Chromosomal assignment of human O6-methylguanine-DNA-methyltransferase gene by hamster-human somatic cell hybrids.

Using an in vitro assay to measure O6-methylguanine-DNA-methyltransferase (MT) activity in cell extracts from a panel of human-hamster cell hybrids, we were able to locate the human MT gene on chromosome 10. Chinese hamster cells have little or no MT activity and the presence of human chromosome 10 was a necessary condition for MT activity in cell hybrids. In some cell hybrids carrying chromosome 10, however, MT activity was not higher than that of hamster cells. As an explanation for this result, genetic determinants repressing MT expression and/or activity might be present in other human chromosomes carried by MT-negative cell hybrids. Partial hyperploidy of the hamster karyotype, variable activity of the parental human cell lines and changes during subculturing of the cell hybrids might also account for the lack of enzymatic activity in chromosome 10 containing hybrids.     

T lymphocytes in children with recurrent respiratory infections: effect of the use of thymostimulin on the alterations of T-cell subsets

OKT3-, OKT4- and OKT8-positive cells were estimated in 303 children with recurrent respiratory infections. The patients (selected by a score method) had experienced 13 or more infections a year. Modifications in T-cell subsets were observed in 154 patients (50.8%). Decreased OKT3- and OKT4-positive cells were present in 80 children (26.4%), while 74 patients (24.4%) showed normal values of OKT3-positive cells but decreased OKT4- and increased OKT8-positive cells. An attempt at treatment with thymostimulin was undertaken in a group of randomly chosen children with modifications in T-cell subsets. The use of thymostimulin induced the treated children more readily than the untreated ones to show improvement in both the score for respiratory infections and the distribution of T-cell subsets.     

NO enhances presynaptic currents during cerebellar mossy fiber-granule cell LTP

Nitric oxide (NO) is a candidate retrograde messenger in long-term potentiation (LTP). The NO metabolic pathway is expressed in the cerebellar granule cell layer but its physiological role remained unknown. In this paper we have investigated the role of NO in cerebellar mossy fiber-granule cell LTP, which has postsynaptic N-methyl-d-aspartate (NMDA) receptor-dependent induction. Pre- and postsynaptic current changes were simultaneously measured by using extracellular focal recordings, and NO release was monitored with an electrochemical probe in P21 rat cerebellar slices. High-frequency mossy fiber stimulation induced LTP and caused a significant NO release (6.2 +/- 2.8 nM; n = 5) in the granular layer that was dependent on NMDA receptor as well as on nitric oxide synthase (NOS) activation. Preventing NO production by perfusing the NOS inhibitor 100 microM NG-nitro-l-arginine (L-NNA), blocking extracellular NO diffusion by 10 microM MbO2, or inhibiting the NO target guanylyl cyclase (sGC) with 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-dione (ODQ) prevented LTP. Moreover, the NO donor 10 microM 2-(N,N-diethylamino)-diazenolate-2-oxide.Na (DEA-NO) induced LTP, which was mutually occlusive with LTP generated by high-frequency stimulation, prevented by ODQ, and insensitive to NMDA channel blockade (50 microM APV + 25 microM 7-Cl-kyn) or interruption of mossy fiber stimulation. Thus NO is critical for LTP induction at the cerebellar mossy fiber-granule cell relay. Interestingly, LTP manipulations were accompanied by consensual changes in the presynaptic current, suggesting that NO acts as a retrograde signal-enhancing presynaptic terminal excitability.     

FINGERPRINTING OF HLA CLASS I GENES FOR IMPROVED SELECTION OF UNRELATED BONE MARROW DONORS

The degree of matching of HLA genes between the selected donor and recipient is an important aspect of the selection of unrelated donors for allogeneic bone marrow transplantation (UBMT). The most sensitive methods currently used are serological typing of HLA class I genes, mixed lymphocyte culture (MLC), IEF and molecular genotyping of HLA class II genes by direct sequencing of PCR products. Serological typing of class I antigenes (A, B and C) fails to detect minor differences demonstrated by direct sequencing of DNA polymorphic regions. Molecular genotyping of HLA class I genes by DNA analysis is costly and work-intensive. To improve compatibility between donor and recipient, we have set up a new rapid and non-radioisotopic application of the ‘fingerprinting PCR’ technique for the analysis of the polymorphic second exon of the HLA class I A, B and C genes. This technique is based on the formation of specific patterns (PCR fingerprints) of homoduplexes and heterodu-plexes between heterologous amplified DNA sequences. After an electrophoretic run on non-denaturing polyacrylamide gel, different HLA class I types give allele-specific banding patterns. HLA class I matching is performed, after the gel has been soaked in ethidium bromide or silver-stained, by visual comparison of patients’ fingerprints with those of donors. Identity can be confirmed by mixing donor and recipient DNAs in an amplification cross-match. To assess the technique, 10 normal samples, 22 related allogeneic bone marrow transplanted pairs and 10 unrelated HLA-A and HLA-B serologically matched patient-donor pairs were analysed for HLA class I polymorphic regions. In all the related pairs and in 1/10 unrelated pairs, matched donor-recipient patterns were identified. This new application of PCR fingerprinting may confirm the HLA class I serological selection of unrelated marrow donors.     

Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones

The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH.     

Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.     

Rush desensitization in heparin hypersensitivity: a case report

Among all the known drug intolerances, adverse reactions to heparin are not very common. No desensitization in patients with heparin hypersensitivity has ever been attempted. We report the case of a 55–year-old female patient with mitral stenosis and insufficiency, and tricuspid and aortic insufficiency. The patient underwent heparin treatment, and urticaria occurred with either s.c. calcium heparin or i.v. sodium heparin. Allergy testing (skin tests and patch tests) was negative. A pseudoallergic intolerance was diagnosed. Mitral valve replacement with the extracorporeal circulation method was necessary; therefore, heparin treatment was administered. A heparin rush desensitization together with antihistamine drugs (4 mg clorpheniramine maleate for 3 d) was started: 50 UI (0.5 mg) s.c. sodium heparin were first administered; within 4 d, 5000 UI (50 mg) sodium heparin was administered i.v. with no side-effects. A full-dosage heparin treatment was then administered and heart surgery was easily performed. During the postsurgical course, i.v. sodium heparin was smoothly replaced with s.c. calcium heparin (25000 UI s.c. per day) and with oral anticoagulants (sodium warfarin).     

Age-dependent tendency to become sensitized to other classes of aeroallergens in atopic asthmatic children.

BACKGROUND: Several longitudinal studies report that allergic sensitization increases with age from childhood to adulthood. OBJECTIVE: To evaluate whether an age-dependent tendency to become sensitized to new classes of allergens is present in atopic children, we studied retrospectively the changes in allergic sensitization in 165 asthmatic patients, monosensitized (ie, sensitized to only one class of allergens) in the first survey. METHODS: All the children (18 months to 8 years at enrollment), attended our outpatient clinics twice, at time intervals ranging from 2 to 10 years. On each visit, sensitization to house dust mites, pollens, animal danders, and molds was determined by skin prick test. RESULTS: We found that 43.6% (n = 72) of the patients became polysensitized on the second survey. According to age on first survey, the patients were further divided into two age groups: (1) group 1 = 18 months to < 5 years old (n = 98) and (2) group 2 = 5 to 8 years (n = 67). The transition from monosensitization to polysensitization observed in the entire population was present in both groups: 47 (47.9%) of the 98 children in group 1 and 25 (37.3%) of the 67 children in group 2 showed to be sensitized to more classes of allergens, as compared with first survey. Both in the whole population and in the two age subgroups, the changes in the frequency of monosensitization between the two evaluations were time-dependent (P < .05, each Chi(2)). Finally, to investigate whether monosensitization to a specific class of allergens could favor the development of polysensitization, we evaluated the frequency of polysensitization in the second survey in patients originally monosensitized to house dust mites or to pollens. We found that of the 130 patients originally monosensitized to house dust mites, 59 became polysensitized (45.4%), while of the 28 patients originally monosensitized to pollens, 9 became polysensitized (32.1%) (P > . 1). Similar results were obtained when patients were divided into age groups. CONCLUSION: These data demonstrate that (1) monosensitized children are likely to become polysensitized and (2) house dust mite sensitization and, at a lower degree, pollen sensitization, apparently seem to play a "triggering" role in the development of polysensitization, since a high proportion of children originally monosensitized to house dust mites or to pollens became polysensitized.