Inhibition of hepatitis C virus infection in cell culture by small interfering RNAs.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and hepatocellular carcinoma, yet fully efficacious treatments are missing. In this study, we investigated RNA interference (RNAi), a specific gene silencing process mediated by small interfering RNA (siRNA) duplexes, as an antiviral strategy against HCV. Synthetic siRNAs were designed to target conserved sequences of the HCV 5' nontranslated region (NTR) located in a functional, stem-loop structured domain of the HCV internal ribosome entry site (IRES), which is crucial for initiation of polyprotein translation. Several siRNAs dramatically reduced or even abrogated the replication of selectable subgenomic HCV replicons upon cotransfection of human hepatoma cells with viral target and siRNAs, or upon transfection of cells supporting autonomous replication of HCV replicon with siRNAs. Importantly, three siRNAs also proved capable of strongly inhibiting virus production in cell culture. One siRNA, targeting a sequence that is highly conserved across all genotypes and forms a critical pseudoknot structure involved in translation, was identified as the most promising therapeutic candidate. These results indicate that the HCV life cycle can be efficiently blocked by using properly-designed siRNAs that target functionally important, highly conserved sequences of the HCV IRES. This finding offers a novel approach towards developing IRES-based antiviral treatment for chronic HCV infections.     

Cell culture systems for the hepatitis C virus

Since the discovery of HCV in 1989, the lack of a cell culture system has hampered research progress on this important human pathogen. No robust system has been obtained by empiric approaches, and HCV cell culture remained hypothetical until 2005. The construction of functional molecular clones has served as a starting point to reconstitute a consensus infectious cDNA that was able to transcribe infectious HCV RNAs as shown by intrahepatic inoculation in a chimpanzee. Other consen- sus clones have been selected and established in a hu- man hepatoma cell line as replicons, i.e. self-replicating subgenomic or genomic viral RNAs. However, these repli- cons did not support production of infectious virus. Inter- estingly, some full-length replicons could be established without adaptive mutations and one of them was able to replicate at very high levels and to release virus particles that are infectious in cell culture and in vivo. This new cell culture system represents a major breakthrough in the HCV field and should enable a broad range of basic and applied studies to be achieved.     

Assessment of Coagulation Disorders in Patients with Acute Leukemia Before and After Cytostatic Treatment

Coagulation disorders are often the reason for fatal bleeding in acute promyelocytic leukemia. Their occurrence as well as pathogenesis and prognostic significance in other subtypes of acute myelogenous leukemia and acute lymphoblastic leukemia is less known. Tests were carried out in 70 patients including 49 with AML and 21 with ALL. In all patients throm-bin-antithrombin complexes (TAT), D-dimer (DD) and plasmin-antiplasmin complexes (PAP), antithrombin 111 activity, fibrinogedfibrin degradation products, AFlT and PT were determined. The tests were performed on diagnosis and after cytostatic treatment.

The level of TAT, DD and PAP was elevated in 83% of the patients on diagnosis and in 90% after treatment. The highest values were observed in AML M3 patients. Among leuke-mic patients with normal levels of TAT, DD and PAP at diagnosis, cytostatic treatment had a negligible effect on the level of these markers. During remission the levels of these markers returned to the normal values while in patients without remission they were either elevated or returned to normal values. No correlation between the levels of activation markers and remission rate was reported. DIC was diagnosed in 13 patients including three after chemotherapy. The DIC was acute or subacute in AML and chronic in ALL patients.

In the majority of acute leukemia patients there were already changes on diagnosis indicating coagulation activation. Except for AML M3, these usually had a subclinical course. The TAT, DD and PAP tests are not reliable markers of remission in acute leukemias.     

Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C

Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease. A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40. As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used. Of 38 chronically infected patients positive for anti-E2 antibodies by IFA, 26 were positive in the Western blot assay (68%) and 25 in the INNO-LIA test (66%). Thus, the detection of anti-envelope antibodies is highly dependent on the antigen formulation, and a native glycosylated form of the proteins is probably needed for their efficient detection. This study shows that the antibody response to HCV envelope proteins depends on the phase of infection. A few acutely infected patients displayed a response to E1 or E2 (36% by Western blot, 7% by IFA), and these antibodies seem to develop in patients evolving toward chronicity. The high prevalence in chronically infected subjects (62% to E2 by Western blot, 90% by IFA), particularly in subjects with essential mixed cryoglobulinemia (68% and 100%), confirms that the resolution of infection involves more than these antibodies. The antienvelope response in patients treated with interferon was investigated, but no significant relationship was found between antibody level prior to treatment and the evolution of hepatitis. The detection of anti-envelope antibodies, therefore, is not predictive of the response to antiviral therapy. © 1996 Wiley-Liss, Inc.     

Surgery or conservative treatment in children with traumatic intracerebral haematoma

In contrast to the case of extracerebral haematomas, the criteria for operative treatment of traumatic intracerebral haematoma (TIH) are not clear. The purpose of this study was to find factors that would be helpful in reaching a decision for surgical or conservative treatment of TIH. We performed a retrospective analysis of 31 consecutive cases of TIH treated in our department. The following factors were estimated: age, mechanism of injury, initial GCS or CCS score, neurological deficits, coexistence of arterial hypotension and respiratory disturbances, and localisation and size of the haematoma. The outcome was evaluated according to a modified GOS. Treatment was surgical for 20 patients and conservative for 11. Patients with GCS or CCS scores of 3–8 were treated surgically significantly more often than those with higher scores. The other factors did not correlate with type of treatment. It seems, then, that the clinical status of the patient, especially the level of consciousness according to the GCS or CCS score, is the most important predictor of the need for surgery in children with TIH. Received: 28 November 1998     

Protection of Animal Research Subjects

The use of experimental animals, mostly rodents, in biomedical research and especially in oncology and immunology should be acknowledged with respect, recognizing the contribution of animal experimentation in the fascinating scientific progress in these disciplines of research. It is an obligation of the investigator to justify the scientific and ethical aspects of each study requiring the use of animals. The international guiding principles for using animals in biomedical research are well defined and have been distributed worldwide by the International Council for Laboratory Animal Science （ICLAS） since 1956, when this Organization was founded. In Poland the ICLAS philosophy and principles are highly respected and were implemented firstly by the members of the Commission on Biology of Experimental Animals appointed in 1962 by the Department of Medical Science of the Polish Academy of Science in Warsaw. Animal Protection Acts, first proclaimed in 1928 were gradually modified and improved. Actual legislation （enacted in 1997, 2003 and 2005） is consistent with EU Directives （86/609/EEC） and follows the internationally recommended principles that include ICLAS guidelines concerning animal welfare and care condition in biomedical research. The problem of ＂alternative methods＂ is briefly discussed.     

Zygomatic abscess with temporal myositis - a rare extracranial complication of acute otitis media

Acute mastoiditis is the most common complication of acute otitis media (AOM) and its early recognition and management still poses a challenge due to potentially serious consequences. The incidences of extracranial and intracranial suppurative complications of AOM in children have decreased significantly, yet they remain a serious clinical problem, especially when caused by bacteria resistant to antibiotics. The authors presented a case of rare AOM complication - zygomatic abscess with temporal myositis. A 6-year-old boy was admitted to the ENT Department with 4 weeks of ear pain, treated for AOM with cefuroxime axetyl and amoxicilline, with acute mastoiditis and subsequent abscess formation in zygomatic and preauricular region. The inflammatory process spread through anterior air cells to the zygomatic cells leading to a fistula formation in the zygomatic bone and breakthrough into the temporal muscle. The surgical procedures applied were: myringotomy with drainage, cortical mastoidectomy and revision of zygomatic area and treatment with antibiotics (ceftriaxon). Enterococcus faecalis and Streptococcus viridans were found in the culture of middle ear and mastoid effusion. After half a year of follow-up the child had a normal hearing. Severe complications of AOM are rare today. An early diagnosis in order to promote adequate management and prevent inherently suppurative complications is essential.