Hot spot mutations in morphological transforming region II (ORF 79) of cytomegalovirus strains causing disease from bone marrow transplant recipients

Summary.  Nested polymerase chain reaction (PCR) amplifying the morphological transforming region II (mtrII) of cytomegalovirus (CMV) has been shown to be useful in the detection of CMV DNA in bone marrow transplant (BMT) recipients. However, there has never been any report on mutation hot spots and subtypes of this open reading frame. Using primers derived from sequences upstream and downstream of mtrII (ORF 79), CMV DNA from peripheral blood leukocytes (PBL) and conventional CMV culture of 16 BMT recipients were amplified by PCR, cloned into pUC118, and sequenced. The amino acid sequences were predicted using the standard triplet code. The DNA sequences obtained from direct amplification of CMV in PBL obtained from the 16 patients were 100% identical to the corresponding ones obtained by amplification of CMV DNA extracted from conventional CMV culture. Within mtrII (ORF 79), hot spot single base mutations were observed at positions +40 (G→A), +123 (A→G), +213 (T→C), and +219 (T→C). However, because of third base degeneracy, only amino acid 14 was changed from valine to isoleucine in the predicted protein of 13 patients. This corresponded to the hot spot mutation at position +40 (GTC→ATC), while the rest were silent mutations. An insertion of 3 bases (ACG) was observed in the CMV DNA of 10 patients at positions +91 to +93, leading to a threonine insertion at amino acid 31 in these patients. For patient no. 147 there was a 65 bp deletion in the CMV DNA amplified later in the course of BMT as compared with that early in the course. This gave rise to a frame shift mutation and a change of more than 70% in the predicted amino acid sequence of the protein. Accepted October 14, 1998 Received May 20, 1998     

CTLA-4 is required for the induction of high dose oral tolerance

Mucosal and systemic administrations of high dose antigens induce long- lasting peripheral T cell tolerance. We and others have shown that high dose peripheral T cell tolerance is mediated by anergy or deletion and is preceded by T cell activation. Co-stimulatory molecules B7-1 (CD80)/B7-2 (CD86) and their counter-receptors CD28/CTLA-4 play pivotal roles in T cell activation and immune regulation. In the present study, we examined the roles of the B7 co-stimulation pathway in the generation of high dose peripheral T cell tolerance. We found that blocking B7:CD28/CTLA-4 interaction at the time of tolerance induction partially prevented T cell tolerance, whereas selective blockade of B7:CTLA-4 interaction completely abrogated peripheral T cell tolerance induced by either oral or i.p. antigens. These results suggest that CTLA-4-mediated feedback regulation plays a crucial role in the induction of high dose peripheral T cell tolerance.      

Molecular genetic characterization of XRCC4 function

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.      

Mutations of OCTN2, an organic cation/carnitine transporter, lead to deficient cellular carnitine uptake in primary carnitine deficiency

Systemic primary carnitine deficiency (CDSP, OMIM 212140) is an autosomal recessive disease characterized by low serum and intracellular concentrations of carnitine. CDSP may present with acute metabolic derangement simulating Reye's syndrome within the first 2 years of life. After 3 years of age, patients with CDSP may present with cardiomyopathy and muscle weakness. A linkage with D5S436 in 5q was reported in a family. A recently cloned homologue of the organic cation transporter, OCTN2, which has sodium-dependent carnitine uptake properties, was also mapped to the same locus. We screened for mutation in OCTN2 in a confirmed CDSP family. One truncating mutation (Trp132Stop) and one missense mutation (Pro478Leu) of OCTN2 were identified together with two silent polymorphisms. Expression of the mutant cDNAs revealed virtually no uptake activity for both mutations. Our data indicate that mutations in OCTN2 are responsible for CDSP. Identification of the underlying gene in this disease will allow rapid detection of carriers and postnatal diagnosis of affected patients.     

Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.     

Bacteriological and molecular analysis of rifampin-resistant Mycobacterium tuberculosis strains isolated in Australia

To develop a better understanding of the epidemiology and molecular biology of rifampin-resistant Mycobacterium tuberculosis strains in Australia, 50 clinical isolates (33 rifampin-resistant and 17 rifampin-sensitive strains) cultured between 1990 and 1997 were analyzed by a number of bacteriological and molecular techniques. Examination of the drug resistance profiles of the 33 rifampin-resistant isolates revealed that 91% were resistant to rifampin in combination with resistance to isoniazid, 88% were resistant to rifampin on first isolation, and 81% showed cross-resistance with rifabutin. On the basis of the demographic data provided for the patients infected with the rifampin-resistant strains, 90% of the patients were born overseas. Of these patients, 64% developed clinical symptoms within 5 years of residence in Australia. On a molecular level, analysis of the rpoB gene revealed that 97% of the rifampin-resistant isolates had missense mutations within a conserved region of the gene, and eight types of missense mutations were detected. Of the 31 rifampin-resistant isolates that were typed by restriction fragment length polymorphism (RFLP) analysis, 28 distinct patterns were obtained by RFLP analysis with IS6110, and three clusters of genetically related isolates were identified. All isolates within the clusters were from patients who were born overseas and who had the same country of origin. The results from this study provide an overview of the current situation of rifampin resistance in Australia and can serve as a basis for continued monitoring of drug-resistant M. tuberculosis strains isolated within the country.     

Practical application of the differential Batho method for inhomogeneity correction on kerma in a photon beam

The Batho equation gives a satisfactory method to correct the dose for points in the electronic equilibrium region for a uniform slab of inhomogeneity in a photon beam. In spite of the many investigations, we believe no simple and adequate method has been found for routine clinical dose calculations which require dose correction of a small-volume inhomogeneity in an arbitrary location. In the present report, we combine the values of the two calculation types of the differential Batho method, which we have developed previously, to give a new calculated value for the scatter perturbation due to an annulus of inhomogeneity. The coefficients in the combination, which we derived from a detailed analysis of the scatter perturbation, are simple geometrical ratios. The new calculated values are in good agreement with measured values. We believe this application of the differential Batho method can provide a practical and accurate method of correcting for inhomogeneities of any size and shape in clinical dose calculations.     

Comparison of two automated DNA amplification systems with a manual one-tube nested PCR assay for diagnosis of pulmonary tuberculosis.

Eighty-four specimens of respiratory secretions culture positive for mycobacteria (70 positive for Mycobacterium tuberculosis and 14 positive for nontuberculous mycobacteria) and 120 culture-negative specimens were evaluated by three DNA amplification techniques: a manual in-house single-tube nested PCR (nPCR) and two commercial automated assays (the Cobas Amplicor System [aPCR-h] from Roche Diagnostic Systems and the Abbott LCx Probe System [aLCx-p] from Abbott Laboratories). The overall diagnostic sensitivities of the nPCR, aPCR-h, and aLCx-p were 77.1, 84.3, and 77.1%, respectively, and the sensitivities were 57.9, 57.9, and 36.8%, respectively, for smear-negative specimens. Specimens culture positive for nontuberculous mycobacteria were negative by all three assays. Eight culture-negative specimens which were positive by one or more assays had previously been documented by culture to be positive for M. tuberculosis and were taken from patients who were treated with antituberculosis agents. Retesting of specimens negative by one assay by the other two assays revealed that each test had its unique group of negative specimens. When considering the DNA extraction and amplification steps of these assays separately, it was found that extracts from aPCR-h and aLCx-p were compatible with nPCR amplication, while the two automated assays could only amplify extracts processed with their own reagents. Limiting dilution analysis revealed that the order of analytical sensitivity was nPCR, followed by aLCx-p and then aPCR-h. Comparison of the work flow of each assay revealed that although the aPCR-h demands the least specimen handling, the turnaround time of aLCx-p is the most favorable.     

Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages

Prunella vulgaris L. (Labiatae), a popular Western and Chinese herbal medicine, has long been associated with anti-viral and anti-bacterial effects. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects remain unknown. As a biological response modifier (BRM), the polysaccharides isolated from P. vulgaris have been shown to up-regulate the immune responses of monocytes/macrophages. However, the immune stimulatory effects seem to contradict its well-known anti-inflammatory properties. We hypothesized that the anti-microbial effects exhibited by the polysaccharides isolated from P. vulgaris encompass both anti-inflammatory and immune stimulatory effects. One of the polysaccharide fractions PV2IV markedly stimulated the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2IV stimulation was as high as that stimulated by bacterial endotoxin lipopolysaccharide (LPS) in a dose-dependent manner. In addition, PV2IV also increased cellular protein levels of inducible nitric oxide synthase (iNOS) and mRNA for tumor necrosis factor-alpha (TNFalpha). Similar to the effects of a high dose of LPS, the fraction PV2 could trigger activation-induced cell death (AICD) by stimulating caspase-3 activity and reduction of MTT uptake in monocytes/macrophages. These results may help our understanding of the molecular mechanism of P. vulgaris, which exhibited both immune stimulatory and anti-inflammatory effects against microbial invasion.