Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.     

Effects of tapering doses of oral prednisone on viral load among HIV-infected patients with unexplained weight loss

In an exploratory study of virologic and immunomodulatory effects of corticosteroid therapy for wasting syndrome, four HIV-infected adults with recent unexplained weight loss were given tapering doses of prednisone over a 2-month period. Serum neopterin and TNF receptor II levels decreased from baseline after 7 days. An antiretroviral effect was observed initially, peaking on days 14-21 (mean change in HIV-1 branched chain DNA assay on day 21 of -0.52 log10; mean change, from baseline to nadir for each individual, of -0.63 log10); subsequent bDNA levels returned toward baseline as prednisone was tapered. No patient lost weight and there was a mean weight gain of 3.5 kg. Anecdotal reports of corticosteroid benefits in the wasting syndrome may result in part from decreased T cell activation leading to decreased HIV replication, an effect that may be self-limited or that may occur only at higher prednisone doses. Studies involving more targeted immunomodulatory agents for wasting syndrome are warranted.     

Familial infantile convulsions and paroxysmal choreoathetosis: a new neurological syndrome linked to the pericentromeric region of human chromosome 16

Benign infantile familial convulsions is an autosomal dominant disorder characterized by nonfebrile seizures, with the first attack occurring at age 3-12 mo. It is one of the rare forms of epilepsy that are inherited as monogenic Mendelian traits, thus providing a powerful tool for mapping genes involved in epileptic syndromes. Paroxysmal choreoathetosis is an involuntary-movement disorder characterized by attacks that occur spontaneously or are induced by a variety of stimuli. Classification is still elusive, and the epileptic nature of this movement disorder has long been discussed and remains controversial. We have studied four families from northwestern France in which benign infantile convulsions was inherited as an autosomal dominant trait together with variably expressed paroxysmal choreoathetosis. The human genome was screened with microsatellite markers regularly spaced, and strong evidence of linkage for the disease gene was obtained in the pericentromeric region of chromosome 16, with a maximum two-point LOD score, for D16S3133, of 6.76 at a recombination fraction of 0. Critical recombinants narrowed the region of interest to a 10-cM interval around the centromere. Our study provides the first genetic evidence for a common basis of convulsive and choreoathetotic disorders and will help in the understanding and classification of paroxysmal neurological syndromes.     

The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets

Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.     

Quinolone antibiotic photodynamic production of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in cultured liver epithelial cells

To study the basis for the phototoxicity of quinolones, a class of synthetic antibacterials, the photodynamic ability to mediate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation in cultured cells was measured for lomefloxacin (LMX), which is strongly associated with clinical phototoxicity in humans, and ciprofloxacin (CFX), which has few reports of phototoxicity. Adult rat liver (ARL-18) cells were exposed to the quinolones in the presence of UVA and DNA was extracted and analyzed by HPLC with electrochemical detection. Low levels of 8-oxo-dG were found in the DNA of nonirradiated ARL-18 cells and this was increased up to 6-fold in the presence of either LMX (50-400 microM) or up to 3.6-fold in the presence of CFX (50-400 microM) and UVA (20 J/cm2) when compared to the UVA control. Comparing separate experiments with LMX and CFX, LMX produced greater levels of 8-oxo-dG either after dark exposure or after UVA exposure at 20 J/cm2. Also, LMX and CFX were both shown to photodegrade in the presence of UVA, and it was determined that UVA photoinstability alone does not reflect phototoxic potential. These data suggest that the photodynamic potential of LMX and CFX to produce 8-oxo-dG may relate to their human clinical phototoxicity profile. We suggest that the observed clinical phototoxicity is mediated through a UVA photodynamic effect on the quinolone to form reactive oxygen species in the presence of molecular oxygen. The findings indicate that 8-oxo-dG formation can serve as a marker for the potential phototoxicity of new quinolones.     

Incidence of hepatitis B virus infection in the United States, 1976-1994: estimates from the National Health and Nutrition Examination Surveys

Recurrence is a common sequela of Clostridium difficile-associated diarrhea (CDD) and may increase morbidity, costs, and treatment-related antimicrobial resistance. Because recurrent CDD (RCDD) frequently occurs very soon after an initial episode, our goal was to determine the risk factors for early RCDD (occurring < or = 45 days after the initial episode). We conducted a case-control study, comparing 13 patients with early RCDD (case patients) with 46 patients who had only one CDD episode (control patients) at Centre Hospitalier Angrignon (Québec) during January 1993 through November 1994. Risk factors for early RCDD included a history of chronic renal insufficiency, a white blood cell count of > or = 15 x 10(3)/mm3, and community-acquired diarrhea with the first CDD episode. For seven of eight case patients, C. difficile strains from the first and second CDD episodes were identical, suggesting that relapse is more common than reinfection. These results suggest that treatments should be directed at preventing relapses in patients at high risk for early RCDD.     

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.