Standardized molecular typing of Candida albicans strains

Summary. A method is presented for the standardization of Candida albicans DNA fingerprinting, which is based on Southern hybridization of Eco RI-digested chromosomal DNA with the moderately repetitive DNA element CARE-2 and the subsequent rehybridization of the blots with a molecular size marker also included in each DNA sample. This method resulted in extremely precise alignment of all strain-specific CARE-2 hybridization patterns, even when analysed on different gels, and will enhance the accuracy of genetic relationship determinations in epidemiological studies including large numbers of strains.
Zusammenfassung. Zur Standardisierung des DNA-Fingerprinting von Candida albicans wurde eine Methode entwickelt, die auf der Southern Hybridisierung Eco RI-gespaltener chromosomaler DNA mit dem mittelrepetitiven DNA-Element CARE-2 und der darauffolgenden Rehybridisierung der Blots mit einem auch in den Proben enthaltenen molekularen Größenmarker beruht. Dies resultierte in einer äußerst präzisen Größen-bestimmung der hybridisierenden Fragmente, so daß alle stammspezifischen CARE-2-Hybridisierungsmuster exakt verglichen werden konnten, auch wenn die Isolate auf verschiedenen Gelen analysiert wurden. Die Methode erhöht die Genauigkeit der Bestimmung genetischer Verwandtschaftsbeziehungen in epidemiologischen Untersuchungen, in denen eine große Anzahl von Stämmen analysiert wird.     

A promising technique for transplantation of bone marrow-derived endothelial progenitor cells into rat heart.

OBJECTIVE: To investigate the feasibility of intracoronary application of endothelial progenitor cells and the subsequent distribution within the heart. METHODS: Endothelial progenitors cells (EPCs) cultured from rat bone marrow were identified by double-positive staining with Dil-Ac-LDL and BS1-lectin. Twenty-four hours before cell transplantation, EPCs were labeled with 5-bromo-2'-deoxyuridine (BrdU). Cells (5 x 10(5) in 250-microl medium) were injected into healthy rats, either as intracoronary application (n=11) or as intramyocardial injection (n = 6). At 15 min or 3 days posttransplantation, hearts as well as other organs (lung, liver, kidney, and spleen) were collected and processed for subsequent BrdU immunohistochemistry. The number of BrdU-positive cells per tissue area was counted. RESULTS: Compared to intramyocardial injection, intracoronary administration resulted in more than twice as much positive cells in the heart (P < .05), with no local differences within the heart. Whereas after 15 min, EPCs were equally distributed in all examined organs (except for the spleen), cells that were still present after 3 days, approximately 10%, were selectively restricted to the heart. CONCLUSIONS: Our data indicate that the intracoronary application provides a promising technique for EPC transplantation in the rat heart.     

Requirements for CD4+ cells and gamma interferon in resolution of established Cryptosporidium parvum infection in mice.

The importance of CD4+ cells and gamma interferon (IFN-gamma) in the resolution of established Cryptosporidium parvum infection was investigated with a murine model of cryptosporidiosis in severe combined immunodeficient (SCID) mice. C. parvum-infected SCID mice were reconstituted with spleen cells from immunocompetent donors. The recipients were able to resolve their C. parvum infection by 17 days postreconstitution. Treatment of reconstituted SCID mice with either anti-CD4 monoclonal antibodies to deplete them of CD4+ cells or with anti-IFN-gamma to neutralize IFN-gamma activity reduced or eliminated their ability to resolve C. parvum infection whereas treatment with either anti-CD8 monoclonal antibodies or anti-asialo-GM1 antibodies had no effect. We also found C. parvum-specific antibodies in serum samples from two of four reconstituted SCID mice killed on postreconstitution day 17 but not in unreconstituted SCID mice. Moreover, anti-CD4-treated mice had no detectable specific antibodies to C. parvum, whereas all mice treated with either anti-CD8 or anti-asialo-GM1 had substantial levels of specific antibodies in their serum. Although the role of the specific antibody is not known, these findings clearly indicate that resolution of an established C. parvum infection in immunologically reconstituted SCID mice is dependent on both CD4+ cells and IFN-gamma.     

Factors in the inactivation of Encephalomyocarditis virus in aerosols.

Encephalomyocarditis virus in aerosols is inactivated rapidly at relative humidities below 50%. In glycerol-water mixtures a similar decrease of infectivity occurs when the glycerol concentration exceeds 78% (wt/wt), corresponding to a relative humidity of 50%. The decay in aerosols does not involve oxygen or surface-dependent factors. Variation of temperature shows the inactivation to be a low-energy process with an activation enthalpy of 15 kcal per mol. The damage could be ascribed to dehydration of the virion, presumably proceeding to removal of structurally essential water molecules. This might trigger irreversible changes in the protein coat, resulting in disintegration of the virion.     

Rapid bioassay of human interferon by direct enzyme immunoassay of encephalomyocarditis virus in HEp-2 cell monolayers after a single cycle of infection

Multiplication of encephalomyocarditis virus (EMCV) in human HEp-2 cells, and its suppression by interferon (IFN), was demonstrated by direct enzyme immunoassay (EIA) in cell culture. EMCV was detected in glutaraldehyde fixed HEp-2 cell monolayers, in wells of 96-well plates, with a horse radish peroxidase (HRPO) labelled EMCV specific monoclonal antibody. Multiplication of EMCV (multiplicity of infection: 50) was indicated by a steep rise of absorbance values measured against infected monolayers starting as early as 5 h after infection and reaching relatively high values at 6 and 7 h. The rise in absorbance values did not occur after preincubation of the HEp-2 cells with either Newcastle disease virus-induced IFN, recombinant gamma IFN or recombinant alfa-2a IFN. Absorbance values were inversely dependent on the amount of IFN used. Therefore the EIA was suitable for rapid titration of IFN. The titres of recombinant gamma and alfa-2a IFN determined with EIA proved to be similar to those given by the manufacturers. The described bioassay of human IFN is objective, rapid and easy to perform and suitable for large scale experiments.     

Homology of ab1 and ab3 monoclonal antibodies that neutralize Semliki Forest virus

A noninternal image monoclonal antiidiotypic antibody (ab2 mAb), designated 1.13A321, that had proved its efficacy as vaccine against infection with Semliki Forest virus (SFV) in BALB/c mice, was used as immunogen to generate a panel of SFV-neutralizing monoclonal anti-anti-idiotypic antibodies (ab3 mAbs) to compare them genetically with ab1 mAb 1.13 (IgG2a). There are various studies that compare ab1 and ab3 mAbs but none that compare virus-neutralizing ab1 and ab3 mAbs. Five SFV-neutralizing ab3 MAbs, all IgG1, were obtained. The Vh gene (36-60), the D gene (Sp2), and the J gene (Jh2) encoding the heavy chain variable regions of all six mAbs, were similar and showed a high homology in the nucleotide sequence. The CDR3 amino acid sequences of four of five ab3 mAbs were identical to that of mAb1. One ab3 differed one amino acid in the CDR3 region. The results suggest that a strict selection criterion (virus neutralization) is sufficient to reach complete homology in the CDR3 region of mAb3. Future experiments are focused on selection of synthetic peptides in the CDR3 region as neutralizing mini-antibodies.     

Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora.

Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.     

Waterzuiveringsmethoden en de toepassing van omgekeerde osmose in de apotheek

For different purposes in the pharmacy we need a different quality of water. After having looked at the usual feed water (tap water) the different water purification methods,i.e. demineralisation, distillation and reverse osmosis are briefly reviewed. The microbiological and chemical quality of reverse osmosis water is discussed. The disinfection and inspection of the integrity of the reverse osmosis membrane is emphasised.Samenvatting De vraag komt aan de orde welke kwaliteit water voor de verschillende toepassingen in de apotheek nodig is. Na kort de aandacht te hebben gegeven aan het gebruikelijke voedingswater, namelijk het drinkwater, komen achter-eenvolgens aan de orde demineralisatie, destillatie en omgekeerde osmose als waterzuiveringsmethoden. De microbiologische en de chemische kwaliteit van met name omgekeerde-osmosewater komen ter sprake, alsmede de wijze waarop de desinfectie en de controle van de inte-griteit van de omgekeerde-osmosemembraan plaatsvindt.

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Samengesteid uit voordrachten gehouden voor de Nederlandse Vereniging van Ziekenhuisapothekers (12 februari 1977) en op het symposium Technologie van de waterbereiding voor farmaceutische doeleinden (15 april 1980).