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.     

Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps.

A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA.     

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.     

Specific T-cell response to a Pneumocystis carinii surface glycoprotein (gp120) after immunization and natural infection.

T cells have been shown to be important in recovery from Pneumocystis carinii pneumonitis, although no specific antigen of P. carinii has been defined as containing T-cell epitopes. P. carinii has an abundant mannosylated surface glycoprotein of approximately 120 kDa (gp120) which induces a prominent host antibody response in experimental animals after exposure to P. carinii in the environment or after recovery from P. carinii pneumonitis. P. carinii gp120 was purified from infected lungs by lectin affinity chromatography. Standard in vitro lymphocyte stimulation assays using purified gp120 and control normal lung preparations were performed on isolated T cells obtained from BALB/c mice after immunization with P. carinii-infected crude lung homogenates or lectin-purified gp120. Lymphocytes from reconstituted severe combined immunodeficient mice which had recovered from naturally acquired P. carinii pneumonitis were also tested. A specific T-cell response was elicited by gp120 after immunization with P. carinii gp120 and after recovery from P. carinii pneumonitis. In addition, the mice developed a strong antibody response to gp120 as ascertained by Western blot (immunoblot). These data suggest that gp120 may be important in the recognition of P. carinii by T cells.     

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.     

IgVH determined genetic restriction of a non-internal image monoclonal anti-idiotypic vaccine against Semliki Forest virus.

Two monoclonal anti-idiotypic antibodies (ab2 mAb), designated 1.13A112 (IgG2a) and 1.13A321 (IgG1) and induced against Semliki Forest virus (SFV)-neutralizing mAb UM 1.13, were investigated with regard to their vaccine potential. 1.13A321 was coupled with glutaraldehyde to keyhole limpet haemocyanin (KLH) and mixed with the adjuvant Quil A. Then when injected subcutaneously into BALB/c mice, it evoked high levels of SFV-neutralizing anti-anti-idiotypic antibodies in serum. In contrast, 1.13A112 had to be indirectly cross-linked to KLH with anti-mouse immunoglobulin to induce a low neutralizing antibody response. Competition binding assay revealed that 1.13A112 and 1.13A321 were completely competitive. Furthermore, SFV neutralization by UM 1.13 and anti-anti-idiotypic (ab3) serum was blocked equally well by either ab2 mAb. These results indicate that ab1 (UM 1.13) and ab3 share at least one antigen-combining site-related idiotope. Induction of SFV-neutralizing antibodies is genetically restricted. Rabbit anti-anti-idiotypic sera against 1.13A321 and 1.13A112 contained no SFV-neutralizing activity. Moreover, in DBA/2, C57BL/6J, CAL-20, and CB-20 mice 1.13A321 did not develop SFV-neutralizing ab3 antibodies in contrast to BALB.K, 129, SWISS, and BAB-14 mice. CAL-20, CB-20, and BAB-14 mice are congenic strains with an inbred background of BALB/c. CB-20 mice derived both IgCH and IgVH from donor strain C57BL/Ka, while BAB-14 mice derived IgCH from C57BL/Ka mice but retained IgVH from BALB/c mice. Clearly, induction of SFV-neutralizing antibodies by 1.13A321 in BAB-14 mice is dependent on IgVH of BALB/c origin. The results suggest that 1.13A321 binds to a paratope-associated recurring idiotope and almost certainly does not bear the internal image of the discontinuous neutralization epitope recognized by mAb UM 1.13. The latter suggestion is sustained by the observation that 1.13A112 and 1.13A321 do not bind to cell receptors.     

Passive intranasal monoclonal antibody prophylaxis against murine Pneumocystis carinii pneumonia

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.     

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.