[The specific enzyme inhibitors for potential therapeutic use]. / Specyficzne inhibitory enzymów o potencjalnym zastosowaniu terapeutycznym.

Therapy for hepatitis C virus (HCV) initially consisted on administering ribavirin - having a broad spectrum of action - and pegylated interferon, and was only effective in 40-50% of patients. Appropriate was to find effective inhibitors of viral replication e.g. by inhibition of a viral enzyme, NTPase/helicase required in the process of translation and RNA replication of the HCV. We developed methods of synthesis of many compounds belonging to different groups - derivatives of nucleosides, benzotriazole, benzimidazole, tropolone and epirubicine. Some of the derivatives inhibit HCV helicase activity at low concentrations and reduces replication of the viral RNA in subgenomic replicon system. In the process of HCV replication casein kinase CK2 plays an important role. It regulates the level of phosphorylation of HCV protein NS5A, which affects the production of infectious virions of HCV. Effective and selective inhibitors of kinase CK2 could be of use in the treatment of HCV in combination with other drugs. CK2 kinase phosphorylates approximately 300 proteins that affect the growth, differentiation, proliferation or apoptosis. Elevated CK2 kinase activity has been observed in several types of cancer and other diseases, therefore, inhibitors of this enzyme are potential therapeutic importance, particularly for anti-cancer treatment. Research carried out in collaboration with prof. Shugar led to the synthesis of one of the most selective inhibitors of this enzyme which is 4,5,6,7-tetrabromo-1H-benzotriazole, used for the study of the role of kinase CK2 in a number of metabolic processes in tumor cells.

Back to the origin of HCV 2c subtype and spreading to the Calabria region (Southern Italy) over the last two centuries: a phylogenetic study.

Circulation of HCV genotype 2 has been described in European Countries where numerous subtypes and unclassified HCV 2 lineages have been reported. In Italy, subtype 1b is the most prevalent, followed by genotype 2. In the present study, phylogeny of HCV 2c was investigated. The phylogeny of HCV 2c isolated from 54 Italian patients in the Calabria region (Southern Italy) was investigated by analyzing a fragment of the NS5B gene. Patients came from 5 metropolitan areas and a small village (Sersale). These areas were geographically dispersed throughout the entire region. A Bayesian coalescent-based framework was used to estimate origin and spreading of HCV 2c in this region. Phylogenetic analysis showed that 28 Italian sequences were intermixed with foreign HCV 2c reference sequences and grouped into 3 major clades: A, B, and C. Nineteen inter-clade sequences were associated uniquely with surgery as risk factor for HCV acquisition. By contrast, a sub-cluster within clade B was associated with blood transfusion. Moreover, sequences from Sersale village grouped in the Italian sub-cluster and were intermixed with 10 sequences from metropolitan areas. The three isolates with the longest branch came from Sersale and belonged to patients who had glass syringes as risk factor. HCV 2c isolates from the Calabria region shared a common ancestor whose origin was traced back to 1889. Our results suggest that, after its introduction - possibly as a result of population movements between Italy and African Countries during Italian colonialism - HCV 2c spread through multiple risk factors, not including intravenous drug use. So, transmission chains followed a pathway different from other European Countries. Although HCV incidence is decreasing, these ways are still ongoing, possibly justifying stability in the relative prevalence of HCV 2c.

Spreading of hepatitis C virus subtypes 1a and 1b through the central region of Argentina.

The recent history of the hepatitis C virus (HCV) subtypes 1a and 1b in the central region of Argentina is hypothesized by phylogeographic reconstruction using coalescent based Bayesian analyses. Direct partial E2 sequences from HCV 1a and 1b infected patients attending different health-care centers of the country were analyzed. The inferred date of the most recent common ancestor (tMRCA) for HCV-1a was: 1962 (between 1943 and 1977) and for HCV-1b was earlier: 1929 (between 1895 and 1953). Diverse ancestral populations were inferred from both subtypes in Córdoba and in Buenos Aires cities and after that, HCV spread within and between larger cities and to other smaller cities. The analyses suggested that HCV-1b was dispersed first and it is currently in a stationary phase whereas HCV-1a was dispersed latter and it is still in a growth phase. Finally, as it was observed in the developed countries, while the transmission of HCV-1b appears to have been somehow prevented, the HCV-1a may still represent a concern in the public health. Further work should be carried out to address their current transmission rate (and its main transmission route) in the Argentinean population.

Otimização de protocolos de testes moleculares para detecção e quantificação do vírus da Hepatite C em sangue coletado em papel de filtro / Optimization of protocols of molecular tests for etection and quantification of hepatitis c virus in blood coellected in filter papers

O objetivo deste estudo foi otimizar protocolos e padronizar métodos de diagnóstico molecular para infecção pelo HCV em amostras de sangue coletado empapel de filtro (SPF) para facilitar o acesso ao diagnóstico em áreas remotas ou com recursos limitados. Para isto, 99 indivíduos forneceram amostras pareadas de soro e SPF, dentre os quais 59eram anti-HCV reagente e 40 eram não reagentes em suas amostras de soro. As amostras anti-HCVreagentes foram submetidas à técnica de quantificação comercial. Para o desenvolvimento da RTPCRquantitativa (RT-qPCR) in house, uma curva padrão interna foi construída utilizando umplasmídeo contendo o inserto do HCV e iniciadores e sonda foram desenhados para a região 5´NCdo HCV. Para otimização da técnica, a concentração de cDNA, transcriptase reversa e númerosde ciclos de reação foram avaliados. Para a extração de RNA de HCV em amostras de SPF, setemétodos foram avaliados. A sensibilidade, especificidade, correlação, reprodutibilidade epresença de inibidores da RT-PCR quantitativa também foram determinados. O conjunto deQIAamp DNA Mini Kit foi o método mais eficiente para extração do RNA do HCV em SPF. A RTqPCRin house desenvolvida foi capaz de quantificar o HCV no soro de 44 amostras onde a medianade carga viral foi inferior aquela observada na técnica comercial (log10 4,94 e log10 6 cópias deHCV/mL, respectivamente)...

The aim of this studywas to optimize protocols and standardize molecular diagnostic methods for HCV infection in driedblood samples (DBS) to facilitate access to diagnosis in remote areas or with limited resources. Forthis, 99 individuals provided paired serum and DBS samples, where 59 of them were anti-HCVreactive and 40 were non-reactive in their serum samples. Anti-HCV positive samples were subjectedto commercial quantification technique. For the development of quantitative RT-PCR (RT-qPCR) inhouse, an internal standard curve was constructed using a plasmid containing the insert and HCVprimers and probe designed for the 5' non coding (NC) region of HCV. For optimization of thesetechniques, the concentration of cDNA, reverse transcriptase and numbers of cycles of reaction wereevaluated. For the extraction of HCV RNA in DBS samples, seven methods were evaluated. Thesensitivity, specificity, correlation, reproducibility and presence of inhibitors in quantitative RT-PCRwere also determined. QIAamp DNA Mini Kit was the most efficient method for HCV RNA extractionamong DBS samples. The in house RT-qPCR was able to quantify HCV among 44 serum sampleswhere the median viral load was lower than that observed in commercial technique (4.94 log10 and 6log10 copies of HCV / mL, respectively). The range of HCV detection using in house RT-qPCR was 10-109 copies of HCV per reaction...

[Historical consideration of the widespread infection of the hepatitis C virus in Japan and use of a fishbone diagram to investigate the cause].

About 75% of Japanese liver cancer is caused by hepatitis C. Widespread infection of the virus resulted from inadequate medical knowledge, as well as the political, economic and administrative conditions of the time. We investigated the association between the widespread infection of the hepatitis C virus and the historical events. We used a fishbone diagram to investigate the cause of widespread infection of the hepatitis C virus and considered the issue from a historical standpoint. We found causes including treatment (medical care), transfusion (medicine), economy (expense) and people (infection route). These causes are explained in further detail below. 1) Treatment (medical care). The initial large-scale infection occurred following attempts to eradicate Schistosoma japonicum involving mass vaccination in schools and public health centers. 2) Transfusion (medicine). The use of non-heated fibrinogen for massive postpartum hemorrhage spread the virus further. In 1987, it resulted in a mass outbreak of hepatitis in Aomori Prefecture. 3) Economy (expense). Recognition of the benefit of disposable syringes was delayed. As a result, disposable syringes were too expensive to be widely available, and did not become low-priced. 4) People (infection route). The second wave of dissemination of the hepatitis C virus was stimulant abuse after World War II. Prior to the discovery of the hepatitis C virus, transmission resulted from repeated use of contaminated syringes. Although we initially thought that these four causes occurred independently on a historical chronology, associations between the causes were found when we investigated the problem with a fishbone diagram.

The history of the "natural history" of hepatitis C (1968-2009).

In the late 1960's, only types A and B hepatitis were believed to exist, distinguished by circumstances of exposure and incubation periods. In the early 1970's, studies of transfusion recipients were begun with the belief that hepatitis B would be responsible should transfusion-associated hepatitis develop. After discovery of the viruses of hepatitis A and B, neither agent was found responsible, hence non-A, non-B (NANB) hepatitis. Initial follow-up of these cases showed that approximately 50% developed chronic hepatitis based on persistence of serum enzymes for at least 6 months. Approximately 15 years later, after the hepatitis C virus had been identified as the cause for NANB hepatitis, chronic hepatitis was found to develop more frequently as indicated by persistent viral infection in over 80% of infected adults but in only about 50% of infected children or young women. Follow-up over 2 to 4 decades indicated that many infected persons developed progressive hepatic fibrosis, sometimes culminating in cirrhosis and/or liver cancer. Long-term natural history studies have proved to be challenging because disease onset is often silent and progression extremely slow. Differing strategies have been used to determine the natural history, the descriptions and results of which are presented in this review.

Scotblood 2008 celebrates 60th anniversary of the NHS through past, present, and future of transfusion medicine, encompassing Hepatitis C Scottish Odyssey, nursing, translational medicine and trauma.

On the occasion of the 60th anniversary of the UK national health service (NHS), Scotblood 2008 featured an opulent array of presentations encompassing the beginnings of the scottish national blood transfusion service (SNBTS), through featuring progress on hepatitis C, to the advancing role of nursing in transfusion medicine, translational medicine, and trauma encountered in military transfusion. This commentary comprises summaries of the presentations, based in part on the abstracts provided by the speakers.

Los virus hepatotrópicos GB y G / Hepatotrophic viruses GB and G

Se hace una revisión del descubrimiento de los virus GB (GB-A, GB-B y GB-C) y se menciona la relación que hay entre el virus GB-C y el virus G. Los virus GB-Ay GB-B al parecer no son patógenos para el hombre. El virus GB-C, G o GB-C/G se trasmite por vía parenteral, generalmente a través de transfusiones sanguíneas o agujas contaminadas; produce infección crónica; su prevalancia varía de menos del 1 por ciento en donadores voluntarios de sangre a más de 10 por ciento en sujetos que presentan infección frecuente por virus C y B, y patogenicidad no está bien definida

Hepatitis C virus: a historical perspective.

Identification and diagnosis of the infecting agent responsible for hepatitis C have only recently occurred. Recognition of an infecting agent distinct from that resulting in hepatitis A or B was made approximately 50 years ago. However, the ability to screen and detect this agent was possible only after molecular biology studies which led to the cloning of parts of the hepatitis C virus (HCV) and the development of a diagnostic antibody test reported by Michael Houghton and colleagues in 1989. The discovery and cloning of HCV has led to a greater understanding of its relationship to acute and chronic hepatitis, cirrhosis, primary liver cancer, and extrahepatic conditions including essential cryoglobulinemia, glomerulonephritis, and serum autoantibody positivity. New antibody tests and quantitation of HCV-RNA have allowed better diagnosis of infectivity and monitoring of treatment effects. HCV genotypes are being related to the natural history of the disease and the effects of treatment. Research continues on HCV hepatitis and other newly identified viral hepatitis agents.