A novel potent Fas agonist for selective depletion of tumor cells in hematopoietic transplants.

There remains a clear need for effective tumor cell purging in autologous stem cell transplantation (ASCT) where residual malignant cells within the autograft contribute to disease relapse. Here we propose the use of a novel Fas agonist with potent pro-apoptotic activity, termed MegaFasL, as an effective ex-vivo purging agent. MegaFasL selectively kills hematological cancer cells from lymphomas and leukemias and prevents tumor development at concentrations that do not reduce the functional capacity of human hematopoietic stem/progenitor cells both in in vitro and in in vivo transplantation models. These findings highlight the potential use of MegaFasL as an ex-vivo purging agent in ASCT.

Rapid PCR-single-strand conformation polymorphism method to differentiate and estimate relative abundance of Pneumocystis carinii special forms infecting rats.

A rapid method that uses PCR-single-strand conformation polymorphism analysis of the intron of the nuclear 26S rRNA gene was shown to differentiate the two Pneumocystis carinii special forms that infect rats, P. carinii f. sp. carinii and P. carinii f. sp. ratti. The method also provides a means for estimation of the relative abundance of the two special forms in the case of a coinfected rat. The results suggest that the method described will help to further standardize the immunosuppressed rat model of P. carinii infection and, thus, contribute to a better understanding of P. carinii infection in humans.

Prophylaxis failure is associated with a specific Pneumocystis carinii genotype.

To investigate the possible association between Pneumocystis carinii types and various clinical and demographic parameters, we used molecular typing to analyze 93 bronchoalveolar lavage specimens from patients with P. carinii pneumonia (PCP). Multivariate regression analysis revealed an association between being infected with a specific P. carinii genotype and receiving anti-PCP prophylaxis (odds ratio, 4.4; 95% confidence interval, 1.0-18.6; P=.05), although no association with a specific drug was detected.

Genetic diversity of Pneumocystis carinii in HIV-positive and -negative patients as revealed by PCR-SSCP typing.

OBJECTIVE: To describe the epidemiology of severe Pneumocystis carinii pneumonia (PCP) in HIV-infected and non HIV-infected patients. METHODS: Bronchoalveolar lavage specimens from 212 European patients with PCP were typed using PCR--single strand conformation polymorphism analysis of four genomic regions of P. carinii f. sp. hominis. Demographic and clinical information was obtained from all patients. RESULTS: Twenty-three per cent of the patients were presumably infected with a single P. c. hominis type. The other patients presented with two (50%) or more (27%) types. Thirty-five genetically stable and ubiquitous P. c. hominis types were found. Their frequency ranged from 0.4% to 10% of all isolates, and up to 15% of those from a given hospital. There was no significant association between the P. c. hominis type or number of co-infecting types per patient and geographical location, year of collection, sex, age, or HIV status. No more than three patients infected with the same type were observed in the same hospital within the same 6 month period, and no epidemiological link between the cases was found. CONCLUSIONS: The broad diversity of types observed seems to indicate that multiple sources of the pathogen co-exist. There was no evidence that in our study population inter-human transmission played a significant role in the epidemiology of P. carinii.

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors.

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.