Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Use of recombinant human antibody fragments for detection of cytomegalovirus antigenemia.

The determination and quantitation of peripheral blood leukocytes (PBLs) expressing human cytomegalovirus (HCMV) antigens is widely employed in clinical virology for rapid diagnosis of HCMV-related infections. We describe how CMV antigenemia may be accurately detected by means of human recombinant monoclonal Fab fragments rescued from a combinatorial phage display library prepared from an HCMV-infected donor. Fourteen recombinant Fabs were tested against HCMV-positive PBLs from a patient with ongoing HCMV infection. Three clones were found to react specifically with the nuclei of these cells. These three recombinant Fabs were subsequently tested, individually and pooled together, against 60 PBL samples taken from immunosuppressed patients. The reactivity observed was comparable to that obtained with mouse monoclonal antibodies commercially available for this purpose. The three recombinant Fabs were shown to react specifically with the 65-kDa viral tegument phosphoprotein encoded by UL83 (pUL83), which is the most abundant viral antigen in HCMV-infected PBLs.

Comparative analysis of subcellular distribution of protein antigens in Mycobacterium bovis bacillus Calmette-Guérin.

The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.

Construction of a polyepitope fusion antigen of human cytomegalovirus ppUL32 and detection of specific antibodies by ELISA.

We have previously shown that single linear epitopes of the major human cytomegalovirus (HCMV) antigens, expressed as fusion proteins or synthesized as oligopeptides can be valuable diagnostic material in the serology of HCMV infection (5, 6, 13). In this work we fused sequences expressing two different epitopes (aa 1005-1048 and aa 595-614) contained in the basic phosphoprotein of 150 KD coded by UL32 (1, 2), (ppUL32), which has repeatedly been shown to be the strongest immunogen present in the viral particle. The fusion protein was tested by ELISA with HCMV-positive human sera in comparison with other fusion proteins of ppUL32. We found that the double epitope fusion protein was recognised by IgM present in a larger number of sera and with more intense reactions than all the other ppUL32 fusion proteins. The double epitope reacted positively with 81.3% and, when denatured, with 94.7% of IgM-positive sera respectively. IgG reactivity was also high, reaching a percentage of 90.7.

An in vivo study on active cytomegalovirus infection in relation to active HIV replication in HIV-I infected drug addicts.

Human cytomegalovirus (CMV) is a major cause of severe disease in HIV-infected persons and some findings suggest that it may accelerate HIV disease. In this study, a total of 621 blood samples from patients with LAS-ARC and AIDS were analysed in parallel for CMV and HIV-I antigenaemias. Results indicate that the presence of CMV antigenaemia and the presence of HIV-I p24 in the blood are highly correlated statistically and encourage other studies on the role of CMV in the evolution of AIDS. In a smaller group of cases, CMV was also isolated from saliva and/or urine. The correlation with HIV replication was positive (although much lower) with CMV detected in saliva and completely negative with CMV isolated from urine.

Enzyme-linked immunoadsorbent assay for the detection of cytomegalovirus-IgM: comparison between eight commercial kits, immunofluorescence, and immunoblotting.

Eight commercially available enzyme-linked immunoadsorbent assays (ELISA) for the detection of cytomegalovirus (CMV)-specific IgM were used in parallel to determine the presence of CMV-IgM in 123 serum samples from pregnant women. The results obtained with the eight kits were compared. Based on concordance of six or more of the eight kits, we assessed sensitivity, specificity, and overall agreement, as well as incidence of false-positive and -negative results for each kit. The results obtained by ELISA were then compared with those obtained by immunofluorescence (IF) and immunoblotting (IB). Our study did not single out one outstanding ELISA kit among the eight evaluated, nor did it suggest that IF or IB are better than ELISA. Furthermore our results indicate that IB might be useful in several cases as, beside its good sensitivity, most IB-false-positive sera are easily recognized as reacting exclusively with pp150, the unique reactivity to pp150 not being among the IB profiles of IB-true-positive sera. Nevertheless 14.6% of sera remained CMV-IgM-indeterminate.

[Rehabilitation of burned patients. II. Therapeutic exercise]. / La riabilitazione del paziente ustionato. Parte Seconda: L'esercizio terapeutico.

After illustrating some major ideas concerning disability and the role played by the Rehabilitation Medicine, with particular reference to burns, the Authors consider rehabilitation. The main therapeutical rehabilitation measures used in the various stages of the disease are described. The Authors conclude by underlying the importance of an early and adequate rehabilitation as well as the need for a multidisciplinary approach since patients suffering from burns present a variety of medical, surgical and rehabilitation problems.

[Rehabilitation of burned patients. I. General aspects]. / La riabilitazione del paziente ustionato. Parte prima: Aspetti generali.

The authors discuss the general aspects of problems connected with pathology caused by burns from a rehabilitation standpoint. The motivations that render all the rehabilitation effort more and more important for assisting the patient suffering from burns are reported. Then, brief considerations follow regarding the physiopathologic aspects of the healing process evolution. The authors also report about the main functional deficiencies caused by the pathologic involvement of the motion as well as of the respiratory system.

[Usefulness of transcutaneous electric nerve stimulation in the treatment of headache of cervical origin]. / Utilità della T.E.N.S. nel trattamento della cefalea di origine cervicale.

Forty-one patients suffering cervical headache and evaluated in accordance with French Manual Medicine School criteria were treated with T.E.N.S. Results demonstrated significant decrease of pain improvement in physical function in these patients, which was also accompanied by changes in self-report of pain complaints. We think that T.E.N.S. can be a technique extremely reliable, practical and effective in the treatment of cervical headache syndrome and that manual semeiotic data may play an important role for screening cervical headache and for evaluating the results of treatment.

[Algoneurodystrophy]. / L'algoneurodistrofia.

The authors consider the extensive literature concerning a quite frequently observed clinical picture, that has been named in different way by each researcher. Algoneurodystrophy is the name used in this analysis. They make a review of the most important works appeared in literature. It points the contrasting aspects of the research made till now. Therefore, in authors' opinion, it's necessary for them to investigate with further studies various aspects of the disease (etiological, pathogenetic, clinical, therapeutic ones, etc.) associated with a prevention of the secondary and tertiary damage the earliest as possible.

Glucosamine sulphate: a controlled clinical investigation in arthrosis.

Efficacy and tolerance of a new preparation of pure glucosamine sulphate, in injectable and oral form, were investigated in 30 patients with osteoarthrosis. Two groups of in-patients with chronic degenerative articular disorders received daily for 7 days either 400 mg glucosamine sulphate or a piperazine/chlorbutanol combination by intravenous or intramuscular injection. During the 2 following weeks, the patients receiving glucosamine had oral glucosamine capsules (6 x 250 mg daily); the other group had placebo. Efficacy was tested by semi-quantitative scoring of pain at rest and during active and passive movements, as well as limitation of articular function, before and after 7 and 21 days of treatment. Patients were positively questioned daily for possible intolerance symptoms. Haematology, circulatory data and urine analysis were tested before and after treatment. During both initial parenteral treatments, each symptom significantly improved, but to a faster and greater extent in the group treated with glucosamine. During the maintenance period, a further improvement was recorded in the patients treated with glucosamine, whereas in those on placebo the symptom scores increased almost to the pre-treatment level. This was considered the major difference between basic therapy, such as with glucosamine, as purely symptomatic treatment. Clinical and biological tolerance were excellent with both treatments, and no definitely drug-related complaints were recorded. It is suggested that parenteral and/or oral treatment with pure glucosamine sulphate should be considered as basic therapy for the management of primary or secondary degenerative osteoarthrosis disorders.