Evaluation of a Diagnostic Algorithm Using Immunoglobulin M Enzyme-Linked Immunosorbent Assay To Differentiate Human West Nile Virus and St. Louis Encephalitis Virus Infections during the 2002 West Nile Virus Epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was ≥1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Characterization of a novel breast carcinoma xenograft and cell line derived from a BRCA1 germ-line mutation carrier

A human tumor xenograft (L56Br-X1) was established from a breast cancer axillary lymph node metastasis of a 53-year-old woman with a BRCA1 germ-line nonsense mutation (1806C>T; Q563X), and a cell line (L56Br-C1) was subsequently derived from the xenograft. The xenograft carries only the mutant BRCA1 allele and expresses mutant BRCA1 mRNA but no BRCA1 protein as determined by immunoprecipitation or Western blotting. The primary tumor, lymph node metastasis, and xenograft were hypodiploid by DNA flow cytometry, whereas the cell line displayed an aneuploidy apparently developed via polyploidization. Cytogenetic analysis, spectral karyotyping, and comparative genomic hybridization of the cell line revealed a highly complex karyotype with numerous unbalanced translocations. The xenograft and cell line had retained a somatic TP53 missense mutation (S215I) originating from the primary tumors, as well as a lack of immunohistochemically detectable expression of steroid hormone receptors, epidermal growth factor receptor, human epidermal growth factor receptor 2 (HER-2), and keratin 8. Global gene expression analysis by cDNA microarrays supported a correlation between the expression profiles of the primary tumor, lymph node metastasis, xenograft, and cell line. We conclude that L56Br-X1 and L56Br-C1 are useful model systems for studies of the pathogenesis and new therapeutic modalities of BRCA1-induced human breast cancer.     

Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction

In view of the increasing number of cases of human microsporidiosis, simple and rapid methods for clear identification of microsporidian parasites to the species level are required. In the present study, the polymerase chain reaction (PCR) was used for speciesspecific detection ofEncephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon (Septata) intestinalis, andEnterocytozoon bieneusi in both tissue and stool. Using stool specimens and intestinal biopsies of patients infected withEnterocytozoon bieneusi (n=9),Encephalitozoon spp. (n=2), andEncephalitozoon intestinalis (n=1) as well as stool spiked with spores ofEncephalitozoon cuniculi andEncephalitozoon hellem and tissue cultures ofEncephalitozoon cuniculi andEncephalitozoon hellem, three procedures were developed to produce PCR-ready DNA directly from the samples. Specific detection of microsporidian pathogens was achieved in the first PCR. The subsequent nested PCR permitted species determination and verified the first PCR products. Without exception, the PCR assay confirmed electron microscopic detection ofEnterocytozoon bieneusi andEncephalitozoon intestinalis in stool specimens and their corresponding biopsies and in spiked stool samples and tissue cultures infected withEncephalitozoon cuniculi andEncephalitozoon hellem. Moreover, identification ofEncephalitozoon spp. could be specified asEncephalitozoon intestinalis. Whereas standard methods such as light and transmission electron microscopy may lack sensitivity or require more time and special equipment, the PCR procedure described facilitates speciesspecific identification of microsporidian parasites in stool, biopsies, and, probably, other samples in about five hours.     

Chromosome number distribution and cellular DNA content in colorectal adenomas from polyposis and nonpolyposis patients

Ploidy analyses of colorectal adenomas were performed by combined flow cytometric DNA analysis of unfixed isolated nuclei and direct chromosome preparation after Colcemid incubation for 9-20 hours. Ten of 18 adenomas from nonpolyposis patients and 4 of 13 adenomas from patients with familial adenomatous polyposis yielded a mean of 25 countable metaphases (range 7-44) per tumor. Of 343 metaphases, only 38% had 46 chromosomes, and 62% were nondiploid. All but one adenoma had diploid or peridiploid modes in the range of 46-50 chromosomes. One adenoma was hyperploid, with a mode of 74 chromosomes and a correspondingly increased nuclear DNA content. In another two adenomas, the DNA analyses showed small hyperploid populations constituting 6% and 2% of the cells. The most striking difference between the DNA analyses and chromosome number distributions was that 13% of all metaphases were hyperploid with chromosome numbers outside the perimodal range but, except in one adenoma, without indication in the DNA histogram of corresponding hyperploid cell populations. We propose that these aberrant metaphases indicate an early acquired genetic instability of the neoplastic epithelium, which may be instrumental in generation of hyperploid, invasive clones, which constitute most colorectal carcinomas.     

Smooth muscle differentiation in cultured human breast gland stromal cells

We analyzed in cultures from the human breast the potential of stromal cells resembling fibroblasts to undergo smooth muscle differentiation. The cellular components of the breast tissue from 10 biopsies were disaggregated by collagenase digestion and further purified by differential centrifugation into suspensions of single cells and intact blood vessels. These two fractions of stromal cells were plated in culture and their phenotypic traits analyzed within 24 hours. During this time, the blood vessel fraction gave rise to stromal cells with smooth muscle differentiation as judged immunocytochemically using monoclonal antibodies to alpha-/gamma-muscle actins, to alpha-smooth muscle actin, to type IV collagen, and to laminin. Furthermore, the cells of this fraction resembled smooth muscle cells based on 2D gel electrophoresis and immunoblotting determination of isoactin content. After 24 hours in culture, the single-cell fraction consisted of an almost pure population of cells not exhibiting smooth muscle differentiation but rather resembling fibroblasts. Maintenance of fibroblast-like cells without smooth muscle differentiation was possible for more than 14 days on chemically defined medium. These cells remained quiescent, as measured by cell quantification and immunoreactivity to the proliferation-associated antigen, Ki-67. Growth of these cells could be stimulated by adding serum at any time during the experimental period. Single-cell fractions from seven biopsies were allowed to grow exponentially in the presence of serum for up to 10 days, and the kinetics of smooth muscle differentiation were monitored immunocytochemically and biochemically. These experiments showed that alpha-smooth muscle actin synthesis was induced in 10 to 80% of the fibroblast-like cells after 4 to 11 days in culture. Both the final number of alpha-smooth muscle actin-positive cells and the onset of synthesis varied with the initial seeding density. Dose-response experiments (at constant cell density) revealed that serum exerted maximal effect at concentrations above 10%. It was therefore concluded that elements of smooth muscle differentiation may arise in non-smooth muscle stromal cells taken directly from human breast tissue.     

Ensemble noise and current relaxation analysis of K+ current in single isolated salivary acinar cells from rat

The K⁺ channel in rat parotid gland acinar cells were investigated by ensemble current noise analysis in single isolated cells employing the giga-seal whole cell current recording mode. Sets of 20–40 identical de- and hyperpolarization voltage steps were applied and the resultant current records were processed by computer to obtain the mean and the variance of the current. The time-course of the mean current could be fitted by the sum of two exponentials, suggesting a 3-state model. The simplest plausible hypothesis is a model with one open and two closed states. Assuming this model, the relationship between the variance (₂) and the mean current (I) could be fitted by the function ₂/I=i–I/N. The estimated single channeli/V-relations were similar to those taken from single channel current recordings, and the size of the population of channels per cell (N) was 76±26 (n=12). The validity of the model was tested by a successful simulation of the time-course of the variance.