Cytokines in human immunodeficiency virus type 1 inefction

Cilj ovog istraživanja bio je usporediti ekspresiju citokina na razini gena i proteina u akutnoj i kroničnoj fazi infekcije HIV-om-1, te analizirati učinak dugotrajne supresije virusne replikacije u periodu duljem od dvije godine na citokinsku imunost. U dio istraživanja koji se bavi usporedbom citokinske imunosti na proteinskoj razini u akutnoj i kroničnoj fazi infekcije uključila sam 34 ispitanika, dok sam u istraživanje učinka dugotrajne supresije virusne replikacije na ekspresiju citokina u infekciji HIV-om-1 uključila 80 ispitanika. Ekspresiju 84 citokinska gena analizirala sam u tri ispitanika u akutnoj i tri u kroničnoj infekciji HIV-om-1 metodom „PCR array“. Za kvantifikaciju Th1/Th2/Th9/Th17/Th22 citokina primjenila sam citometriju pomoću kuglica (bead-based cytometry). U kroničnoj infekciji HIV-om-1 dokazala sam statistički značajno povećanje ekspresije 13 citokinskih gena (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11,14 i 8), te sniženje ekspresije il12a. U akutnoj fazi je u odnosu na kontrolnu skupinu došlo do povećanja koncentracija IL-10, IL-4 i TNF-α. Povećane koncentracije IL-10 i TNF-α, te uz njih IL-2, IL-6, IL-13 i IL-22 otkrivene su u kroničnoj infekciji u odnosu na kontrolu. Usporedbom ekspresije citokina između dviju faza infekcije HIV-om-1 vidljivo je smanjenje koncentracija IL-9. Koncentracija IL-17A snižena je u ispitanika prije primjene antiretrovirusne terapije, a kod kojih je dokazana supresija virusne replikacije. Ekspresija aktivacijskih biljega CD38 i HLA-DR snižena je u kroničnoj u odnosu na akutnu infekciju, te u supresiji virusne replikacije u odnosu na uzorke prije primjene terapije. U ovom je istraživanju dokazana promjena citokinskih profila u infekciji HIV-om-1 između akutne i kronične faze na razini genske i proteinske ekspresije.The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 infection and to analyze the effect of long-term suppression of viral replication in period longer than 2 years on cytokine immunity. To analyze cytokine expression on protein level in acute and chronic phase of HIV type 1 infection 34 patients were enrolled and the effect of long-term suppression of viral replication was determined in 80 HIV-positive patients. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic phase of HIV type 1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and down regulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed significant decrease in IL-9 concentration. Level of IL-17 was lower in therapy-naive patients with a suppression of viral replication. Expression of activation markers CD38 and HLA-DR was lower in a chronic stage of infection compared to acute infection as well as in samples with suppression of viral replication compared to samples before therapy initiation. This study showed change in cytokine profile in both gene and protein expression in different stages of HIV-infection

The comparison of Th1, Th2, Th9, Th17 and Th22 cytokine profiles in acute and chronic HIV-1 infection

The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 (HIV-1) infection. Thirty four patients were enrolled for cytokine expression analysis on protein level in acute and chronic stage of HIV-1 infection. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic stage of HIV-1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and downregulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed a significant decrease in IL-9 concentration. This study showed changes in cytokine profiles on both gene and protein levels in different stages of HIV-infection

HIV subtype diversity and primary resistance to antiretroviral drugs in newly diagnosed HIV-infected persons from Croatia in 2013

Cilj ovog istraživanja bio je odrediti heterogenost podtipova HIV-a te analizirati primarnu rezistenciju virusa na antiretrovirusne lijekove u 30 prethodno neliječenih osoba koje su uključene u kliničku skrb Referentnog centra za dijagnostiku i liječenje HIV/AIDS-a tijekom 2013. g. Sekvenciranjem pol gena HIV-a te primjenom bioinformatičkih algoritama određeni su podtipovi virusa te detektirane mutacije povezane s primarnom rezistencijom na antiretrovirusne lijekove. Većina ispitanika (86,7%) bila je zaražena podtipom B. Otkriveno je 12 mutacija povezanih s primarnom rezistencijom HIV-a na antiretrovirusne lijekove, tj. 9 mutacija povezanih s primarnom rezistencijom na nukleozidne analoge inhibitore reverzne transkriptaze (NRTI) te 3 mutacije povezane s rezistencijom na nenukleozidne inihibitore reverzne transkriptaze (NNRTI). Primarna rezistencija na dvije klase antiretrovirusnih lijekova (NRTI i NNRTI) otkrivena je u jednog ispitanika. Najzastupljenija mutacija povezana s primarnom rezistencijom virusa na lijekove bila je T215S (rezistencija na NRTI). Mutacije značajne za nastanak primarne rezistencije detektirane su u 4 osobe zaražene podtipom B, jedne osobe zaražene podtipom A1 te jedne osobe zaražene rekombinantom podtipova A1 i B. U ovom je istraživanju po prvi puta dokazana pojava primarne rezistencije HIV-a na antiretrovirusne lijekove u osoba zaraženih non-B podtipovima virusa iz Hrvatske.The aim of this study was to analyse HIV subtype diversity as well as primary resistance to antiretroviral drugs in 30 treatment-naive individuals entering clinical care at the National Refence Center for HIV/AIDS during 2013. Sequencing the pol region of the viral genome and bioinformatic algorithms were used to determine HIV subtypes and to analyse transmitted drug resistance mutations. The majority of individuals (86,7 %) were infected with HIV-1 subtype B. Twelve mutations associated with primary resistance to antiretroviral drugs were detected in the study: 9 mutations conferring resistance to NRTI (nucleotide reverse-transcriptase inhibitors) and 3 mutations associated with resistance to NNRTI (non-nucleotide reverse-transcriptase inhibitors). Primary resistance to two antiretroviral drug classes (NRTI and NNRTI) were detected in one individual. The most frequent mutation was T1215S associated with primary resistance to NRTI. Drug resistance mutations were detected in 4 subtype B and 2 non-B HIV-infected individuals. This study showed, for the first time, the presence of mutations associated with primary resistance of HIV to antiretroviral drugs in individuals infected with non-B subtypes in Croatia

Interleukin-28B polymorphism in persons with chronic hepatitis C in Croatia

Polimorfizam jednog nukleotida (engl. single nucleotide polymorphism, SNP) rs12979860 u blizini promotora gena za interleukin-28B (IL-28B) tj. genotipizacija IL-28B povezana je s prirodnim tijekom infekcije virusom hepatitisa C kao i s odgovorom na antivirusno i imunomodulacijsko liječenje kombinacijom pegiliranog interferona alfa i ribavirina (tzv. dvojna terapija). U eri direktno djelujućih antivirusnih lijekova genotip IL-28B više nije značajan prediktor ishoda liječenja no i dalje je važan parametar koji određuje prirodni tijek povijesti HCV infekcije. U zemljama koje nemaju univerzalni pristup novim terapijskim opcijama genotipizacija IL-28B i dalje se primjenjuje u pred-terapijskoj obradi bolesnika koji se liječe dvojnom terapijom. Cilj ovog rada bio je analizirati rezultate genotipizacije IL-28B (udio homozigota za T i C alel te heterozigota) u 595 bolesnika s kroničnim hepatitisom C koji su bili uključeni u kliničku skrb Zavoda za virusni hepatitis Klinike za infektivne bolesti "Dr. Fran Mihaljević" u Zagrebu od siječnja 2013. do prosinca 2015. g. SNP rs12979860 detektiran je metodom lančane reakcije polimerazom u stvarnom vremenu. Analiza SNP-a rs12979860 u genu za IL-28B pokazala je da su 335 od 595 (56,3%) ispitanika bili CT heterozigoti, 173 od 595 ispitanika (29,1%) C homozigoti i 87 od 595 ispitanika (14,6%) T homozigoti. Distribucija C i T alela u SNP-u rs12979860 gena za IL-28B sukladni su literaturnim podatcima analize prevalencije ovih alela u osoba bijele rase europskog porijekla.Single nucleotide polymorphism (SNP) rs12979860 in the promotor region of the gene coding for interleukin-28B (IL-28B) is associated with the natural history of hepatitis C virus (HCV) infection and response to antiviral and immunomodulatory therapy with combination of pegylated interferon alpha and ribavirin (dual therapy). Despite the fact that the predictive role IL-28B genotyping is decreasing in the era of highly successful direct acting antiviral (DAA)-based treatment, it remains to be useful as a tool for predicting a natural course of HCV infection. In resource-limited settings with restricted access to new treatment options IL-28B polymorphism typing is still a part of pre-treatment workup before admission of dual therapy. The aim of this study was to analyse the results of IL-28B genotyping (percentages of C and T homozygotes and heterozygotes) for 595 Caucasian patients with chronic hepatitis C receiving clinical care at the Department of Viral Hepatitis of the University Hospital for Infectious Diseases, Zagreb and Croatian Reference Center for Viral Hepatitis since January 2013 till December 2015. Detection of single nucleotide polymorphism (SNP) rs12979860 was determined by using a real-time PCR test. Analysis of rs12979860 SNP in the IL-28B gene showed that 335 of 595 (56.3%) patients were CT heterozygotes, followed by C homozygotes (173 of 595 patients, 29.1%) whereas T homozygotes were the least frequent (87 of 595 patients, 14.6%). The patterns of IL-28 polymorphism in Croatian patients with chronic hepatitis C is in accordance with other results for Caucasians

The comparison of Th1, Th2, Th9, Th17 and Th22 cytokine profiles in acute and chronic HIV-1 infection

The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 (HIV-1) infection. Thirty four patients were enrolled for cytokine expression analysis on protein level in acute and chronic stage of HIV-1 infection. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic stage of HIV-1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and downregulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed a significant decrease in IL-9 concentration. This study showed changes in cytokine profiles on both gene and protein levels in different stages of HIV-infection

Human Cytomegalovirus (HCMV) Genetic Diversity, Drug Resistance Testing and Prevalence of the Resistance Mutations: A Literature Review

Human cytomegalovirus (HCMV) is a pathogen with high prevalence in the general population that is responsible for high morbidity and mortality in immunocompromised individuals and newborns, while remaining mainly asymptomatic in healthy individuals. The HCMV genome is 236,000 nucleotides long and encodes approximately 200 genes in more than 170 open reading frames, with the highest rate of genetic polymorphisms occurring in the envelope glycoproteins. HCMV infection is treated with antiviral drugs such as ganciclovir, valganciclovir, cidofovir, foscarnet, letermovir and maribavir targeting viral enzymes, DNA polymerase, kinase and the terminase complex. One of the obstacles to successful therapy is the emergence of drug resistance, which can be tested phenotypically or by genotyping using Sanger sequencing, which is a widely available but less sensitive method, or next-generation sequencing performed in samples with a lower viral load to detect minority variants, those representing approximately 1% of the population. The prevalence of drug resistance depends on the population tested, as well as the drug, and ranges from no mutations detected to up to almost 50%. A high prevalence of resistance emphasizes the importance of testing the patient whenever resistance is suspected, which requires the development of more sensitive and rapid tests while also highlighting the need for alternative therapeutic targets, strategies and the development of an effective vaccine

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

HEPATITIS B VIRUS GENOTYPING, DETECTION OF REVERSE TRANSCRIPTASE RESISTANCE AND IMMUNE ESCAPE MUTATIONS IN PERSONS WITH CHRONIC HEPATITIS B FROM CROATIA

Approximately 257 million people worldwide live with chronic hepatitis B virus (HBV) infection, which, if left untreated, can lead to liver cirrhosis or hepatocellular carcinoma. The hepatitis B virus is a DNA virus with a reverse transcriptase that has no exonuclease activity, which results in a high mutation rate. Reverse transcriptase inhibitors, which interfere with viral replication, are used to treat the infection. Mutations in the A-B reverse transcriptase interdomain can be associated with resistance to antiviral drugs, as well as immune escape. The aim of this study was to analyze HBV genotypes circulating in the Croatian population and analyze resistance as well as immune escape mutations. A selected A-B reverse transcriptase interdomain was sequenced using the Sanger method. HBV genotypes, subtypes, drug resistance as well as immune escape mutations were analyzed using the Geno2Pheno algorithm in 30 patients with chronic hepatitis B. Genotype A (subtype A2) was detected in 20% and genotype D (subtypes D1, D2 and D3) in 80% of viral isolates. Drug resistance mutations rtL180M and rtM204V were detected only in genotype A isolates. Immune escape mutations R122K and sT131N were detected in all genotype A isolates, while mutations sD144E, sM133I, sM133L, sP120S, sQ101H and sR122K were detected in 8 genotype D isolates. Genotype distribution and the prevalence of mutations observed in this study are in accordance with data from the majority of other European countries