Comparative analysis of genetic variability among Candida albicans isolates from different geographic locales by three genotypic methods.

The objective of the present study was to conduct a comparative genotypic analysis of Candida albicans isolates from the United States, Europe, and Southeast Asia to determine whether differences between isolates might be associated with geographic locations. The genotypes of 86 unrelated isolates of C. albicans (from the United States and Europe) and 26 isolates from Singapore were examined by three DNA typing methods. Computer-assisted methods were used to analyze the gel patterns for all isolates. A dendrogram based on the overall similarity of the patterns obtained by restriction endonuclease analysis (REA) with EcoRI clustered the U.S. and European isolates into two major groups (groups A and B). The Singaporean isolates demonstrated unique REA profiles, with nine isolates having both or neither of the REA-characteristic 3.7- and 4.2-kb bands present in groups A and B. By REA profiles, the Singaporean isolates were related to each other with similarity values (S(AB)s) of > 0.80, but only one isolate mixed with the U.S. and European isolates at this S(AB) (an arbitrary threshold for genetic similarity). Randomly amplified polymorphic DNA (RAPD) analysis generated DNA profiles that clustered the C. albicans isolates into approximately the same number of distinct typing groups as REA. However, isolates identical to each other by REA were generally different from each other by RAPD analysis. In a composite dendrogram prepared from the results obtained by RAPD analysis, the isolates from the United States and Europe clustered in major groups with S(AB)s of > 0.85, while Singaporean isolates connected to these clusters at S(AB)s of > or = 0.75. Pulsed-field gel electrophoresis was less discriminatory, discerning about one-third as many distinct subtypes as REA or RAPD analysis; the Singaporean isolates were distributed randomly with the U.S. and European isolates. These results suggest that a high degree of genetic diversity exists between C. albicans isolates from Southeast Asia and those from the United States and Europe.     

Different pH requirements for entry of the two picornaviruses,human rhinovirus 2 and murine encephalomyocarditis virus

The entry into cells of human rhinovirus 2 (HRV 2) and murine encephalomyocarditis (EMC) virus was studied by the use of light-sensitive virus grown in the presence of acridine orange (HRV 2) and neutral red (EMC). HeLa cells were protected against infection with HRV 2 by NH₄Cl, monensin, and other compounds known to increase the pH of intracellular vesicles. Preincubation of the cells with the same compounds reduced the ability of the cells to bind [³⁵S]methionine-labeled HRV 2, apparently due to inhibition of recycling of endocytosed receptors back to the cell surface. The cells were also protected against infection when HRV 2 was bound to cells on ice and the cells were then incubated at 37° with the different compounds. This indicates that low pH is also necessary for some event in the entry process taking place after the virus is bound to the cells. In contrast, compounds which increase the pH in acidic intracellular compartments did not protect mouse L-cells against infection with EMC-virus, and the entry of the virus was inhibited by low pH in the medium. This inhibition was partly overcome by the presence of the ionophore monensin, which elevates the pH in endosomes and lysosomes. Possibly, EMC virus enters the cytosol from vesicles with neutral or slightly alkaline pH.     

Serological studies of actionomyces israelii by crossed immunoelectrophoresis: standard antigen-antibody system for A. israelii.

Standard preparations of crude cytoplasmic and whole cell-associated antigen mixtures of Actinomyces israelii were analyzed by crossed immunoelectrophoresis (CIE), with a standard polyvalent antiserum comprising purified and concentrated immunoglobulin G antibodies to formolized whole cells of A. israelii serotypes 1 and 2. The standard antigens provided four antigen-antibody systems for A. israelii. The immunoprecipitation patterns of the system were compared, and the immunochemical characteristics of individual precipitates were analyzed. Each system contained specific precipitates, but also one or two precipitates which were immunochemically identical to precipitates of the other systems. The standard system for A. israelii based on cytoplasmic antigens was best reproducible and revealed the highest number of immunoprecipitates. These precipitates possessed immunochemical properties which made them suitable for CIE studies. The cytoplasmic antigen mixture of A. israelii was, therefore, adopted as the most suitable for further development of a crossed immunoelectrophoretic system for A. israelii. In subsequent assays the cytoplasmic antigen mixture was raised in rabbit against cell lysates of A. israelii, serotypes 1 and 2. A standard antigen-antibody system for A. israelii was obtained which revealed an immunoprecipitation pattern of 10 distinguishable precipitates. The resolving power and separation by CIE of this standard system for A. israelii was compared with that of crossed immunoelectrofocusing. The results suggest that these methods supplement each other. Crossed immunoelectrofocusing appeared to be a useful tool for separation of specific components of the protein-antigen complex of A. israelii for analytic serology. The CIE in conjunction with a standard reference antigen-antibody system for A. israelii based on cytoplasmic antigens offers great potentialities in diagnostic A. israelii serology.     

Evaluation of human seroreactivity to Bartonella species in Sweden

Among the species that compose the expanding genus Bartonella, thus far only B. henselae and B. quintana have reportedly been isolated from humans in Europe. To evaluate the prevalence of Bartonella infection in Sweden, we conducted a retrospective serological examination of 126 human serum samples. These samples were analyzed for antibodies to B. henselae, B. quintana, and B. elizabethae. Serum samples from 100 blood donors, who spanned the ages of 20 to 60 and had no apparent clinical signs of illness, were also studied as a control group. An immunoglobulin G indirect fluorescence antibody assay revealed 4 and 8.3% Bartonella positivity rates for the blood donor and patient group, respectively, when a cutoff titer of >/=64 was chosen. Among the blood donors, four were seropositive to B. elizabethae; one of these also had concordant positive titer to B. henselae. In the patient group, 14 serum samples were positive against Bartonella spp. These serum specimens represented nine patients. In three of these seropositive patients, paired serum samples displayed a fourfold increase in antibody titer to at least one of the three antigens. These three patients are discussed. In this report we also present a case study of a 60-year-old Swedish male with fatal myocarditis. Postmortem serological analysis revealed a high titer against B. elizabethae. PCR and nucleotide sequencing of the myocardial tissue from this patient, and of liver tissue from one of the other three patients, showed sequences similar to B. quintana. The age, geographical origin, animal contacts, and serological response pattern to the different Bartonella antigens differed among the four patients. This study substantiates the presence of Bartonella spp. in Sweden, documents the seroreactivity to three Bartonella antigens in Swedish patients, and reports the first two cases of B. quintana-like infections in Sweden.     

Idiotypic characterization of antibody-induced antibody responses

Anti-idiotypic antisera were produced in syngeneic (C57BL/6) mice against a monoclonal anti-Dextran B512 (Dex) antibody (38-13). In radioimmunoassays, anti-idiotypic antibodies were shown to react with the homologous idiotype, while failing to recognize another monoclonal anti-Dex antibody, independently derived from C57BL/6 mice (D-16). Plaque inhibition tests confirmed the specificity of the anti-idiotypic antibodies and revealed that the 38-13 idiotype is expressed by about half of all anti-Dex antibodies produced in C57BL/6, but not in CBA mice. Injection of normal (but not athymic) C57BL/6 mice with low doses of 38-13 monoclonal antibodies, contained culture supernatants or ascitic fluids, resulted in a 10-20 fold increase in the numbers of anti-Dex PFC detected in the spleen 5 days later, the majority of which carried the 38-13 idiotype.     

Testicular cancer susceptibility in the 129.MOLF-Chr19 mouse strain: additive effects,gene interactions and epigenetic modifications

Testicular germ cell tumors (TGCTs) are the most common solid cancers affecting young men. Although the evidence for genetic predisposition to TGCTs in humans is compelling, the genetic control of susceptibility is poorly understood. The 129S1/SvImJ (129/Sv) inbred strain of mice is an excellent model for studying TGCT susceptibility. We previously reported a new mouse strain, the 129.MOLF-Chr19 chromosome substitution strain, which develops spontaneous TGCTs at a high frequency (70-80%) as compared with the much lower rate in the 129/Sv strain (5%). To characterize the genetic control of TGCT susceptibility, we created a panel of single- and double-congenic strains derived from 129.MOLF-Chr19. The frequency of TGCTs in these strains suggests that several genes with additive and epistatic effects located at distinct sites on chromosome 19 control susceptibility. However, an alternative interpretation involving epigenesis is based on a striking correlation between TGCT frequency and the length of the MOLF-derived congenic segment, regardless of their chromosomal location on Chr 19 in each congenic strain. We also show that bilateral TGCT cases result from the coincidental co-occurrence of unilateral TGCTs rather than from the action of distinct genes that control susceptibility to bilateral versus unilateral TGCT cases. Finally, we propose that these TGCTs result from disrupted testicular and spermatogenic developmental programs.