Biomedical subjects
Publications and source records attributed to R Cattaneo.
Lengthening of the humerus has been considered to be particularly risky because of the danger of neurological damage. The Ilizarov technique makes it possible to avoid complications. The authors have performed eight such lengthenings, with gains in length of 8 to 16 cms. Only one case was complicated by an ulnar and radial paralysis which recovered secondarily.
We have used a novel approach called expression selection to precisely define the hepatitis B virus (HBV) enhancer. Expression selection is based on a shuttle vector containing an enhancerless SV40 T antigen gene, the SV40 origin of replication and a plasmid replicon. This vector is linearized, ligated with the sonicated DNA to be analyzed and transfected into eukaryotic cells, where only plasmids which have incorporated an enhancer can express T antigen and therefore replicate. Vectors amplified by replication are selectively rescued in E. coli and their inserts analyzed. When we performed this protocol with HBV DNA we rescued two overlapping fragments of 166 and 214 bp which in HBV DNA map about 500 bp upstream of the core antigen mRNA initiation site and 1150 bp downstream of the surface antigen mRNA initiation site. These results were confirmed by conventional deletion mapping. When compared to the SV40 enhancer in nonhepatic cell lines, the HBV enhancer is only 5 to 10% as active; nevertheless, it also acts in an orientation-independent manner and in a position downstream of a gene. The HBV enhancer is situated in the coding region of the potential reverse transcriptase, and thus is the first enhancer identified to map in a protein-coding region.
The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA.
The treatment of non-union of long bones is one of the best indications for the use of Ilizarov external fixation. The method is somewhat expensive but without risk. In 20 patients, use of the technique achieved healing in cases in which traditional methods such as bone grafts, plates or other types of external fixator had failed. The main advantages are avoidance of further vascular disturbance, a stable and elastic fixation and the possibility of early weight-bearing with consequent compression.
Purified IgM rheumatoid factors (RF; 3 monoclonal and 2 polyclonal) were shown to inhibit, in a dose-dependent manner, 2 complement-mediated functions, i.e., the immune complex solubilization capacity and the inhibition of immune precipitation. Inhibition of immune complex solubilization capacity occurred only if RF was added at the same time as, but not after, addition of the complement source. Experimental evidence suggests that the effects of RFs were not related to their anticomplementary activity, but rather required the attachment of RF to the Fc region of the IgG molecule. Although no clinical data are available so far, it might be plausible that these newly described properties of RF have biologic relevance.
Hepatitis B virus (HBV) transcription was studied in the liver of an infected chimpanzee and compared with HBV transcription in heterologous systems. Besides the well characterized 2.3-kb surface antigen mRNA produced in most systems, a second major transcript was identified in the liver. This 3.8-kb transcript (+/- 300 bases) is slightly larger than the HBV genome and is probably involved both in core/e antigen synthesis and in HBV replication via reverse transcription. In addition, minor variants of the 2.3-kb surface antigen mRNA were characterized as probably being involved in the expression of HBsAg-related minor proteins. Finally, several potential transcription signals, identified on the HBV genome using heterologous expression systems, were found to be poorly active if at all in the infected liver, thereby stressing the importance of HBV transcription studies performed with liver material.
Recombinant DNA molecules consisting of the simian virus 40 (SV40) early region and different subgenomic hepatitis B virus DNA fragments were constructed in vitro and packaged in vivo into SV40 capsids by using a complementing SV40 helper virus. Upon infection with these virus stocks the three known hepatitis B-specific antigens were expressed under SV40 control. The surface antigen was released into the medium, and the core antigen and its derivative hepatitis B e antigen were only detected intracellularly. Size analysis of the core gene product(s) by immunoblotting revealed the presence of a single protein species identical with the 21-kilodalton core antigen isolated from human liver. The hepatitis B core antigen expressing construct did not contain a putative precore sequence, indicating that such a sequence is not needed for hepatitis B core antigen synthesis in animal cells. S1 analysis demonstrated the use of SV40 signals for initiation and polyadenylation of the core gene transcripts. In addition, a processing-polyadenylation signal was identified within the core gene.
Lymphocyte subsets were analyzed in peripheral blood and lymph nodes from a drug-addict with acquired immunodeficiency syndrome (AIDS) presenting with disseminated lymphadenopathic and oropharyngeal Kaposi's sarcoma. At the onset of disease, hypergammaglobulinemia, increase of OKT8+ T cell subset and reversal of OKT4/OKT8 ratio were found in the blood. At the same time, lymph nodes displayed, besides Kaposi's sarcoma, marked follicular hyperplasia, plasmocytosis and increase of OKT8+/Leu 2a+ T cells within follicular centers. These results are interpreted to indicate that at an early stage of disease the major tissue alterations took place within follicular centers and consisted of both B cell activation and T suppressor cell reaction. These changes correlated with immunological abnormalities observed in peripheral blood. Immunohistochemical investigation of lymphoid tissue may be useful to detect AIDS patients at an early stage.
A cDNA clone of Foot and Mouth Diseases Virus (FMDV), strain C1, has been sequenced. The limits of the structural genes were defined by comparison with the available protein data. We identified two potential translation initiation sites for the viral polyprotein separated by 84 nucleotides. We suggest that these two initiation sites could be used to express two proteins differing only at the N-terminal, P16 and P20a. This model is supported by the fact that antiserum against a bacterially synthesized polypeptide corresponding to the anterior region of the polyprotein precipitates specifically both P16 and P20a. Comparison of the C1 sequence with two other serotypes, O1K and A10 revealed variability in the major immunogenic structural protein, VP1, and also in two other capsid proteins, VP2 and VP3. P16/P20a, VP4, and the N-terminal part of the precursor of the nonstructural genes, P52, are rather conserved between the different FMDV strains.
Plasmids containing hepatitis B virus (HBV) DNA and a 232-bp SV40 DNA fragment encoding the origin of replication were constructed. When introduced by transfection into COS cells, these plasmids directed the synthesis of hepatitis B surface antigen. S1 mapping of the mRNAs covering the S gene showed that transcriptional initiation was promoted by the interaction of HBV sequences with an SV40 promoter element: transcription started on HBV DNA but had several properties of SV40 late transcription. The detection of a promoter element in an SV40 origin fragment commonly used in the COS system is important for the interpretation of data deriving from expression studies in COS cells.
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Con A induced suppressor cell activity was evaluated in 10 patients with Systemic Lupus Erythematosus and in 20 normal subjects. The suppressor activity, calculated according to Shou et al. (1976), was markedly reduced in SLE patients (mean value 45.2% vs 72.9% P less than 0,01). No statistical difference was evidentiated between the patients with mild and those with severe disease, though the last ones showed the lowest values. Low degree of suppression was found related to low hemolytic complement activity (P less than 0.02) but not to anti-DNA antibody titre or immune complex presence.
A preliminary map of the hepatitis B virus genome has been derived from the nucleotide sequence of cloned human hepatitis virus (HBV) DNA. The genes for two viral antigens were identified and their expression was studied using SV40 vector systems. HBV DNA cloned and amplified in bacteria induces hepatitis in chimpanzees demonstrating that the cloned HBV DNA is functionally intact.
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The possibility of cloning large segments of DNA in cosmid vectors offers distinct advantages, in particular for the study of multigene families. Large size fragments of mouse embryo DNA were successfully cloned in the cosmid pHC 79. Twelve recombinants hybridizing specifically to an immunoglobulin kappa chain variable region probe were identified. In 9 of these recombinants, the size of the insert ranges from 30 to 43 kilobases. Factors affecting the cloning efficiency of a complex mammalian genome in cosmids were studied. The stability of these recombinant cosmids and the preparation of recombinant cosmid DNA are also discussed.
Circulating immune complexes (CIC) in the sera of 60 newly diagnosed leukaemic patients were investigated by two methods, 125I-C1q binding test (C1q-BA) and conglutinin binding assay (KgB-SP). Positivity percentages were respectively 20.0% (C1q-BA) and 28.3% (KgB-SP). The small overlap between the results of the two methods suggests the occurrence of different types of CIC. The presence of CIC was found to be related only to clinical haemorrhage and thrombocytopenia; it did not prove to affect the prognosis and the survival of leukaemic patient.
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