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1.  Sleep duration and incidence of colorectal cancer in postmenopausal women 
British Journal of Cancer  2013;108(1):213-221.
Background:
Sleep duration is dependent on circadian rhythm that controls a variety of key cellular functions. Circadian disruption has been implicated in colorectal tumorigenesis in experimental studies. We prospectively examined the association between sleep duration and risk of colorectal cancer (CRC).
Methods:
In the Women's Health Initiative Observational Study, 75 828 postmenopausal women reported habitual sleep duration at baseline 1993–1998. We used Cox proportional hazards regression model to estimate the hazard ratio (HR) of CRC and its associated 95% confidence interval (CI).
Results:
We ascertained 851 incident cases of CRC through 2010, with an average 11.3 years of follow-up. Compared with 7 h of sleep, the HRs were 1.36 (95% CI 1.06–1.74) and 1.47 (95% CI 1.10–1.96) for short (⩽5 h) and long (⩾9 h) sleep duration, respectively, after adjusting for age, ethnicity, fatigue, hormone replacement therapy (HRT), physical activity, and waist to hip ratio. The association was modified by the use of HRT (P-interaction=0.03).
Conclusion:
Both extreme short and long sleep durations were associated with a moderate increase in the risk of CRC in postmenopausal women. Sleep duration may be a novel, independent, and potentially modifiable risk factor for CRC.
doi:10.1038/bjc.2012.561
PMCID: PMC3553538  PMID: 23287986
sleep; colorectal neoplasms; risk factors; women; prospective studies
2.  The relationship between physical activity and low back pain outcomes: a systematic review of observational studies 
European Spine Journal  2010;20(3):464-474.
Although clinical guidelines advocate exercise and activity in the management of non-specific low back pain (NSLBP), the link between levels of physical activity and outcomes is unclear. This systematic review investigated the relationships between free living activity levels after onset of low back pain (LBP) and measures of pain, and disability in patients with NSLBP. Cohort and cross-sectional studies were located using OVID, CINAHL, Medline, AMED, Embase, Biomed, PubMed-National Library of Medicine, Proquest and Cochrane Databases, and hand searches of reference lists. Studies were included if a statistical relationship was investigated between measures of free living physical activity (PA) in subjects with LBP and LBP outcome measures. Twelve studies (seven cohort and five cross-sectional) were included. One prospective study reported a statistically significant relationship between increased leisure time activity and improved LBP outcomes, and one cross-sectional study found that lower levels of sporting activity were associated with higher levels of pain and disability. All other studies (n = 10) found no relationship between measures of activity levels and either pain or disability. Heterogeneity of study designs, particularly in terms of activity measurement, made comparisons between studies difficult. These data suggest that the activity levels of patients with NSLBP are neither associated with, nor predictive of, disability or pain levels. Validated activity measurement in prospective research is required to better evaluate the relationships between PA and LBP.
doi:10.1007/s00586-010-1616-2
PMCID: PMC3048226  PMID: 21053026
Physical activity; Low back pain; Systematic review; Outcomes; Guidelines
3.  Incidence of diabetic retinopathy in people with type 2 diabetes mellitus attending the Diabetic Retinopathy Screening Service for Wales: retrospective analysis  
Objectives To determine the incidence of any and referable diabetic retinopathy in people with type 2 diabetes mellitus attending an annual screening service for retinopathy and whose first screening episode indicated no evidence of retinopathy.
Design Retrospective four year analysis.
Setting Screenings at the community based Diabetic Retinopathy Screening Service for Wales, United Kingdom.
Participants 57 199 people with type 2 diabetes mellitus, who were diagnosed at age 30 years or older and who had no evidence of diabetic retinopathy at their first screening event between 2005 and 2009. 49 763 (87%) had at least one further screening event within the study period and were included in the analysis.
Main outcome measures Annual incidence and cumulative incidence after four years of any and referable diabetic retinopathy. Relations between available putative risk factors and the onset and progression of retinopathy.
Results Cumulative incidence of any and referable retinopathy at four years was 360.27 and 11.64 per 1000 people, respectively. From the first to fourth year, the annual incidence of any retinopathy fell from 124.94 to 66.59 per 1000 people, compared with referable retinopathy, which increased slightly from 2.02 to 3.54 per 1000 people. Incidence of referable retinopathy was independently associated with known duration of diabetes, age at diagnosis, and use of insulin treatment. For participants needing insulin treatment with a duration of diabetes of 10 years or more, cumulative incidence of referable retinopathy at one and four years was 9.61 and 30.99 per 1000 people, respectively.
Conclusions Our analysis supports the extension of the screening interval for people with type 2 diabetes mellitus beyond the currently recommended 12 months, with the possible exception of those with diabetes duration of 10 years or more and on insulin treatment.
doi:10.1136/bmj.e874
PMCID: PMC3284424  PMID: 22362115
4.  Spatial organisation of microbiota in quiescent adenoiditis and tonsillitis 
Journal of Clinical Pathology  2006;60(3):253-260.
Background
The reasons for recurrent adenotonsillitis are poorly understood.
Methods
The in situ composition of microbiota of nasal (5 children, 25 adults) and of hypertrophied adenoid and tonsillar tissue (50 children, 20 adults) was investigated using a broad range of fluorescent oligonucleotide probes targeted to bacterial rRNA. None of the patients had clinical signs of infection at the time of surgery.
Results
Multiple foci of ongoing purulent infections were found within hypertrophied adenoid and tonsillar tissue in 83% of patients, including islands and lawns of bacteria adherent to the epithelium, with concomitant marked inflammatory response, fissures filled with bacteria and pus, and diffuse infiltration of the tonsils by bacteria, microabscesses, and macrophages containing phagocytosed microorganisms. Haemophilusinfluenzae mainly diffusely infiltrated the tissue, Streptococcus and Bacteroides were typically found in fissures, and Fusobacteria,Pseudomonas and Burkholderia were exclusively located within adherent bacterial layers and infiltrates. The microbiota were always polymicrobial.
Conclusions
Purulent processes persist during asymptomatic periods of adenotonsillitis. Most bacteria involved in this process are covered by a thick inflammatory infiltrate, are deeply invading, or are located within macrophages. The distribution of the bacteria within tonsils may be responsible for the failure of antibiotic treatment.
doi:10.1136/jcp.2006.037309
PMCID: PMC1860565  PMID: 16698947
5.  Effects of interpregnancy interval and outcome of the preceding pregnancy on pregnancy outcomes in Matlab, Bangladesh 
Bjog   2007;114(9):1079-1087.
Objective
To estimate the effects on pregnancy outcomes of the duration of the preceding interpregnancy interval (IPI) and type of pregnancy outcome that began the interval.
Design
Observational population-based study.
Setting
The Maternal Child Health–Family Planning (MCH–FP) area of Matlab, Bangladesh.
Population
A total of 66 759 pregnancy outcomes that occurred between 1982 and 2002.
Methods
Bivariate tabulations and multinomial logistic regression analysis.
Main outcome measures
Pregnancy outcomes (live birth, stillbirth, miscarriage [spontaneous fetal loss prior to 28 weeks], and induced abortion).
Results
When socio-economic and demographic covariates are controlled, of the IPIs that began with a live birth, those <6 months in duration were associated with a 7.5-fold increase in the odds of an induced abortion (95% CI 6.0–9.4), a 3.3-fold increase in the odds of a miscarriage (95% CI 2.8–3.9), and a 1.6-fold increase in the odds of a stillbirth (95% CI 1.2–2.1) compared with 27- to 50-month IPIs. IPIs of 6–14 months were associated with increased odds of induced abortion (2.0, 95% CI 1.5–2.6). IPIs ≥ 75 months were associated with increased odds of all three types of non-live-birth (NLB) outcomes but were not as risky as very short intervals. IPIs that began with a NLB were generally more likely to end with the same type of NLB.
Conclusions
Women whose pregnancies are between 15 and 75 months after a preceding pregnancy outcome (regardless of its type) have a lower likelihood of fetal loss than those with shorter or longer IPIs. Those with a preceding NLB outcome deserve special attention in counselling and monitoring.
Please cite this paper as: DaVanzo J, Hale L, Razzaque A, Rahman M. Effects of interpregnancy interval and outcome of the preceding pregnancy on pregnancy outcomes in Matlab, Bangladesh. BJOG 2007;114:1079–1087.
doi:10.1111/j.1471-0528.2007.01338.x
PMCID: PMC2366022  PMID: 17617195
Birth spacing; fetal loss; induced abortion; interpregnancy intervals; miscarriage; pregnancy outcomes; pregnancy spacing; stillbirth
6.  Bacterial biofilm within diseased pancreatic and biliary tracts 
Gut  2005;54(3):388-395.
Background: Bacterial community structures in human pancreatic and biliary tracts were evaluated.
Methods: Gall bladder stones from 153 patients, 20 gall bladder walls, six common duct stones, 52 biliary stents, 21 duodenal biopsies, nine pancreatic duct biopsies, and five bile ducts were investigated using fluorescence in situ hybridisation (FISH) with ribosomal RNA targeted Cy3/Cy5 (carbocyanine) labelled oligonucleotide probes.
Result: Duodenal, gall bladder, and bile duct walls were free of bacteria. A dense multispecies bacterial biofilm was present within the pancreatic duct of patients with calcific pancreatitis and within biliary stents, irrespective of diagnosis. The concentration, density, and amenability of the biofilm to FISH and DNA staining declined progressively with the grade of stent occlusion. The lowest detectable bacterial concentrations were found by FISH in completely occluded stents and brown/mixed gall stones. Bacteria were not detectable with FISH in cholesterol gall stones.
Conclusions: A wide range of different branches and groups of bacteria participate in the development of biofilms on the surfaces of foreign bodies, such as biliary stents, mixed gall stones, or calcific pancreatic ducts, but not on the surface of pure cholesterol gall stones. Occlusion of stents leads to progressive extinction of the biofilm and mummification of its components. Deposition of cholesterol or other substances within the biofilm matrix may be a novel mechanism of host defence against bacteria present in these biofilms.
doi:10.1136/gut.2004.043059
PMCID: PMC1774423  PMID: 15710988
bacterial biofilms; cholesterol; pancreas; biliary tract; gall stone pathogenesis; stent occlusion
7.  Neocentromere formation in a stable ring 1p32-p36.1 chromosome 
Journal of Medical Genetics  1999;36(12):914-918.
Neocentromeres are functional centromeres formed in chromosome regions outside the normal centromere domains and are found in an increasing number of mitotically stable human marker chromosomes in both neoplastic and non-neoplastic cells. We describe here the formation of a neocentromere in a previously undescribed chromosomal region at 1p32→p36.1 in an oligospermic patient. Cytogenetic GTL banding analysis and the absence of detectable fluorescence in situ hybridisation (FISH) signals using telomeric probes indicate the marker to be a ring chromosome. The chromosome is negative for CBG banding and is devoid of detectable centromeric α satellite and its associated centromere protein CENP-B, suggesting activation of a neocentromere within the 1p32-36.1 region. Functional activity of the neocentromere is shown by the retention of the ring chromosome in 97% of the patient's lymphocytes and 100% of his cultured fibroblasts, as well as by the presence of key centromere binding proteins CENP-E, CENP-F, and INCENP. These results indicate that in addition to CENP-A, CENP-C, and CENP-E described in earlier studies, neocentromere activity can further be defined by CENP-F and INCENP binding. Our evidence suggests that neocentromere formation constitutes a viable mechanism for the mitotic stabilisation of acentric ring chromosomes.


Keywords: neocentromere; centromere proteins; α satellite DNA; chromosome 1
PMCID: PMC1734276  PMID: 10593999
8.  Serum antibody to lipopolysaccharide antigens of Shigella species among U.S. military personnel deployed to Saudi Arabia and Kuwait during Operations Desert Shield and Desert Storm. 
During Operations Desert Shield and Desert Storm, U.S. troops were at high risk of diarrheal disease due to Shigella spp., particularly Shigella sonnei. In order to better understand the serologic response to Shigella infection, 830 male U.S. combat troops were evaluated before and after the deployment to Saudi Arabia and Kuwait for immunoglobulin A (IgA) and IgG anti-Shigella lipopolysaccharide (LPS) (antibody to S. sonnei form I and Shigella flexneri serotypes 1a, 2a, and 3a) in serum. Just before deployment, 10.3% of the subjects were seropositive for IgA and 18.3% were positive for IgG anti-Shigella LPS. IgA and IgG anti-LPS antibody levels in serum prior to deployment were significantly associated with nonwhite race and ethnicity, birth outside the United States, and antibody to hepatitis A virus and Helicobacter pylori. During the deployment, which lasted for a mean of 131 days, 60% of the subjects reported at least one episode of diarrhea and 15% reported an episode of diarrhea with feverishness; also, 5.5% of the subjects exhibited IgA seroconversion to Shigella LPS and 14.0% exhibited IgG seroconversion. A significant association between the development of diarrheal symptoms and either positive predeployment anti-LPS antibody or seroconversion was not found. These data indicate that in this population of U.S. Desert Storm troops who were at high risk of Shigella infection, there was no apparent relation between IgA or IgG anti-Shigella LPS in serum and diarrheal disease.
PMCID: PMC170224  PMID: 8574833
9.  Protection against local Shigella sonnei infection in mice by parenteral immunization with a nucleoprotein subcellular vaccine. 
Infection and Immunity  1995;63(7):2762-2765.
Nucleoprotein subcellular (NPS) vaccine, consisting of ribosome-bound O polysaccharide, was prepared from avirulent Shigella sonnei. NPS vaccine was tested for safety and protective activity in the mouse intranasal challenge model of Shigella infection. The vaccine was nontoxic when injected in doses up to 10,000 micrograms, and a single subcutaneous injection of as little as 0.1 micrograms gave significant protection against a lethal intranasal challenge with S. sonnei. These data demonstrate the induction of local protective immunity by parenteral immunization, support the concept of the ribosome as a potent vaccine vector, and give additional evidence for the protective activity of the NPS vaccine against Shigella infection.
PMCID: PMC173369  PMID: 7790095
10.  Intransal or intragastric immunization with proteosome-Shigella lipopolysaccharide vaccines protects against lethal pneumonia in a murine model of Shigella infection. 
Infection and Immunity  1995;63(6):2382-2386.
Mice immunized intransally or intragastrically with proteosome vaccines containing either Shigella sonnei or S. flexneri 2a lipopolysaccharide were protected against lethal pneumonia caused by homologous organisms in an experimental murine intranasal challenge model of Shigella infection. Histopathological analysis demonstrated that immunization also protected against the progressive lesions resulting from invasion of the pulmonary mucosa by S. sonnei. These data show that mucosal proteosome-lipopolysaccharide vaccines can protect against lethal bacterial pneumonia and indicate that such vaccines are promising candidates for protection against intestinal shigellosis.
PMCID: PMC173317  PMID: 7768627
11.  Antibody and cytokine responses in a mouse pulmonary model of Shigella flexneri serotype 2a infection. 
Infection and Immunity  1995;63(5):1947-1954.
A murine pulmonary model was used to study the mucosal immune response to Shigella flexneri serotype 2a infection. Inoculation of BALB/cJ mice with shigellae via the intranasal route resulted in bacterial invasion of bronchial and alveolar epithelia with concomitant development of acute suppurative bronchiolitis and subsequent development of lethal pneumonia. The pathology of pulmonary lesions resembled the colitis that characterizes shigellosis in humans and primates. Significant protection against a lethal dose of S. flexneri 2a was observed in mice previously infected with two sublethal doses of the homologous strain. Immunity against lethal challenge was associated with decreased bacterial invasion of the mucosal epithelium. Over the course of two sublethal challenges, which constituted primary and secondary immunizations, mice developed pulmonary and serum immunoglobulin G and A antibody recognizing both lipopolysaccharide and invasion plasmid antigens IpaB and IpaC. Immune mice and naive control mice differed in lung lavage cytokine levels following lethal challenge. Immune mice developed significantly elevated levels of pulmonary gamma interferon within 6 h of challenge, while naive control mice developed elevated levels of this cytokine later during the initial 24-h period. Both groups had elevated levels of gamma interferon during the 24- to 48-h period of infection. Both groups also had elevated levels of tumor necrosis factor alpha within 6 h of challenge, but the control mice had significantly higher levels at the 48- and 72-h time points. Elevated levels of interleukin-4 were observed only in immunized mice. This cytokine appeared within 24 h and receded between 48 and 72 h. Fluorescence-activated cell sorter analysis of lung parenchymal cells showed that both groups experienced an initial influx of monocytes, but the proportion of this cell type began to recede in immunized mice after 48 h of infection, while peak levels were maintained in the control animals. These studies suggest that elements of local B lymphocyte activity, as well as Th1 and Th2 lymphocyte activity, may contribute to the survival of immune mice after intranasal challenge with shigellae.
PMCID: PMC173248  PMID: 7729907
12.  Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K-12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen. 
Infection and Immunity  1992;60(6):2218-2224.
A live, oral Shigella vaccine, constructed by transfer of the 140-MDa invasiveness plasmid from Shigella flexneri 5 and the chromosomal genes encoding the group- and type-specific O antigen of S. flexneri 2a to Escherichia coli K-12, was tested in humans. Designated EcSf2a-1, this vaccine produced adverse reactions (fever, diarrhea, or dysentery) in 4 (31%) of 13 subjects who ingested a single dose of 1.0 x 10(9) CFU, while at better-tolerated doses (5.0 x 10(6) to 5.0 x 10(7) CFU), it provided no significant protection against challenge with S. flexneri 2a. A further-attenuated aroD mutant derivative, EcSf2a-2, was then tested. Rhesus monkeys that received EcSf2a-2 in three oral doses of ca. 1.5 x 10(11) CFU experienced no increase in gastrointestinal symptoms compared with a control group that received an E. coli K-12 placebo. Compared with controls, the vaccinated monkeys were protected against shigellosis after challenge with S. flexneri 2a (60% efficacy; P = 0.001). In humans, EcSf2a-2 was well tolerated at inocula ranging from 5.0 x 10(6) to 2.1 x 10(9) CFU. However, after a single dose of 2.5 x 10(9) CFU, 4 (17%) of 23 subjects experienced adverse reactions, including fever (3 subjects) and diarrhea (209 ml) (1 subject), and after a single dose of 1.8 x 10(10) CFU, 2 of 4 subjects developed dysentery. Recipients of three doses of 1.2 to 2.5 x 10(9) CFU had significant rises in serum antibody to lipopolysaccharide (61%) and invasiveness plasmid antigens (44%) and in gut-derived immunoglobulin A antibody-secreting cells specific for lipopolysaccharide (100%) and invasiveness plasmid antigens (60%). Despite its immunogenicity, the vaccine conferred only 36% protection against illness (fever, diarrhea, or dysentery) induced by experimental challenge (P = 0.17). These findings illustrate the use of an epithelial cell-invasive E. coli strain as a carrier for Shigella antigens. Future studies must explore dosing regimens that might optimize the protective effects of the vaccine while eliminating adverse clinical reactions.
PMCID: PMC257146  PMID: 1587589
13.  Genetic basis of virulence in Shigella species. 
Microbiological Reviews  1991;55(2):206-224.
Shigella species and enteroinvasive strains of Escherichia coli cause disease by invasion of the colonic epithelium, and this invasive phenotype is mediated by genes carried on 180- to 240-kb plasmids. In addition, at least eight loci on the Shigella chromosome are necessary for full expression of virulence. The products of these genes can be classified as (i) virulence determinants that directly affect the ability of shigellae to survive in the intestinal tissues, e.g., the aerobactin siderophore (iucABCD and iutA), superoxide dismutase (sodB), and somatic antigen expression (rfa and rfb); (ii) cytotoxins that contribute to the severity of disease, e.g., the Shiga toxin (stx) and a putative analog of this toxin (flu); and (iii) regulatory loci that affect the expression of plasmid genes, e.g., ompR-envZ, which mediates response to changes in osmolarity, virR (osmZ), which mediates response to changes in temperature, and kcpA, which affects the translation of the plasmid virG (icsA) gene which is associated with intracellular bacterial mobility and intracellular bacterial spread. A single plasmid regulatory gene (virF) controls a virulence-associated plasmid regulon including virG (icsA) and two invasion-related loci, i.e., (i) ipaABCD, encoding invasion plasmid antigens that may be structural components of the Shigella invasion determinant; and (ii) invAKJH (mxi), which is necessary for insertion of invasion plasmid antigens into the outer membrane.
Images
PMCID: PMC372811  PMID: 1886518
14.  Expression in Escherichia coli K-12 of the 76,000-dalton iron-regulated outer membrane protein of Shigella flexneri confers sensitivity to cloacin DF13 in the absence of Shigella O antigen. 
Infection and Immunity  1989;57(9):2794-2798.
One of the chromosomal segments associated with virulence in Shigella flexneri encodes the production of aerobactin and the synthesis of an iron-regulated 76-kilodalton outer membrane protein believed to be the ferric-aerobactin receptor. However, S. flexneri expressing this putative aerobactin receptor, which is slightly larger than that encoded by pColV, is insensitive to the killing action of cloacin DF13, a bacteriocin which binds to other aerobactin receptor proteins and kills the cells. In this paper we show that the conjugal transfer of DNA encoding the iron-regulated 76-kilodalton protein from S. flexneri to Escherichia coli K-12 conferred cloacin DF13 sensitivity on the recipients. However, E. coli K-12 which had also inherited genes specifying Shigella O-antigen biosynthesis remained cloacin insensitive. The data suggest that it is unwise to use cloacin DF13 sensitivity alone to screen transconjugants or clinical isolates for the expression of aerobactin receptor proteins.
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PMCID: PMC313528  PMID: 2474501
15.  Plasmid-associated adherence of Shigella flexneri in a HeLa cell model. 
Infection and Immunity  1989;57(8):2580-2582.
The initial interaction of Shigella flexneri with HeLa cells was studied at 4 degrees C, a temperature that inhibits parasite-directed endocytosis. It was found that invasive strains were 10-fold more adherent to HeLa cells than were isogenic, noninvasive strains which had lost a 140-megadalton plasmid. Adherent strains were also more hydrophobic than were nonadherent strains.
PMCID: PMC313491  PMID: 2663729
16.  Characterization of virulence marker antigen of Shigella spp. and enteroinvasive Escherichia coli. 
Journal of Clinical Microbiology  1989;27(3):561-563.
Antisera produced in rabbits immunized with an enteroinvasive O143 strain of Escherichia coli were absorbed with an avirulent derivative of the same strain. The resulting sera have been previously shown to recognize enteroinvasive pathogens when used in an enzyme-linked immunosorbent assay. In the current study, Western blots (immunoblots) showed that such an absorbed rabbit antiserum recognized two proteins (IpaB and IpaC) which are encoded by a large, virulence-associated plasmid. These proteins are the apparent constituents of the virulence marker antigen which is expressed by shigellae and enteroinvasive E. coli.
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PMCID: PMC267358  PMID: 2654184
17.  Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. 
Infection and Immunity  1989;57(2):477-486.
Escherichia coli K-12 hybrids carrying both the 220-kilobase plasmid and the purE-linked kcpA locus from Shigella flexneri expressed a 140-kilodalton (kDa) protein which was recognized by convalescent sera from monkeys infected with S. flexneri. These hybrids were tested for the ability to produce plaques in HeLa cell monolayers. Hybrid strains which carried both the 220-kilobase plasmid and the kcpA locus had a plaque-forming efficiency of at least 10(-4) PFU/CFU, whereas the plaque-forming efficiency of hybrids that carried only the shigella invasion plasmid ranged from undetectable to 10(-6). Variants were purified from the rare plaques formed by E. coli hybrids that carried only the shigella invasion plasmid. These plaque-purified strains also expressed the 140-kDa protein, and they had a plaque-forming efficiency of at least 10(-4). Transduction of the purE locus from a plaque-purified hybrid into a non-plaque-forming E. coli K-12 strain did not alter the phenotype of the recipient, but conjugation of the shigella invasion plasmid into this transductant reconstituted both expression of the 140-kDa protein and the plaque-forming phenotype. Invasive E. coli K-12 hybrids carrying only the shigella invasion plasmid remained localized within discrete areas of the HeLa cell cytoplasm, whereas hybrids that also carried the S. flexneri kcpA locus grew in a dispersed pattern throughout the host cell cytoplasm. The dispersal of these organisms was inhibited by cytochalasin D, which suggested that host cell microfilaments may play a role in the intracellular spread of enteroinvasive pathogens.
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PMCID: PMC313121  PMID: 2643571
18.  Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella spp. 
Infection and Immunity  1986;53(1):57-63.
The serum antibody response to proteins encoded by the virulence-associated plasmid of Shigella flexneri was determined in monkeys challenged with virulent S. flexneri serotype 2a. With water-extractable antigen in an enzyme-linked immunosorbent assay, a significant increase in antibody titer against proteins from a plasmid-carrying, virulent strain of S. flexneri serotype 5 could be demonstrated in convalescent sera. There were minimal antibody titers against proteins from an avirulent (plasmid-free) organism. Previously identified plasmid-coded polypeptides a, b, c, and d were predominant antigens recognized by a majority of the convalescent sera in immunoblots. An additional 140-megadalton plasmid-coded polypeptide was also recognized by half of the sera. Convalescent serum from an infected monkey recognized antigens on the bacterial surface in several different plasmid-containing Shigella species and in an enteroinvasive Escherichia coli strain. A survey of sera obtained from children 5 to 10 years of age who had been infected with S. flexneri or S. sonnei revealed high enzyme-linked immunosorbent assay titers in both acute and convalescent sera against a water extract from a virulent Shigella strain. In contrast, children under 3 years of age had no antibody titer in either acute or convalescent sera against the virulence-associated shigella proteins, while 3- to 4-year-old children mounted an immune response against these proteins only in convalescence.
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PMCID: PMC260075  PMID: 3721580
19.  Effect of guinea pig or monkey colonic mucus on Shigella aggregation and invasion of HeLa cells by Shigella flexneri 1b and 2a. 
Infection and Immunity  1986;51(3):975-978.
The effects of guinea pig and rhesus monkey colonic mucus preparations on Shigella aggregation and invasion of HeLa cell monolayers by Shigella flexneri serotype 1b, 2a, and 5 strains were investigated. Guinea pig mucus caused agglutination of S. flexneri serotype 1b but not of S. flexneri serotype 2a or 5. Guinea pig mucus also inhibited HeLa cell invasion by S. flexneri serotypes 1b and 2a. Monkey mucus neither agglutinated any Shigella strain nor inhibited HeLa cell invasion.
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PMCID: PMC260999  PMID: 3081449
20.  Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli. 
Infection and Immunity  1985;50(3):620-629.
Seven plasmid-coded polypeptides, designated a through g, were identified by two-dimensional nonequilibrium pH gradient electrophoresis of radiolabeled extracts from minicells of virulent Shigella flexneri serotypes 2a and 5 and enteroinvasive Escherichia coli O143. These polypeptides were deemed to be products of 140-megadalton (MDa) virulence-associated plasmids because they were not synthesized in minicells which were not harboring a 140-MDa plasmid or in minicells which were carrying an F lac plasmid of the same incompatibility group. Synthesis of these polypeptides was repressed in minicells incubated at 30 degrees C and in minicells isolated from a noninvasive opaque colonial variant, even though these strains harbored a 140-MDa plasmid. Enriched fractions of polypeptides b, c, and d were obtained from S. flexneri serotype 5 by preparative isoelectric focusing, and polyclonal rabbit antisera recognizing each polypeptide were raised. These antisera were able to detect cross-reacting plasmid-coded polypeptide antigens in S. flexneri serotype 3, Shigella sonnei, and enteroinvasive E. coli O143. In addition, Western blots of minicell extracts from S. flexneri serotype 5 or E. coli O143 indicated that plasmid-coded polypeptides a through d were recognized by convalescent antiserum from a monkey infected with S. flexneri serotype 2a.
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PMCID: PMC261123  PMID: 3905608
21.  Cloning of plasmid DNA sequences involved in invasion of HeLa cells by Shigella flexneri. 
Infection and Immunity  1985;49(1):164-171.
A large plasmid is found in virulent isolates of Shigella sp. and encodes functions essential for invasion of mammalian cells. To identify plasmid sequences necessary for invasion, we isolated a series of Tn5 insertions in pWR100, the virulence plasmid of Shigella flexneri serotype 5. These insertions demonstrated that three separate EcoRI fragments of pWR100 were required for invasion of HeLa cells. However, the corresponding native EcoRI fragments, when cloned into pBR325, did not restore virulence to plasmidless strains. Construction of a lambda-sensitive, plasmidless Shigella recipient enabled us to shotgun clone plasmid DNA directly into S. flexneri by using the cosmid vector pJB8 and score for expression of invasive functions. In this fashion, we succeeded in isolating six independent recombinants which restored invasion of HeLa cells in plasmidless Shigella recipients. The cloned inserts all contained a common core of ca. 37 kilobases, thus defining a minimum sequence necessary for invasion of HeLa cells. Virulence-associated peptides produced by wild-type S. flexneri were also produced by the recombinants. Expression of these peptides and expression of invasiveness by the clones were regulated by growth temperature, as is expression of these traits in wild-type S. flexneri. A complete invasive phenotype was not expressed by the recombinants in that they failed to produce a positive Sereny test. Possible explanations for this behavior as it relates to the mechanism of bacterial invasion are discussed.
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PMCID: PMC262074  PMID: 2989179
22.  Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli. 
Infection and Immunity  1985;49(1):67-71.
One of the chromosomal segments associated with the virulence of Shigella flexneri and transferred to Escherichia coli K-12 by conjugation has been shown to code for the production of aerobactin and for the synthesis of an iron-regulated 76,000-dalton (76K) outer membrane protein. Analysis of various E. coli K-12-S. flexneri transconjugants showed that the genes involved with the synthesis of aerobactin and with the production of the 76K protein were linked to the mtl region of the S. flexneri chromosome. S. flexneri itself synthesized a 76K protein in its outer membrane under iron restriction as well as traces of 81K and 74K proteins. An examination of four enteroinvasive strains of E. coli showed that each produced aerobactin and a 76K outer membrane protein during iron-restricted growth. The profile of the iron-regulated proteins expressed by the enteroinvasive strains of E. coli was virtually identical to that expressed by the laboratory-constructed E. coli K-12-S. flexneri hybrids under the same growth conditions.
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PMCID: PMC262059  PMID: 3159680
23.  Expression of lipopolysaccharide O antigen in Escherichia coli K-12 hybrids containing plasmid and chromosomal genes from Shigella dysenteriae 1. 
Infection and Immunity  1984;46(2):470-475.
The requirement for both plasmid and chromosomal genes in the biosynthesis of Shigella dysenteriae 1 lipopolysaccharide O antigen was demonstrated in Escherichia coli-Shigella hybrids. A 6-megadalton S. dysenteriae 1 plasmid, designated pWR23, was phenotypically tagged with the Tn3 ampicillin-resistance transposon. The tagged plasmid, designated pWR24, was transferred by transformation or conjugal mobilization to a rough E. coli K-12 recipient. Although the resultant hybrids were agglutinated in S. dysenteriae 1 antiserum, they did not remove all of the anti-Shiga agglutinins in absorption experiments. Modified lipid A core structure was detected in these hybrids, but Shiga O antigen was not expressed. When the his+ locus of the S. dysenteriae 1 chromosome was transferred by transduction to E. coli K-12 containing pWR24, complete Shiga O antigen was expressed. Lipopolysaccharide extracted from these hybrids was indistinguishable chemically, electrophoretically, and serologically from native S. dysenteriae 1 lipopolysaccharide.
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PMCID: PMC261557  PMID: 6389345
24.  Oral vaccination of monkeys with an invasive Escherichia coli K-12 hybrid expressing Shigella flexneri 2a somatic antigen. 
Infection and Immunity  1984;46(2):465-469.
A living oral vaccine, designed to protect against Shigella flexneri 2a infections, was constructed by using Escherichia coli K-12 as a carrier strain. The hybrid strain, designated EC104, contained both chromosomal and plasmid genes from S. flexneri donor strains. In addition to expressing the S. flexneri 2a somatic antigen, it had inherited the property of epithelial-cell invasion. After the oral administration to rhesus monkeys, EC104 was isolated from the feces for up to 3 days, but by day 4 all stool cultures were negative. The serum antibody response against S. flexneri 2a somatic antigen was variable, but the vaccine conferred significant protection against an oral challenge with virulent S. flexneri 2a.
PMCID: PMC261556  PMID: 6389344
25.  Heterogeneity of molecular size and antigenic expression within lipooligosaccharides of individual strains of Neisseria gonorrhoeae and Neisseria meningitidis. 
Infection and Immunity  1984;45(3):544-549.
We determined the Mr of neisserial lipooligosaccharides (LOS) by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis, minimal loading concentrations, and Salmonella isogenic rough mutant LOS as Mr standards. Salmonella LOS were resolved into three components. The migration distance of each component was linearly related to its theoretical Mr (r = 0.99). Neisserial LOS also contained multiple components whose calculated Mr ranged from 3,200 to 7,100. The relative abundance of components and their MrS varied greatly among strains. Meningococcal LOS were composed almost exclusively of two closely migrating components; gonococcal LOS were more heterogeneous. LOS from a gonococcus selected for resistance to a Pseudomonas pyocin contained only a single component that was different from and of intermediate Mr among the three components of the parent strain. A monoclonal antibody directed against the meningococcal L8 LOS epitope was used to determine whether heterogeneity of antigen expression reflected Mr heterogeneity. Single components of the L8 meningococcal LOS and of the LOS of 3 of 19 gonococcal strains bound the monoclonal antibody. Gonococcal LOS components that expressed the L8 epitope were of a similar Mr (4,800). Smaller components of these same LOS did not express the epitope.
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PMCID: PMC263327  PMID: 6432693

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