Determination of Copy Number of rRNA Genes in Pneumocystis carinii f. sp. hominis

Differential PCR was performed to determine the copy number of rRNA genes in Pneumocystis carinii f. sp. hominis. Two different reference genes, thymidylate synthase (TS) and beta-tubulin (BTU) genes, were used. Primers for the internal transcribed spacer (ITS) region of nuclear rRNA genes and either the TS or BTU gene were mixed together to perform PCR on seven different bronchoalveolar lavage specimens from patients with P. carinii pneumonia. The radioactivity derived from the incorporated radioactive nucleotides of each PCR product band was then used to calculate the copy number of the ITS relative to that of the TS or BTU gene. The copy number ratio between the ITS and the TS gene was determined to be 0.8, and that between the ITS and the BTU gene was also 0.8. These results suggest that the ITS has the same copy number as the TS or BTU gene. Since the copy number of the TS or BTU gene is presumed to be 1, the results also suggest that P. carinii f. sp. hominis has only one copy of the ITS and thus one copy of the nuclear rRNA genes. Therefore, two types of ITS sequences derived from a specimen would indicate that the patient is infected by two types of P. carinii f. sp. hominis.     

Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.

The clinical and pathological features of carcinomas of the pancreas with DNA replication errors (RER+) have not been characterized. Eighty-two xenografted carcinomas of the pancreas were screened for DNA replication errors using polymerase chain reaction amplification of microsatellite markers. Cases with microsatellite instability in at least two markers of a minimum of five tested were considered RER+. RER status was correlated with histological appearance, karyotype of the carcinomas when available, K-ras mutational status, and patient outcome. Three (3.7%) of the eighty-two carcinomas were RER+. In contrast to typical gland-forming adenocarcinomas of the pancreas, all three RER+ carcinomas were poorly differentiated and had expanding borders and a prominent syncytial growth pattern. Neither a Crohn's-like lymphoid infiltrate nor extracellular mucin production were prominent. Ductal adenocarcinomas of the pancreas typically contain a mutant K-ras gene, yet all three RER+ carcinomas had wild-type K-ras. One of the three RER+ carcinomas was karyotyped and showed a near diploid pattern. All three of the RER+ tumors were removed via Whipple resection. One of the three patients is free of disease 16 months after pancreaticoduodenectomy, one is alive and free of tumor at 52 months but developed two colon carcinomas during this period, and the third died of pancreatic cancer at 4 months. None of the three patients had a family history of colorectal carcinoma. A review of the K-ras wild-type carcinomas in a previously characterized series of pancreatic carcinomas with known K-ras mutational status identified two additional cancers with poor differentiation, a syncytial growth pattern, and pushing borders. Both of the cancers were diploid and both patients were longterm survivors (over 5 years). The inclusion of such patients in previous prognostic studies of pancreas cancer may explain the failure of histological grade to be a predictor of prognosis. These data suggest that DNA replication errors occur in a small percentage of resected carcinomas of the pancreas and that wild-type K-ras gene status and a medullary phenotype characterized by poor differentiation, and expanding pattern of invasion, and syncytial growth should suggest the possibility of DNA replication errors in carcinomas of the pancreas.     

Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients

Quantitative monitoring of human cytomegalovirus (HCMV) infection is helpful in determining appropriate antiviral management of transplant recipients. Quantitative PCR technologies have demonstrated accuracy in measuring systemic HCMV loads. A total of 298 consecutive whole-blood specimens submitted to the Clinical Virology Laboratory at Vanderbilt University Medical Center from 15 February to 31 October 1999 were included in the study. In addition to a qualitative colorimetric microtiter plate PCR assay (MTP-PCR) and a semiquantitative pp65 antigenemia assay, each specimen was measured for HCMV loads by a quantitative PCR assay performed on an ABI PRISM 7700 Sequence Detection System (TaqMan). Compared to results of the MTP-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value were 70.5, 97.5, 87.8, and 92.8% for the antigenemia assay and were 96.7, 92.0, 75.6, and 99.1% for the TaqMan assay, respectively. There was a high correlation between antigenemia values and HCMV loads as determined by the TaqMan (r = 0.989; P < 0.001). Antigenemia values of 0, 1 to 10, 11 to 100, 101 to 1,000, and over 1,000 positive cells per 2 x 10(5) leukocytes corresponded to median HCMV loads measured by TaqMan of 125, 1,593, 5,713, 16,825, and 5,425,000 copies/ml, respectively. Corresponding to antigenemia values of 1 to 2, 10, and 50 positive cells per 2 x 10(5) leukocytes, HCMV viral loads of 1,000, 4,000, and 10,000 copies/ml are proposed as cutoff points for initiating antiviral therapy in patient groups with high, intermediate, and low risk of CMV diseases.     

A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes.

A mutant toxin, R7, of thermostable direct hemolysin (TDH) with a single amino acid substitution at glycine 62 was analyzed. The hemolytic activity of R7 decreased to less than 1/1,000 of that of wild-type TDH, and its mouse lethality was undetectable. This mutant toxin, however, showed a marked inhibitory effect on hemolysis by wild-type TDH. Enzyme immunoassay and flow cytometric analysis demonstrated that R7 retained approximately 50% of the ability to bind to erythrocytes compared with that of wild-type TDH, suggesting that its inhibition of hemolysis by wild-type TDH might be due to blocking the binding sites on the erythrocyte membrane. Wild-type TDH affected the erythrocyte membrane by causing an influx of calcium and propidium iodide, while R7 showed no detectable effects of these kinds. These results suggest that hemolysis by TDH consists of at least two steps, binding and postbinding, and that R7 is likely to be a postbinding activity-deficient mutant toxin of TDH.     

冲击伤肺肋面的平行出血条纹

本文采用部分肋骨切除术,切除15只家兔双侧5、6、7、8肋中任一肋距脊柱约2cm处一段长约1cm的肋骨。一周后对受冲击波致伤的肺肋面的出血情况进行解剖观察,发现肺肋面的出血条纹呈“工”字形,从而为冲击伤肺肋面的平行出血条纹是肋间压痕这一观点找到了直接的实验证据。     

Microsatellite analysis of synchronous and metachronous tumors: a tool for double primary tumor and metastasis assessment.

Despite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. In many cases, the classification of these lesions as either two separate primary tumors or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. To establish the origin of tumors, we assessed tumor cell clonality using PCR-based microsatellite analysis on microdissected archival tissues for loss of heterozygosity (LOH) and microsatellite instability (MSI) in a series of 19 paired synchronous and metachronous tumors from several organs. As a control group, 15 autopsy cases with an unequivocally recognizable primary tumor and associated metastases were also examined. Based on LOH and MSI findings, and using a panel of 4 to 12 (median 7) microsatellite markers, we were able to establish the clonal pattern of microsatellite changes in 17 out of 19 (89%) biopsy cases and thus determine if they were either double primary tumors (41%) or metastases (59%). Of interest, identical or similar pattern of microsatellite abnormalities were detected in 15 primary tumors and corresponding metastasis from autopsies. Our results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms and metastases in synchronous and metachronous tumors.