Interleukin-1 dysregulation is an intrinsic defect in macrophages from MRL autoimmune-prone mice

Macrophages (M?) from pre-diseased autoimmune-prone MRL mice (both MRL/+ and MRL/1pr) dramatically underproduce the cytokine interleukin-1 (IL-1) in comparison to M? from a number of normal strains. In this study we show that IL-1 dysregulation by MRL M? is fully expressed at birth, and that this defect does not change with time or the development of disease. We also constructed adult irradiation chimeras (consisting of A/J → MRL and MRL → A/J mice), and show that M? isolated from these chimeras display a pattern of IL-1 production indistinguishable from that of the donor strain controls. Moreover, when we constructed a mixed chimera (A/J + MRL → A/J), the A/J and MRL M? coexisting within the same animal retained their individual patterns of IL-1 production when isolated by negative selection. Taken together, these results provide the first substantive evidence for an intrinsic defect (IL-1 dysregulation) in M? from MRL autoimmune-prone mice.     

Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.     

Volunteer studies of deletion mutants of Vibrio cholerae O1 prepared by recombinant techniques

Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.     

Increased Escherichia coli enterotoxin detection after concentrating culture supernatants: possible new enterotoxin detectable in dogs but not in infant mice.

The heat-stable enterotoxin (ST) of Escherichia coli can be detected by infant mouse or dog intestinal loop tests. These tests differ in that the dog assay uses concentrated culture supernatants and is based on measurements of net intestinal absorption, whereas the mouse test uses unconcentrated supernatants and depends on gross fluid accumulation. To compare the relative sensitivities of these assays, culture supernatants of randomly selected E. coli isolates from 34 Bangalee diarrhea patients were tested for ST in dog loops and infant mice. Supernatants were also tested for heat-labile enterotoxin (LT) in dog loops, Y-1 adrenal cells, and Chinese hamster ovary cells. E. coli supernatants that produced positive responses for both ST and LT in the dog loop assay (ST+/LT+) also produced positive responses when tested for ST in infant mice and for LT in cell lines. Supernatants of strains negative for ST and LT in dog loop (ST-/LT) were also negative in other assays. Of 10 strains positive for just ST in the dog loop test (ST+/LT-), only 5 were ST positive in the standard infant mouse test. Supernatants of the other five strains (dog loop positive, mouse test negative) were then concentrated 100-fold and retested in mice. Three of these five gave consistently positive results after concentration, and two were only intermittently positive. Concentrated supernatants of negative control strains (ST-/LT-) were all negative in mice. The dog assay detects more strains producing ST than the infant mouse test. The infant mouse test, which detects only gross fluid accumulation, failed to detect approximately half of the 10 strains which produced ST alone (ST+/LT-; P = 0.025). Concentrating supernatants for the mouse assay increases sensitivity for detection of ST, but certain E. coli strains produce a variety of ST to which infant mice do not respond.     

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.     

Characterization of defined ompR mutants of Salmonella typhi: ompR is involved in the regulation of Vi polysaccharide expression.

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.     

Tobacco withdrawal in women and menstrual cycle phase

Because negative mood is a characteristic of both tobacco withdrawal and menstrual discomfort, withdrawal may vary by menstrual cycle phase. Tobacco withdrawal, mood, and menstrual discomfort were assessed in premenopausal women who quit smoking during either the follicular (Days 1-14 postmenstrual onset; n = 41) or luteal (Day 15 or longer postmenstrual onset; n = 37) phase of the menstrual cycle and maintained biochemically verified smoking abstinence during the postquit week. Women quitting during the luteal phase reported significantly greater increases in tobacco withdrawal and self-reported depressive symptoms than women quitting during the follicular phase. These results indicate that selecting a quit-smoking day early in the follicular phase may attenuate withdrawal and negative affect in premenopausal female smokers.     

Purification and characterization of the serotype c antigen from Actinobacillus actinomycetemcomitans.

The serotype c antigen from Actinobacillus actinomycetemcomitans was purified with fractional ethanol precipitation of cell-free culture supernatant, sequential ion-exchange chromatography, and gel filtration chromatography. The preparation obtained demonstrated a single precipitin line in immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis when rabbit antisera to serotype c whole bacterial cells were used. No immunological reaction was detected with antisera to serotype c lipopolysaccharide, indicating that lipopolysaccharide was not present in the preparation. The serotype c antigen was composed of 95% carbohydrate, 2% protein, and 3.1% phosphate. Gas chromatographic analysis of the antigen obtained from growth in either complex or chemically defined media revealed that the carbohydrate constituent was composed of 84 to 90.1% mannose, 4.8 to 16% glucose, 1.9% N-acetylglucosamine, 1.4% fucose, and 0.2% galactose. The present data suggest that A. actinomycetemcomitans serotype c antigen is predominantly a mannose-containing carbohydrate suggestive of a mannan.     

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.