 years using microarray analysis. An increased expression of genes associated with an immune response was observed in long-term treated patients with well preserved peritoneal function, when compared to patients treated less than 2 years. From 100 genes highly expressed in long-term patients, a significant up-regulation of six was found by RT-qPCR: LY9 (lymphocyte antigen 9), TNSFR4 (tumor necrosis factor receptor superfamily, member 4), CD 79A (CD79a molecule), CCR7 (chemokine C-C receptor 7), CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) and IL2RA (interleukin 2 receptor alpha chain). Furthermore, the effluent cell population was analysed. A positive relationship between the number of granulocytes and NK cells on one hand, and duration of PD treatment on the other, was shown. We conclude, that the mechanisms of adaptive immunity promoting T helper 2 cells response are activated in the long-term before functional alterations develop. It consequently might trigger the fibrosis promoting processes.

Macrophage phenotypes in the adipose tissue of postmenopausal women.

Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration and monocyte/macrophage presence within the sub-endothelial space of the artery. In this project, phenotypes of macrophages connected with subclinical inflammation in adipose tissue of living kidney donors were studied. Samples of subcutaneous adipose tissue of living kidney donors (n=36) were exposed to collagenase. Stromal vascular fraction (SVF) was eluted from the samples, then labeled with monoclonal antibodies (anti-CD14 and anti-calprotectin), conjugated with fluorochromes and analyzed by flow cytometry. The positive correlation between the number of total macrophages and calprotectin-positive macrophages with BMI in the subcutaneous adipose tissue of postmenopausal women was demonstrated (p<0.05; R=0.43 and p<0.01; R=0.60), whereas no positive correlation in premenopausal women and men was shown. In conclusion, we documented a significant effect of BMI increase on the presence of total macrophages in adipose tissue of postmenopausal women, in contrast to premenopausal women. This difference was much more pronounced when proinflammatory macrophages with membrane-bound calprotectin were analyzed.

Chemokines induced in human respiratory epithelial cells by IL-1 family of cytokines.

IL-1-related cytokines share similarities in their receptor distribution and signalling pathways; however, overlapping actions of these cytokines have not been clearly demonstrated. The aim of our study was to compare the capacity of different IL-1-related cytokines to stimulate production and release of multiple CC and CXC chemokines by epithelial cells. The chemokine gene expression was studied using a cDNA array system in human alveolar type-II like cells A549 stimulated by IL-1ß, IL-18, and IL-33. The chemokine levels in culture supernatants were measured using multiplex immunoluminometric assay or by ELISA. In repetitive experiments, in response to IL-1ß epithelial cells expressed mRNA for CCL2, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, and CXCL11. In contrast, induction of epithelial cells by IL-33 and IL-18 resulted only in moderate up-regulation of a few CC or CXC chemokines compared to the potent effect of IL-1ß stimulation. We conclude from our data that individual members of the IL-1 family, although related in molecular structure and signalling pathways, widely differ in their capacity to stimulate epithelial production of both CXC and CC chemokines.

A prospective study in children with a severe form of atopic dermatitis: clinical outcome in relation to cytokine gene polymorphisms.

BACKGROUND AND OBJECTIVE: The course of atopic dermatitis (AD) in childhood is characterized by typical changes in phenotype, including a shift from skin involvement to respiratory allergy usually around the third year of age. We thus designed a prospective study to monitor the outcome of severe AD and to investigate the association between cytokine gene polymorphisms and clinical manifestations. METHODS: Clinical and laboratory follow-up of 94 patients with severe AD and 103 healthy controls was performed using routine methodology. Allele, genotype, and haplotype frequencies of single nucleotide polymorphisms of 13 selected cytokine/receptor genes were analyzed using PCR with sequence-specific primers. RESULTS: In our study, genotypes of 7 polymorphisms--LL-4 -1098G/T and -590C/T, IL-6 -174C/G and nt565A/G, and IL-10 -1082A/G, -819C/T, and -592A/C were significantly associated with atopic AD (P < .05). A significant association was also found for TNF-alpha AA and IL-4 GC haplotypes and AD. We confirm the progressive clinical improvement of AD together with a decrease in the severity index SCORAD (SCORing atopic dermatitis) during childhood (P < .05). We found significant differences between IL-4Ralpha +1902 A/G and positivity of tree pollen-specific IgE (P < .05) in the AD group. Moreover, a weak association was also found between IL-10 -819C/T and IL-10 -590A/C and the appearance of allergic rhinitis (P < 0.1). CONCLUSIONS: We confirmed a clinical shift in allergic phenotype in the first 3 years of life, and showed an association between IL-4, IL-6, and IL-10 polymorphisms and AD. Our data indicate that IL-4alpha and IL-10 polymorphisms may be considered predictive factors of respiratory allergy in children with AD.

Epithelial cells modulate genes associated with NF kappa B activation in co-cultured human macrophages.

Macrophages located in airways and the alveolar space are continually exposed to different signals from the respiratory mucosa. In this respect, epithelial cells represent an important source of cytokines and mediators modulating the state of activation and/or differentiation of mononuclear phagocytes. Many of the proinflammatory genes induced in macrophages during immune and immunopathological reactions are regulated by transcription factor NF kappa B. The aim of our study was to characterize changes in the expression of genes associated with NF kappa B activation and signalling in THP-1 human macrophages co-cultured with A549 respiratory epithelial cells. At least 4-fold upregulation of mRNA level was found in 29 of 84 tested genes including genes for multiple cytokines and chemokines, membrane antigens and receptors, and molecules associated with NF kappa B signalling. The mRNA induction was confirmed at the level of protein expression by evaluating the release of IL-6 and IL-8 and by ICAM-1 expression. Blocking of one NFκB subunit by p65 siRNA inhibited the production of IL-6 in both cell types while IL-8 release from THP-1 cells did not seem to be affected. We conclude from our data that unstimulated respiratory epithelial cells regulate genes associated with NF kappa B dependent immune responses in human macrophages and that these interactions may play a key role in immediate responses in the respiratory mucosa.

Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid.

Uterine fibroid or leiomyoma is a frequent non-malignant tumour with unknown aetiology and pathogenesis. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of uterine fibroid. A large spectrum of Th1/Th2 cytokine gene polymorphisms in 102 patients with uterine leiomyoma was compared with 145 healthy controls. An association between polymorphisms of the IL4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL4 -590 and at position -33 was less frequent in the patient group than in the control group (P = 0.03). Besides IL-4, we observed different genotype distribution of the TNFA gene -308 A/G. The frequency of genotype AA was higher in the younger (≤ 35 years) patient group (P = 0.02). Our study thus suggests that certain cytokine gene polymorphisms, especially of the IL4 and TNFA genes, may be associated with increased risk for development of uterine fibroid. Further investigation would be needed to elucidate the mechanisms responsible for these associations.

Cytokine gene polymorphisms in sarcoidosis.

BACKGROUND: Sarcoidosis is a disease characterized by granuloma formation in many organs, but mostly in lung and lymph nodes. The immunopathogenic background of the disease is probably based on disregulation of immune response to different antigens. The imbalance of immune reactivity might be influenced by genetic background. In our study, we have investigated cytokine genetic polymorphisms in sarcoidosis group and compared the results with that of a group of healthy volunteers. METHODS: Thirty one sarcoidosis patients were enrolled to our study. Basic demographic data were collected. Polymorphisms in the promoter regions of the IL-1alpha, IL-1beta, IL-1R, IL-1RA, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha, IFN-gamma and in the translated regions of the TGF-beta, IL-1 beta, IL-2, IL-4 and IL-4RA genes were characterized. RESULTS: For IL-10, the (-819) and (-592) CC homozygosity was statistically more frequent in the sarcoidosis group compared to healthy controls. According to the haplotypes, the majority of sarcoidosis patients had IL-10 (-1082)(-819)(-592) ACC haplotype 2 compared to controls with ATA in most of the cases. CONCLUSIONS: The results of our study support the hypothesis of a genetically encoded immune regulation imbalance in sarcoidosis. The high-producer IL-10 (-819) and (-592) CC genotypes and intermediate- producer IL-10 (-1082) (-819) (-592) ACC haplotype 2 present in the majority of our sarcoidosis patients could support the role of genetically encoded disregulation of cell- mediated immune response to an unknown antigen.

Hepatocyte growth factor and antibodies to HLA and MICA antigens in heart transplant recipients.

Recent unconfirmed literature data suggest that elevated concentrations of the multifunctional cytokine hepatocyte growth factor (HGF) might be a marker of increased incidence of acute rejection after organ transplantation. The aim of this study was to test the hypothesis that HGF levels may correlate with the rejection and/or with the production of HLA and MHC Class I chain-related antigens A (MICA) specific antibodies. Sixty-three heart transplant recipients were included into the study. Hundred and eighty-five endomyocardial biopsies (EMB) obtained up to 6 months after transplantation were retrospectively analyzed for signs of cellular (CR) and antibody-mediated rejection (AMR). Pre- and post-transplant sera were tested for HGF concentrations and antibodies to HLA class I, class II and MICA antigens by xMap technology (Luminex). Pre-transplant HGF did not correlate with the incidence of CR or AMR. However, higher HGF concentrations correlated significantly with HLA antibody production before and after transplantation (P = 0.006 and P < 0.0001 respectively). Patients with both HLA class I and class II antibodies before transplantation had significantly lower AMR-free survival. Furthermore, recipients with pre-transplant donor-specific antibodies (DSA) had significantly lower AMR-free survival (50%) than recipients without pre-transplant HLA antibodies (90%) and patients with antibodies not specific to donor antigens (92%) (P = 0.005). Post-transplant MICA antibodies tended to be more frequent in patients with AMR (P = 0.063). In conclusion, elevated HGF concentrations in our study were not associated with the incidence of CR and/or AMR but with the presence of HLA-specific antibodies. Testing for DSA before heart transplantation by Luminex may be helpful for the identification of patients with increased risk of AMR.

Bronchoalveolar lavage fluid cellular characteristics, functional parameters and cytokine and chemokine levels in interstitial lung diseases.

Idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP) and sarcoidosis belong to interstitial lung diseases (ILD) where an imbalance of regulatory, profibrotic and antifibrotic cytokines is hypothesized. The relationship of bronchoalveolar lavage (BAL) fluid (BALF) cytokines, BALF cell profile and ILD course is supposed. The aim of our study was to correlate BALF cytokine and chemokine levels with BALF cellular characteristics and lung function parameters in different ILD. Twenty-two sarcoidosis, seven IPF and 11 HP patients underwent lung function tests and BAL. The BALF differential cell counts and superficial cell markers were characterized, and MCP-1, MIP-1alpha, MIP-1beta, RANTES, epithelial neutrophil-activating protein (ENA)-78, FGF, G-CSF, GM-CSF, IFN-gamma, interleukin (IL)-1alpha, IL-1RA, IL-1beta, -2beta, -4beta, -5beta, -6beta, -8beta, -10beta, -17beta, tumour necrosis factor (TNF)-alpha, thromobopoietin (Tpo) and vascular endothelial growth factor (VEGF) values measured. The BALF VEGF values were highest in sarcoidosis (P = 0.0526). IL-1RA values were higher in IPF and HP compared with sarcoidosis (P = 0.0334). IL-8/ENA-78 ratio positively correlated with BALF neutrophil counts in IPF (r = 0.89, P = 0.04). Vital capacity and TL(CO) values positively correlated with VEGF and negatively with IL-8 BALF levels in all ILDs but the correlations were most significant in sarcoidosis group. We suppose that VEGF plays a role in ILDs' early phases and has rather angiogenic than profibrotic effect. On the contrary, IL-8 is probably upregulated in advanced ILDs with prominent fibrosis and marked lung functions decline. We state that BALF VEGF, IL-8 and ENA-78 levels and IL-8/ENA-78 ratio could become useful markers of ILDs' phase, activity and prognosis. They might also be helpful in treatment modality choice.

Distribution of KIR genes in the Czech population.

Killer cells immunoglobulin-like receptors (KIRs) are a family of inhibitory and activating receptors expressed mainly by natural killer (NK) cells and few subsets of T lymphocytes. KIRs regulate NK cells' activity through interactions with specific HLA class I molecules and other yet unknown ligands presented on target cells. At present, 17 KIR genes and pseudogenes have been identified. As the number of KIR genes in different haplotypes varies, a wide range of genotypes in different ethnic populations may be observed. In our study, 125 healthy non-related Czech individuals were KIR typed both by sequence-specific primers and by sequence-specific oligonucleotide KIR genotyping methods. Thirty-eight different genotypes were observed in the Czech population and all 16 KIR genes known to date were found. Framework genes KIR 3DL3, KIR 2DL4, KIR 3DL2 and the pseudogene KIR 3DP1 were present in all individuals. The most frequent non-framework KIR genes detected in the Czech population were: KIR 2DL1 (95%), KIR 3DL1 (94%), KIR 2DS4 (92%) and the pseudogene 2DP1 (94%). Human leucocyte antigen (HLA)-C typing demonstrated prevalence of the C1/C2 heterozygosity (43%) and C1 homozygosity (41%) over the C2 heterozygosity. One hundred and twenty individuals from our panel carried at least one inhibitory KIR for the corresponding HLA-C group found in the genotype. Gene frequencies and found genotypes demonstrated similarity of the Czech population's KIR repertoire with the KIR repertoires of other Caucasian populations studied before.

Polymorphism of interleukin-18 promoter influences the onset of kidney graft function after transplantation.

It has been well recognized that the promoter polymorphisms of interleukin-18 (IL-18) influence the level of cytokine expression. In our previously published data, we showed constitutive IL-18 expression in the epithelium of renal distal tubules in patients after kidney transplantation and significantly elevated IL-18 expression during acute rejection. In this study, we evaluated the clinical significance of two functional promoter polymorphisms of the IL-18 gene at positions -607 A/C (rs1946518) and -137 C/G (rs187238) in patients after kidney transplantation and looked for associations with the onset of graft function and the incidence of rejection episodes. Promoter polymorphisms in 124 patients and 103 unrelated controls were evaluated by sequence-specific primer polymerase chain reaction and the allele, genotype and haplotype frequencies were statistically correlated. We found a statistically different distribution of the allele frequency of -607 A/C polymorphism between patients with immediate or delayed onset of kidney graft function. Data showed that the C allele, which contributes to higher IL-18 expression, is more frequent in patients with delayed onset of function (P = 0.03, odds ratio = 1.93; 95% confidence interval = 1.15-3.25). A/C single nucleotide polymorphisms of the IL-18 promoter at position -607 may influence the onset of early kidney allograft function.

Cytokine gene polymorphisms in the Dutch population.

Numerous studies have shown that variations in the production and activity of cytokines influence the susceptibility and/or resistance to various infectious agents, autoimmune diseases, and cancer, as well as the predisposition to allograft rejection. Differences in the production of cytokines between individuals are often caused by single nucleotide polymorphisms (SNP) in the promoter or coding regions of cytokine genes. The cytokine polymorphisms of 107 unrelated Caucasian individuals originating from various parts of the Netherlands were studied and compared with the results of two European (Czech and Italian) populations. Twenty-two SNPs of 13 different cytokine genes were analysed. To test the Hardy-Weinberg equilibrium, allele frequencies were estimated by direct gene counting. Evaluation of the allele frequencies of the Dutch, Italian and Czech populations showed that five SNPs were significantly different between the Dutch and the Italians, while these SNPs did not vary between the Dutch and the Czechs. This analysis, in combination with other types of immune profiling, may be helpful for prediction of the clinical outcome of various infectious and immune-related disorders, as well as for estimation of the risk for rejection and graft vs. host disease after organ or stem cell transplantation.

Correlation of IL-1alpha and IL-4 gene polymorphisms and clinical parameters in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a serious disease characterized with progressive scarring of the lungs in which a genetic background is supposed. We have tested correlation of promotor regions of IL-1alpha and IL-4 gene polymorphisms with clinical parameters in IPF. We investigated the group of 30 patients with IPF. The correlations of vital capacity (VC) and diffusing capacity for carbon monoxide (DL(CO)), bronchoalveolar lavage (BAL) fluid cell counts and high resolution computed tomography (HRCT) alveolar and interstitial scores with different genotypes of IL-4 at (-1098), (-590) and (-33) positions and IL-1 alpha at (-889) position were tested. The PCR method was used for genotyping. The carriers of CT genotype at IL-1 alpha (-889) position had higher VC at the time of diagnosis. The CC genotype at this position was more frequent in patients with higher counts of HLADR+ T lymphocytes in BAL. The GT genotype at IL-4 (-1098) position correlated with higher counts of CD4(+) T lymphocytes, and inversely the TT genotype with higher counts of CD8(+) T lymphocytes in BAL fluid. According to dynamic changes of HRCT score the CT genotype at IL-4 (-33) was more frequent in patients with progressive disease compared to that with stable disease. We assume from our data that the gene polymorphisms of the promotor region of IL-4 at position (-1098) and (-33) and IL-1 alpha at position (-889) are likely to play a pathogenic role in IPF and in modification of its clinical presentation and severity.

Strategy aimed at reduction of sputum eosinophils decreases exacerbation rate in patients with asthma.

Under Global Initiative for Asthma guidelines, the clinical control of disease activity and the adjustment of treatment in patients with asthma are based on symptoms, use of rescue medication, lung function and peak expiratory flow measurement (standard strategy). We investigated whether a strategy to reduce the number of sputum eosinophils (EOS strategy) gives better clinical control and a lower exacerbation rate compared with the standard strategy. Fifty-five patients with moderate to severe asthma entered this open, randomized, parallel-group study and visited the out-patient department every 3 months for 18 months. The dose of corticosteroids was adjusted according to the standard strategy or the percentage of sputum eosinophils (EOS strategy). During the study period, the EOS strategy led to a significantly lower incidence of asthma exacerbations compared with the standard strategy group (0.22 and 0.78 exacerbations per year per patient, respectively). There were significant differences between the strategies in time to first exacerbation.

Th1/Th2 cytokine gene polymorphisms in patients with idiopathic pulmonary fibrosis.

We investigated 30 patients with idiopathic pulmonary fibrosis (IPF) and 103 healthy volunteers for the cytokines polymorphisms of the IL-1alpha, IL-1beta, IL-1R, IL-1RA, IL-2, IL-4, IL-6, IL-10, IL-12, tumor necrosis factor-alpha, interferon-gamma, transforming growth factor-beta, IL-1beta, IL-2, IL-4, and IL-4RA genes. The strongest correlation of a genotype with the disease was found for gene polymorphisms at the promotor region of IL-4, where the CT genotypes at the positions (-590) and (-33) were more frequent in the IPF group (P < 0.0001, P(corr) < 0.0022; vs P < 0.0001, P(corr) < 0.0022). Our results support the idea of the pathogenic role of cytokine gene polymorphisms in the etiology and pathogenesis of IPF, with emphasize on the IL-4 promotor gene polymorphisms.

[The risk of malignancy after organ transplantation]. / Riziko vzniku malignity po orgáinové transplantaci.

Improvements in immunosuppressive therapy during the past decade brought about improvements of the long term tolerance of organ allografts. However, the long-term immunosuppressive therapy has an important limitation, because it can increase the risk of cardivascular diseases, infections and tumors. As compared with age-matched healthy population, organ-transplant recipients have an increased incidence of tumors.