Rat ileocecal immunocytoma. An ultrastructural study with special attention to the presence of viral particles.

Fifteen spontaneous immunocytomas originating in the ileocecal lymph nodes of Lou/C/Wsl rats were studied by means of electron microscopy. The histology was characteristic, the tumor being formed by an accumulation of large, rounded cells with slightly eccentric ovoid nuclei, large nucleoli, and finely condensed chromatin along the nuclear walls; the cytoplasma was rich in polyribosomes. The appearance of the rough endoplasmic reticulum was apparently the same whether or not the tumor was secretory. Its development varied from one cell to another, and in only a small proportion of cells did it attain any considerable volume. In all the tumors examined, we noted the presence of intracisternal A-particles. In its morphology, the rat immunocytoma resembled the plasmacytomas induced in mice, and it also resembled certain human tumors such as Burkitt's lymphoma.

Rat monoclonal antibodies. IV. Easy method for in vitro production.

A simple technique for in vitro production of monoclonal antibodies is described. Two to 5 X 10(6) hybridoma cells are diluted in 300 ml of medium in a 2 litre 'roller bottle' and incubated for 2 weeks without subsequent adjustment of the cell concentration or renewal of the medium. The method is based on the observation that when hybridoma cell cultures are maintained in the same medium, although cell growth stops after a few days and the percentage of dead cells increases rapidly, the amount of monoclonal antibody present in the culture medium increases up to the 15th day, the yield reaching more than 10 mg per flask. The method can be used both for rat and mouse hybridoma cell culture.

Rat monoclonal antibodies. I. Rapid purification from in vitro culture supernatants.

A technique for purifying rat monoclonal antibodies quickly and efficiently from in vitro culture supernatants is described. It is based on the fact that more than 95% of rat immunoglobulins carry kappa light chains. A mouse monoclonal antibody with suitable binding affinity for rat kappa light chains is immobilized on solid support and used to purify rat immunoglobulins. Milligrams of rat monoclonal antibodies may be rapidly concentrated from culture supernatants with high recovery. Rat monoclonal antibodies expressing lambda light chains (about 5% of the total) may be purified in a similar way with an appropriate anti-rat lambda chain monoclonal antibody.

An anti-HIV-1 gag protein rat monoclonal antibody library.

A set of 18 rat monoclonal antibodies (MAbs) directed against human immunodeficiency virus type 1 (HIV-1) gag proteins was derived from 4 independent fusion protocols. The epitopes recognized were delineated using a random fragment expression library representing the whole HIV-1IIIB genome. This panel of rat MAbs was used to analyze the antigenicities of the HIV-1 CAp24 major core protein and the HIV-1 NCp7 nucleocapsid protein. As a result, a limited set of antigenic domains as defined, 3 on CAp24 between amino acids (aa) 195 and 268, 323 and 329, 329 and 352, and one on NCp7 (aa 382-392). Only 4 mouse anti-CAp24 MAbs appeared to recognize the COOH-terminal domain (aa 329-352) defined by the majority of our MAbs. The rat anti-CAp24 (Q1B10) and the rat anti-NCp7 (I5B11) MAbs described here, defined two newly described epitopes, aa 323-329 and aa 382-392.

HIV-1 detection by nested PCR and viral culture in fresh or cryopreserved postmortem skin: potential implications for skin handling and allografting.

AIMS: To date, the risk relating to the handling or allografting of human immunodeficiency virus type 1 (HIV-1) infected postmortem skin remains hypothetical. While blood screening for HIV antibodies is still the key safety procedure to detect HIV infected cadavers, false negative results are a concern. Conversely, false positive results may hamper the collection of skin allografts. Accordingly, viral culture was used to clarify skin infectivity and the nested polymerase chain reaction (PCR) was used to assess the reliability of skin PCR testing. METHODS: Viral culture and nested PCR performed with gag and pol specific primers were investigated in cadaveric skin and blood from 12 HIV-1 infected patients. Samples were collected repeatedly between one and five days in seven patients. In most cases, analyses were performed on triplicate skin samples: fresh (n = 26); cryopreserved in 5% dimethylsulphoxide (n = 21), or cryopreserved in 30% glycerol (n = 26). RESULTS: HIV was isolated in two of 26 cultures of fresh skin specimens (8%), seven of 47 cryopreserved skin specimens (15%), and eight of 26 blood specimens (31%). The nested PCR detected HIV-1 in all skin samples (n = 73), regardless of the postmortem interval or cryopreservation. In blood, a positive signal was found in eight of 12 patients but two of them had discordant results on successive samples. CONCLUSIONS: These data suggest that nested PCR on postmortem skin samples can detect HIV more reliably than on blood. They also demonstrate the potential viral infectivity of fresh or stored skin postmortem samples in HIV infected patients. They underscore the need for caution during the handling of skin tissue from HIV infected cadavers and confirm the potential risk related to accidental allografting of HIV contaminated skin.

A latex agglutination test for the detection of canine parvovirus and corresponding antibodies.

Latex particles coated with rat monoclonal antibodies directed against a canine parvovirus are agglutinated by the virus antigens. In this study, the reaction was quantitated by a technique which had been described as immunoassay by particles counting. The method is as sensitive as a radioimmunoassay with the same antibody, but has the advantages of simplicity and safety. It allows the detection of parvoviral antigens to 4 ng/ml. Incubation of these antigens with specific antibodies resulted in inhibition of the agglutination reaction. By this procedure the presence of antibodies against canine parvovirus can be detected to a concentration of 150 ng/ml. The system can be easily automated, thus increasing reproducibility. This latex agglutination technique can be performed as a slide test; and although this method is 4-5 times less sensitive, it could be useful in field studies, for the detection of the viral antigens and the corresponding antibodies.

Hepatitis C virus genotypes in hemodialyzed patients: a multicentric study.

The few studies concerning HCV genotypes in hemodialyzed patients concerned small groups of patients, issued from single units. Using the RFLP typing method in the 5' non-coding region (5' NCR), we performed the genotyping of HCV strains of 80 patients issued from 14 Belgian dialysis units. Forty-seven of the 80 patients were also tested by Inno-Lipa II (Innogenetics, Gent, Belgium). Sixty-four of 80 patients (80%) were infected with subtype 1b, 2 (2.5%) with subtype 1a, 6 (7.5%) with subtype 2a and 1 (1%) with subtype 2b; 6 patients (7.5%) showed a type 4 and 2 patients (2.5%) a type 5 infection, respectively. Only 1 patient (1%) showed a mixed infection (1a and 1b). In the 47 patients tested by both methods, we observed a very good agreement (98%) between RFLP and Inno-Lipa II's results. Our multicentric study detected HCV genotypes 4 or 5 in 8/80 (10%) of hemodialyzed patients in Belgium. The substantial prevalence of these genotypes could not be fully explained by a nosocomial spread of HCV infection: indeed, four patients belonged to dialysis units located in different cities, and two appeared infected with distinct subtypes in a same unit. Thus, the discovery of a "rare" HCV genotype in several hemodialyzed patients does not always point to nosocomial HCV transmission.

[Permanent tattoos following injections with Dermo-Jet: anatomoclinical study. Apropos of 5 cases]. / Tatouages permanents consécutifs à des injections par Dermo-Jet: étude anatomo-clinique. A propos de cinq observations.

Blue or black tattoos were observed in five patients who had received several intradermal injections (of a lidocaine solution or of a triamcinolone acetonide suspension) with the Dermo-Jet. A histological examination revealed the presence of black masses, rounded or elongated, different in size, distributed throughout dermal tissue. Additionally, conglomerates of black grains in the cytoplasm of histiocytes and of pericytes are precisely observed in semi-thin sections. It has been proved by several investigations that these foreign particles are not of metallic nature. It can be concluded from electron microscopic studies that the particles are fragments of black rubber, from the upper joint of the reservoir. Indeed, some rubber fragments are leached into the reservoir. Some of these are passing through the wire-mesh filter of the nozzle (with the solution or the suspension) when injections are made. Therefore, they are injected intradermally and are permanently tattooing the skin.

[Rapid detection of viruses by electron microscopy. Interest of negative staining for the diagnosis of some skin lesions of viral origin (author's transl)]. / Détection rapide des virus au microscope électronique. Intérêt de la méthode de coloration négative pour le diagnostic de certaines dermatoses d'origine virale.

Viral particles can be visualized by electron microscopy using negative staining. Such an approach, widely used in research, provides a method for detecting virions which are present in clinical specimens. It is considered to be the method of choice in cases with suspicion of smallpox. Direct visualization has been systematically applied for the diagnosis of viral diseases in dermatology. Negative staining by pseudoreplication appears to be simple, rapid and efficient. It was thus possible to detect viruses in vesicle fluids, in scrapings and in crusts. During the course of this study 66 cases have been analyzed and viruses have been demonstrated in 49. If a skin lesion seems to be of viral origin, direct examination may confirm the clinical impression.

[Construction of nested set of unidirectional deletion of recombinant plasmid DNA for sequencing].

In order to rapidly sequence a batch of large DNA fragments, we developed a method for the construction of nested set of unidirectional deletions. A nested set of unidirectional deletions of the large DNA fragment of a c-type retrovirus gene was successfully constructed by the adoption of this method. The recombinant plasmid DNA was excised by BamHI and SphI at on end of the target DNA to create 5' and 3' overhanging ends. The DNA was digested with Exo III from 5' overhanging end to generate a set of unidirectional deletion. With the use of this method a set of deleted plasmid DNA and the deleted target DNA excised from the deleted constructs were observed on agrose gel electrophoresis. We sequenced all of 24 deleted DNA fragments (forward and reverse orientation). All of them contained overlapping sequences at their ends. These sequences were readily aligned. The results demonstrate this is an efficient method for sequencing large DNA fragment. The influential factors in this method were discussed in this report.