A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates. To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n ‫؍‬ 35), controls; groups B to D (n ‫؍‬ 86), patients with febrile neutropenia, without fungal colonization (group B; n ‫؍‬ 29) and with fungal colonization (group C; n ‫؍‬ 36); and patients with documented invasive fungal infection (IFI) (group D; n ‫؍‬ 21). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood. Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested. For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.

Abstract. The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8 + cytolytic T cells. To gain further understanding of the Fas system, we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.OPTOSIS is an active cell death process that takes place in a spectrum of physiological conditions such as embryonic development and normal cell turnover, but is also the mode of cell death resulting from CD8 ÷ T cell-mediated cytotoxicity (Steller, 1995). The mechanisms that regulate apoptotic cell death are therefore crucial for normal development and homeostasis.The Fas receptor (FasR, Apo-1 CD95) 1 is a cysteine-rich type I membrane protein belonging to the tumor necrosis factor (TNF) NGF receptor family, and shows high sequence similarities to CD27, CD30, CD40, and the lymphotoxin-13 receptor (Nagata and Golstein, 1995;Smith et al., 1994). Upon contact with cross-linking antibodies or the natural ligand (FasL), cells expressing Fas undergo apoptosis rapidly (Itoh et al., 1991;Trauth et al., 1989) by way Please address all correspondence to L

Summary:PCR-based preemptive therapy with ganciclovir has been shown to reduce the incidence of CMV disease after BMT. Failures of this treatment strategy are CMV disease and secondary non-viral infections. Eighty-six consecutive patients at high risk for CMV disease who received PCR-based preemptive therapy with ganciclovir were assessed for treatment failures and possible risk factors. Ganciclovir was initiated in 57 of 86 patients (66%). Only 28 of 86 (32%) patients received 4 or more weeks of ganciclovir. Recurrence of CMV infection after successful treatment was more frequent among recipients of a BMT from an unrelated compared to a sibling donor (P ‫؍‬ 0.004). Three (3.5%) patients developed non-fatal early onset CMV disease and seven of 68 (10.3 %) late onset CMV disease (Ͼ100 days post transplant). Risk factors for late onset CMV disease were cGVHD (P ‫؍‬ 0.0017) and duration of prior antiviral therapy Ͼ4 weeks (P ‫؍‬ 0.0073). The incidence of secondary non-viral infections was 28% with the duration of antiviral treatment being a significant risk factor for secondary bacterial (P ‫؍‬ 0.0045) and invasive fungal infections (P ‫؍‬ 0.006). Thus, PCR-based preemptive treatment with ganciclovir reduces early onset CMV disease, but the duration of antiviral therapy prior to day ؉100 is a significant risk factor for late onset CMV disease as well as secondary non-viral infections. Bone Marrow Transplantation (2000) 25, 757-763.

Summary:Because of this evidence for an important role of CD4 + T cells in HCMV disease we investigated the development of HCMV-specific T cell proliferation after allogeneic Thirty patients undergoing allogeneic BMT were screened post-transplant together with their marrow BMT and its correlation with the occurrence of HCMV disease early (during the first 100 days) and late (Ͼ100 days) donors for CMV-specific T cell proliferation and the occurrence of CMV disease. Twenty-one of these post-transplant. As shown in several studies, long-term use of prophylacpatients received a marrow transplant from an HLAmatched sibling donor, and nine from an HLA-matched tic ganciclovir, 7 or use in a pre-emptive setting 8 is associated with a low rate of early-, but an increased incidence of unrelated donor. All these patients were either CMV seropositive and/or had received a transplant from a late-onset, HCMV disease. Since ganciclovir has been shown to exhibit myelo-and immunosuppressive proper-CMV-seropositive donor. Patients were monitored for CMV-viraemia until day +100 post-BMT by PCR and ties 7,9 long-term use was shown to delay recovery of the HCMV-specific immune reconstitution post-transplant. In virus culture, and thereafter by virus culture only when clinically indicated. The proliferative T cell response spite of ganciclovir prophylaxis in patients receiving a transplant from an HLA-matched unrelated donor (MUD) was investigated at regular monthly intervals beginning on day +30. A proliferative response to HCMV (median, an increased rate of HCMV infection and disease has been documented. 10 We were thus specifically interested in evalday +123) was documented in these patients between day +37 and +730 post-BMT. None of the patients with uating HCMV-specific immune reconstitution in this patient cohort. a documented CMV-specific T cell proliferation on day 120 (n = 17) developed CMV disease in the later postWe used a HCMV-specific proliferation assay to screen patients at regular monthly intervals until at least 1 year transplant period, but of the patients lacking CMVspecific proliferation (n = 13), 30.8% developed CMV post-BMT. We were specifically interested in identifying patients at risk of developing late-onset HCMV disease by disease after day 120. Thus, patients lacking a CMVspecific T-helper cell response might benefit from differentiating cases according to delayed kinetics of HCMV-specific T cell reconstitution after transplantation sensitive screening for CMV infection and pre-emptive therapy after day +100.either from an HLA-identical sibling or from an unrelated bone marrow donor. Keywords: CMV infection; T cell proliferation; late onset CMV disease Materials and methods PatientsHuman cytomegalovirus is still a major cause of morbidity and mortality in recipients of an allogeneic stem cell transThirty consecutive allogeneic bone marrow transplant plant. Cell-mediated immunity has been shown to be of recipients at risk of developing HCMV infection post-transimportance in the host response to cytomegalov...

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One of the hallmarks of apoptosis is the digestion of genomic DNA by an endonuclease, generating a ladder of small fragments of double-stranded DNA. We have examined the nature of the DNA breaks produced in mouse thymocytes triggered to undergo apoptosis by steroids or by stimulation of the T cell receptor. Whereas the typical ladder pattern of oligonucleosomal fragments was observed after agarose gel electrophoresis, numerous single-strand cuts were detected after electrophoresis under denaturing conditions. Single-strand nicks were found to be very frequent in the internucleosomal regions, but also to occur in the core particle-associated DNA. An identical pattern of single-strand nicks was obtained when chromatin DNA was exposed to the single-strand cleaving deoxyribonuclease I. The nicked DNA fragments, extracted from apoptotic thymocytes, were sensitive to the action of S1-nuclease. We propose that DNA fragmentation induced during apoptosis is not due to a double-strand cutting enzyme as previously postulated, but rather is the result of single-strand breaks. This ensures the dissociation of the DNA molecule at sites where cuts are found within close proximity.

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