Polymorphism of HLA-DRB4 gene in Croatia

U ovom istraživanju analiziran je polimorfizam gena HLA-DRB4 i haplotipova HLA-DRB1-DRB4 u Hrvatskoj među kadaveričnim davateljima organa (N=144). Svi uzorci bili su odabrani na temelju prisustva jednog od alela skupine HLA-DRB53 (aleli DRB1*04, DRB1*07 i DRB1*09). Određivanje alela lokusa HLA-DRB4 provedeno je metodom lančane reakcije polimerazom i početnicama specifičnim za jedan ili više alela HLA (engl. Polymerase Chain Reaction–Sequence Specific Primers, PCR-SSP). Aleli lokusa HLA-DRB1 i -DQB1 određeni su metodom lančane reakcije polimerazom i probama specifičnim za jedan ili grupu alela HLA (engl. Polymerase Chain Reaction–Sequence Specific Oligos, PCR-SSO). Na lokusu HLA-DRB4 otkriveno je sedam različitih alela, 5 funkcionalnih alela (DRB4*01:01:01:01, DRB4*01:02, DRB4*01:03, DRB4*01:05 i DRB4*01:08) i dva nul alela (DRB4*01:03:01:02N i HLA-DRB4*03:01N). Među ispitanicim pozitivnim za jedan od alela skupine HLA-DRB1*04 nije otkrivena statistički značajna razlika u raspodjeli alela DRB4 s obzirom na prisustvo alela DQB1. U haplotipovima HLA-DRB1*04-DQB1 najčešći prisutni alel je bio DRB4*01:03 (86,7%). Među HLA-DRB1*07:01 pozitivnim haplotipovima uočena je statistički značajna razlika (p<0,05) u raspodjeli alela HLA-DRB4 s obziroma na prisustvo alela HLA-DQB1. Haplotipovi: HLA-DRB1*07:01-DQB1*03:03 na lokusu HLA-DRB4 u 98,0% slučajeva imali su nul alel DRB4*01:03:01:02N, dok su haplotipovi DRB1*07:01-DQB1*02:02 na lokusu DRB4 u 65,5% slučajeva imali prisutan alel DRB4*01:03. Unutar skupine HLA-DRB1*04 pozitivnih haplotipova nije uočena statistički značajna razlika u raspodjeli alela HLA-DRB4 s obzirom na prisustvo alela HLA-DQB1. Alel HLA-DRB1*09:01 je kod svih ispitanika uočen u kombinaciji s alelima HLA-DRB4*01:03-DQB1*03:03.In this study the polymorphism of HLA-DRB4 alleles and HLA-DRB1-DRB4 haplotypes among Croatian cadaveric organ donors (N = 144) was analyzed. All samples were selected based on the presence of one of the alleles of HLA-DRB53 group (DRB1*04, DRB1*07, and DRB1*09 alleles). Typing of HLA-DRB4 alleles was carried out by polymerase chain reaction sequence specific primers for one or more HLA alleles (method: PCR-SSP). HLA-DRB1 and -DQB1 alleles were determined by polymerase chain reaction sequence specific oligos for one allele or a group of HLA alleles (method: PCR-SSO). On the HLA-DRB4 locus seven different alleles were discovered; 5 functional alleles (DRB4*01:01:01:01, DRB4*01:02, DRB4*01:03, DRB4*01:05, and DRB4*01:08) and two null alleles (DRB4*01:03:01:02N and HLA-DRB4*03:01N). Among individuals positive for one of the HLA-DRB1*04 alleles no statistically significant difference was detected in the distribution of the HLA-DRB4 alleles given the presence of the DQB1 alleles. Among HLA-DRB1*04-DQB1 haplotypes the most present was DRB4*01:03 allele (86.7%). Among HLA-DRB1*07:01 positive haplotypes a statistically significant difference (p<0.05) in the distribution of HLA-DRB4 alleles due to the presence of different HLA-DQB1 alleles was observed. The HLA-DRB1*07:01-DQB1*03:03 haplotype in 98.0% of cases had the DRB4*01:03:01:02N allele on the HLA-DRB4 locus, while DRB1*07:01-DQB1*02:02 haplotype on the HLA-DRB4 locus in 65.5% of cases had the DRB4*01:03 allele (65.5%). Within the group of the HLA-DRB1*04 positive haplotype, there was no statistically significant difference in the distribution of HLA-DRB4 alleles due to the presence of different HLA-DQB1 alleles. The HLA-DRB1*09:01 allele was observed in combination: HLA-DRB4*01:03-DQB1*03:03 in all the cases

Microflora and health

Neke bakterije mliječne kiseline su prilagođene za rast u biljnim hranidbenim podlogama gdje su fenolni spojevi u izobilju. Većina proučavanih fenolnih spojeva ima inhibitorni učinak na rast bakterija mliječne kiseline. Rasvjetljavanje metaboličkih puteva bi dovelo do dobivanja biotehnološki korisnih sojeva i bjelančevina. Ti sojevi ili proteini će biti adekvatni u postupcima razrade za dobivanje hrane s poboljšanim senzorskim i prehrambenih svojstvima. Iako su brojna istraživanja dovela do rezultata u posljednjih nekoliko godina još uvijek nije moguće dobiti uvjerljive kliničke dokaze uloge probiotika u nastanku raka debelog crijeva. Rak debelog crijeva je nepraktičan u smislu broja subjekata, troškova, trajanja istraživanja i etičkih načela. Nekoliko mehanizama bi moglo objasniti preventivno djelovanje probiotika protiv početka nastanka raka debelog crijeva. Najvjerojatnije različiti sojevi probiotika rade sa specifičnim mehanizmima. Također, korištenje probiotičkih bakterija dokazuje pozitivno djelovanje u prevenciji i terapiji akutnih dijareja u dojenčadi i djece, kao i kod dijareja uzrokovanih uzimanjem antibiotika, a to ima prvenstveno značenje u smanjenju troškova liječenja. Daljnja istraživanja jako su potrebna kako bi se utvrdio utjecaj svakog mehanizma i stvarna korist od probiotika u liječenju brojnih bolesti.Some lactic acid bacteria are adapted to growth in plant nutritional substrates where are phenolic compounds in abundance. Most of the studied phenolic compounds is carried the inhibitory effect on the growth of lactic acid bacteria. The elucidation of metabolic pathways would lead to getting biotechnology useful strains and proteins. These strains or proteins will be adequate in the elaboration of procedures for obtaining food with improved organoleptic and nutritional properties. Although numerous studies have led to the results in the last few years, it is still not possible to obtain conclusive clinical evidence of the role of probiotics in the development of colon cancer. Colorectal cancer is impractical in terms of the number of the subject, cost, duration of studies and ethical principles. Several mechanisms could explain the preventive effect of probiotics against the beginning of developing colorectal cancer. Most likely different strains of probiotics work with specific mechanisms. Also, the use of probiotic bacteria proven positive effect in the prevention and treatment of acute diarrhea in infants and children, as well as diarrhea caused by taking antibiotics, and it has meaning primarily in reducing the cost of treatment. Further studies are strongly required to determine the impact of each mechanism and the actual benefit of probiotics in the treatment of many diseases

The Need for Systematic Monitoring and Improved Surveillance of Hepatitis C Patients in Croatia

Aim: The aim of this study was emphasizing the need for a more systematic monitoring of patients diagnosed with HCV in Croatia. Methods: From 2014 to 2018, at the University Hospital for Infectious Diseases, sera from 23,524 patients were tested for HCV. Confirmatory testing was performed by Western Blot. Adult patients with an anti-HCV positive screening test were analysed. HCV RNA was quantified by real-time PCR, while HCV genotypes and subtypes were determined by PCR and the reverse hybridization method. Results: A total of 428 anti-HCV ELISA-positive adults were analysed (68.7% males, 31.3% females, median age 43 years, range 19-88 years). Hepatitis C was confirmed by WB in 390, while 28 patients had borderline WB results. Anti-HCV was not confirmed by WB in 10 patients. HCV RNA was tested in 331 patients and viremia was detected in 218 patients. There was no data on HCV RNA in 97 patients (22.66%). HCV genotypes/subtypes were determined in 185 of 218 anti-HCV WB positive patients. Genotype 1 was detected in 97/185 (52.43%), genotype 2 was detected in 3/185 (1.62%), while subtype 3a was detected in 76/185 (41.08%) and genotype 4 in 9/185 patients (4.86%). Conclusion: In a five-year period, the HCV seroprevalence rate in subjects tested at the University Hospital for Infectious Diseases was 1.81%. According to the data analysed, almost one quarter of patients with detected anti-HCV antibodies were not treated further, which indicates the need for a systematic monitoring of patients diagnosed with HCV. It is necessary to determine viremia after a positive anti-HCV screening result in order to initiate treatment and prevent HCV-related complications. (Radmanić L, Cetinić Balent N, Šimičić P, Vince A, Židovec Lepej S, Đaković Rode O. The Need for Systematic Monitoring and Improved Surveillance of Hepatitis C Patients in Croatia. SEEMEDJ 2020; 4(2); 28-34

Changes in the stage of fibrosis and steatosis in patients with chronic hepatitis C virus treated with direct-acting antiviral drugs who achieved sustained virological response

Hepatitis C vodeći je uzrok kronične bolesti jetre, hepatocelularnog karcinoma i važna indikacija za transplantaciju jetre. Glavni cilj liječenja je postizanje trajnog virološkog odgovora koji označava negativnu viremiju 12 tjedana nakon završetka liječenja (SVR, od engl. sustained virological response). Uvođenjem antivirusnih lijekova s izravnim djelovanjem, povećala se stopa SVR-a, skratilo vrijeme trajanja terapije i utjecalo se na stadij fibroze i steatoze jetre nakon liječenja. Cilj ovog rada bio je analizirati promjenu stadija fibroze (F1-F4) i steatoze (S0-S3) primjenom tranzijentne elastografije u prethodno neliječenih bolesnika s kroničnim hepatitisom C (n=205) koji su postigli trajni virološki odgovor, a bili liječeni isključivo direktno djelujućim antivirusnim lijekovima (DAA).Hepatitis C is the leading cause of chronic liver disease, hepatocellular carcinoma and an important indication for liver transplantation. The main objective of the treatment is to achieve a sustained virological response defined as a negative HCV RNA 12 weeks after completion of therapy (SVR). Direct-acting antivirals have been developed to treat chronic HCV infection. They have resulted in increased SVR rates, shorter and simpler treatment regimens, and minimal adverse effects. The aim of this study was to analyze the change in the stage of fibrosis (F1-F4) and steatosis (S0-S3) using transient elastography in treating naive patients with chronic hepatitis C (n = 205) who achieved a sustained virological response and were treated exclusively with directly acting antiviral drugs (DAA)

The Role of Stem Cell Factor, Epidermal Growth Factor and Angiopoietin-2 in HBV, HCV, HCC and NAFLD

Growth factors play a significant role in the immunopathogenesis of liver diseases, especially in liver fibrosis and cirrhosis. They can also play a role in liver regeneration and tissue repair. The regenerative capacity of the liver has been well established. Molecular mechanisms leading to regeneration involve a complex network of diverse molecules. Chronic liver injury leads to the dysregulation of regenerative mechanisms in the liver that, in addition to molecular oncogenesis, lead to uncontrolled cell proliferation and development of hepatocellular carcinoma (HCC). Stem cell factor (SCF), epidermal growth factor (EGF) and Angiopietin-2 (Ang-2) have been shown to be extremely important in the pathogenesis of liver diseases, and given their role in hepatitis B (HBV) or C virus (HCV), HCC and nonalcoholic fatty liver disease (NAFLD), they seem to be potential targets for future research into antifibrotic drugs. The role of SCF receptor c-kit in the liver is debatable, as it has impact on both liver regeneration and liver disease. EGF is a potential indicator of the survival of patients with HCC and can be a biomarker and therapeutic target structure in HCC. Further research is needed to investigate the potential role of Ang-2 for NAFLD associated with liver damage as a non-invasive circulating biomarker

Molecular Epidemiology and Baseline Resistance of Hepatitis C Virus to Direct Acting Antivirals in Croatia

Molecular epidemiology of hepatitis C virus (HCV) is exceptionally complex due to the highly diverse HCV genome. Genetic diversity, transmission dynamics, and epidemic history of the most common HCV genotypes were inferred by population sequencing of the HCV NS3, NS5A, and NS5B region followed by phylogenetic and phylodynamic analysis. The results of this research suggest high overall prevalence of baseline NS3 resistance associate substitutions (RAS) (33.0%), moderate prevalence of NS5A RAS (13.7%), and low prevalence of nucleoside inhibitor NS5B RAS (8.3%). Prevalence of RAS significantly differed according to HCV genotype, with the highest prevalence of baseline resistance to NS3 inhibitors and NS5A inhibitors observed in HCV subtype 1a (68.8%) and subtype 1b (21.3%), respectively. Phylogenetic tree reconstructions showed two distinct clades within the subtype 1a, clade I (62.4%) and clade II (37.6%). NS3 RAS were preferentially associated with clade I. Phylogenetic analysis demonstrated that 27 (9.0%) HCV sequences had a presumed epidemiological link with another sequence and classified into 13 transmission pairs or clusters which were predominantly comprised of subtype 3a viruses and commonly detected among intravenous drug users (IDU). Phylodynamic analyses highlighted an exponential increase in subtype 1a and 3a effective population size in the late 20th century, which is a period associated with an explosive increase in the number of IDU in Croatia

The Effect of Treatment-Induced Viral Eradication on Cytokine and Growth Factor Expression in Chronic Hepatitis C

In this study, we evaluated the effect of hepatitis C virus eradication using direct-acting antivirals (DAA) on the serum cytokine and growth factor profiles of chronic hepatitis C patients (CHC). Serum concentrations of 12 cytokines and 13 growth factors were measured in 56 patients with CHC before, during the DAA treatment and after sustained virological response using bead-based flow cytometry. Cytokine and growth factor levels were also measured in 15 healthy individuals. The majority of the selected cytokines and growth factors exhibited similar concentrations before, during and after successful DAA treatment, the exceptions being IL-10, EGF, HGF and VEGF. Significantly lower concentrations of IL-10, IL-13, IL-4, IL-4, IL-9, TNF- α and higher levels of Ang-2, HGF and SCF were observed in patients with CHC before and after DAA treatment compared with healthy individuals. Patients with severe fibrosis stages exhibited higher levels of Ang-2 and lower levels of EGF, PDGF-AA and VEGF. Furthermore, IL-4, IL-5 and SCF were characterized as potential biomarkers of DAA treatment using random forest. Additionally, logistic regression characterized EGF as a potential biomarker of severe CHC. Our results suggest inhibition of pro-inflammatory processes and promotion of liver regeneration in CHC patients during DAA treatment

The Effect of Treatment-Induced Viral Eradication on Cytokine and Growth Factor Expression in Chronic Hepatitis C

In this study, we evaluated the effect of hepatitis C virus eradication using direct-acting antivirals (DAA) on the serum cytokine and growth factor profiles of chronic hepatitis C patients (CHC). Serum concentrations of 12 cytokines and 13 growth factors were measured in 56 patients with CHC before, during the DAA treatment and after sustained virological response using bead-based flow cytometry. Cytokine and growth factor levels were also measured in 15 healthy individuals. The majority of the selected cytokines and growth factors exhibited similar concentrations before, during and after successful DAA treatment, the exceptions being IL-10, EGF, HGF and VEGF. Significantly lower concentrations of IL-10, IL-13, IL-4, IL-4, IL-9, TNF- &alpha; and higher levels of Ang-2, HGF and SCF were observed in patients with CHC before and after DAA treatment compared with healthy individuals. Patients with severe fibrosis stages exhibited higher levels of Ang-2 and lower levels of EGF, PDGF-AA and VEGF. Furthermore, IL-4, IL-5 and SCF were characterized as potential biomarkers of DAA treatment using random forest. Additionally, logistic regression characterized EGF as a potential biomarker of severe CHC. Our results suggest inhibition of pro-inflammatory processes and promotion of liver regeneration in CHC patients during DAA treatment

Distinct Expression Patterns of Genes Coding for Biological Response Modifiers Involved in Inflammatory Responses and Development of Fibrosis in Chronic Hepatitis C: Upregulation of SMAD-6 and MMP-8 and Downregulation of CAV-1, CTGF, CEBPB, PLG, TIMP-3, MMP-1, ITGA-1, ITGA-2 and LOX

Background and Objectives: The aim of this study was to analyze the expression of genes on transcriptomic levels involved in inflammatory immune responses and the development of fibrosis in patients with chronic hepatitis C. Materials and Methods: Expression patterns of 84 selected genes were analyzed with real-time quantitative RT PCR arrays in the peripheral blood of treatment-naive patients with chronic hepatitis C and healthy controls. The panel included pro- and anti-fibrotic genes, genes coding for extracellular matrix (EMC) structural constituents and remodeling enzymes, cell adhesion molecules, inflammatory cytokines, chemokines and growth factors, signal transduction members of the transforming growth factor- beta (TGF-ß) superfamily, transcription factors, and genes involved in epithelial to mesenchymal transition. Results: The expression of SMAD-6 coding for a signal transduction TGF-beta superfamily member as well as MMP-8 coding for an ECM protein were significantly increased in CHC patients compared with controls. Conclusions: Chronic hepatitis C was also characterized by a significant downregulation of a set of genes including CAV-1, CTGF, TIMP-3, MMP-1, ITGA-1, LOX, ITGA-2, PLG and CEBPB encoding various biological response modifiers and transcription factors. Our results suggest that chronic hepatitis C is associated with distinct patterns of gene expression modulation in pathways associated with the regulation of immune responses and development of fibrosis

Distinct Cytokine Profiles in Severe COVID-19 and Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is identified as a risk factor for developing severe COVID-19. While NAFLD is associated with chronic low-grade inflammation, mechanisms leading to immune system hyperactivation remain unclear. The aim of this prospective observational study is to analyze cytokine profiles in patients with severe COVID-19 and NAFLD. A total of 94 patients with severe COVID-19 were included. Upon admission, clinical and laboratory data were collected, a liver ultrasound was performed to determine the presence of steatosis, and subsequently, 51 were diagnosed with NAFLD according to the current guidelines. There were no differences in age, sex, comorbidities, and baseline disease severity between the groups. Serum cytokine concentrations were analyzed using a multiplex bead-based assay by flow cytometry. Upon admission, the NAFLD group had higher C-reactive protein, procalcitonin, alanine aminotransferase, lactate dehydrogenase, and fibrinogen. Interleukins-6, -8, and -10 and CXCL10 were significantly higher, while IFN-γ was lower in NAFLD patients. Patients with NAFLD who progressed to critical illness had higher concentrations of IL-6, -8, -10, and IFN-β, and IL-8 and IL-10 appear to be effective prognostic biomarkers associated with time to recovery. In conclusion, NAFLD is associated with distinct cytokine profiles in COVID-19, possibly associated with disease severity and adverse outcomes