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1.  Severely Fibrotic Pancreases from Young Patients with Chronic Pancreatitis: Evidence for a Ductal Origin of Islet Neogenesis 
Acta diabetologica  2011;50(5):807-814.
While it is known that islet cell mass increases considerably after birth, general uncertainty surrounds the source of new beta cells in humans. Chronic pancreatitis (CP) presents a natural injury model for studying postnatal beta-cell regeneration in the human pancreas. In this report, we present histological evidence from human CP pancreases to support the theory that islet neogenesis can occur from ductal precursor cells after birth. Three young patients (ages 16, 12, and 28 years) underwent total pancreatectomy for the management of CP followed by islet isolation and autologous transplantation to prevent or minimize post-surgical diabetes. In all cases, the pancreases had extensive fibrosis, a rock-like consistency, and calcifications in the ducts. During islet isolations, we observed the unusual release of islets with many ductal fragments. In histopathological evaluation of these pancreases, solid cords of cells sometimes formed islet like structures intraductally or extending from ductal structures. Immunofluorescence staining for chromogranin, insulin, proinsulin, PDX1, glucagon and cytokeratins confirmed these structures to be composed of chromogranin-positive endocrine cells which included both β-cells and α-cells. Labeling for Ki67 to demonstrate mitotic activity showed frequent labeling of duct epithelial cells and of some periductal cells. Using insulin and wide-spectrum cytokeratin double-immunofluorescent labeling, we found insulin positive cells to be present within the ductal lumens, among the cytokeratin positive ductal epithelium, and extending from the ductal epithelium into surrounding connective tissues, providing evidence for a ductal origin of islet neogenesis.
PMCID: PMC4124082  PMID: 21773756
islet neogenesis; chronic pancreatitis; islet precursor cell; beta cell; islet progenitor cell
2.  Oral Intoxication of Mice with Shiga Toxin Type 2a (Stx2a) and Protection by Anti-Stx2a Monoclonal Antibody 11E10 
Infection and Immunity  2014;82(3):1213-1221.
Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD50) of 2.9 μg, but no morbidity occurred after oral intoxication with up to 157 μg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.
PMCID: PMC3957995  PMID: 24379294
3.  Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia 
Journal of Medical Microbiology  2012;61(Pt 10):1380-1392.
Current vaccine approaches to combat anthrax are effective; however, they target only a single protein [the protective antigen (PA) toxin component] that is produced after spore germination. PA production is subsequently increased during later vegetative cell proliferation. Accordingly, several aspects of the vaccine strategy could be improved. The inclusion of spore-specific antigens with PA could potentially induce protection to initial stages of the disease. Moreover, adding other epitopes to the current vaccine strategy will decrease the likelihood of encountering a strain of Bacillus anthracis (emerging or engineered) that is refractory to the vaccine. Adding recombinant spore-surface antigens (e.g. BclA, ExsFA/BxpB and p5303) to PA has been shown to augment protection afforded by the latter using a challenge model employing immunosuppressed mice challenged with spores derived from the attenuated Sterne strain of B. anthracis. This report demonstrated similar augmentation utilizing guinea pigs or mice challenged with spores of the fully virulent Ames strain or a non-toxigenic but encapsulated ΔAmes strain of B. anthracis, respectively. Additionally, it was shown that immune interference did not occur if optimal amounts of antigen were administered. By administering the toxin and spore-based immunogens simultaneously, a significant adjuvant effect was also observed in some cases. Thus, these data further support the inclusion of recombinant spore antigens in next-generation anthrax vaccine strategies.
PMCID: PMC3541767  PMID: 22767539
4.  Surgical Protocol Involving the Infusion of Paramagnetic Microparticles for Preferential Incorporation within Porcine Islets 
Transplantation proceedings  2010;42(10):4209-4212.
Despite significant advances, widespread applicability of islet cell transplantation (ICT) remains elusive. Refinement of current islet isolation protocols may improve ICT outcomes. Islet purification by magnetic separation (MS) has shown early promise. However, surgical protocols must be optimized to maximize the incorporation of paramagnetic microparticles (MP) within a greater number of islets. The objective of this study is to explore the impact of MP concentration and infusion method on optimizing MP incorporation within islets.
Five porcine pancreata were procured from donors following cardiac death. Splenic lobes were isolated and infused with varying concentrations of MP (8, 16 and 32 × 108 MP/L of cold preservation solution) and using one of two delivery techniques (hanging bag versus hand-syringe). Following procurement and infusion, pancreata were stored at 0–4°C during transportation (< 1 hour), fixed in 10% buffered formalin and examined by standard MRI and histopathology.
T2*-weighted MRI illustrated homogeneous distribution of MP in all experimental splenic lobes. In addition, histologic analysis confirmed that MP were primarily located within the microvasculature of islets (82–85%), with few MP present in acinar tissue (15–18%), with an average of 5–7 MP per islet (within a 5 μm thick section). The highest MP incorporation was achieved at a concentration of 16 × 108 MP/L using the hand-syringe technique.
This preliminary study suggests that optimization of a surgical protocol, MP concentrations and applied infusion pressures may enable more uniform distribution of MP in the porcine pancreas and better control of MP incorporation within islets. These results may have implications in maximizing the efficacy of islet purification by MS.
PMCID: PMC3035915  PMID: 21168666
5.  Speriolin is a novel human and mouse sperm centrosome protein 
Human Reproduction (Oxford, England)  2010;25(8):1884-1894.
Oocytes in humans, mice and other mammals lack identifiable centrioles. The proximal centriole brought in by the fertilizing sperm in humans and most other mammals appears to gives rise to the centrioles at the spindle poles in the zygote, and is believed to indicate that centrioles are inherited through the paternal lineage. However, both the proximal and distal sperm centrioles degenerate in mice and other rodents. A bipolar mitotic spindle nucleates from multiple centrosome-like structures in the mouse zygote and centrioles are not seen until the blastocyst stage, suggesting that centrioles are inherited through the maternal lineage in mice. We previously identified speriolin as a spermatogenic cell-specific binding partner of Cdc20 that co-localizes with pericentrin in mouse spermatocytes and is present in the centrosome in round spermatids.
The nature and localization of speriolin in mouse and human sperm and the fate of speriolin following fertilization in the mouse were determined using immunofluorescence microscopy, immunoelectron microscopy and western blotting.
Speriolin surrounds the intact proximal centriole in human sperm, but is localized at the periphery of the disordered distal centriole in mouse sperm. Human speriolin contains an internal 163-amino acid region not present in mouse that may contribute to localization differences. Speriolin is carried into the mouse oocyte during fertilization and remains associated with the decondensing sperm head in zygotes. The speriolin spot appears to undergo duplication or splitting during the first interphase and is detectable in 2-cell embryos.
Speriolin is a novel centrosomal protein present in the connecting piece region of mouse and human sperm that is transmitted to the mouse zygote and can be detected throughout the first mitotic division.
PMCID: PMC2907228  PMID: 20542897
centriole; flagellum; fertilization; paternal inheritance; zygote
6.  Rapid Quantitative Assessment of the Pig Pancreas Biopsy Predicts Islet Yield 
Transplantation proceedings  2010;42(6):2036-2039.
The cost of islet procurement from donor pigs is increased by the use of organs that produce low yields. We developed an assessment system using dithizone-stained pig pancreas biopsies to enable the preselection of donor organs.
Pig pancreas biopsy slices were soaked in dithizone solution. The islets were evaluated before islet isolation by converting the islet counts (IC) to islet equivalents (IE), then determining the IE/cm2, IE/IC, % >150 μm islets, and % >200 μm islets. These parameters were evaluated in 3 different areas of pancreas (duodenal, splenic, and connecting lobe; n = 42 each). Stepwise multivariate linear regression analysis was performed to assess for correlations with islet yield and decide which area of the pancreas had the most predictive value. To identify other predictors, including donor and islet isolation variables, we performed binary logistic regression analysis with significant variables from the univariate analysis (n = 67). For this analysis, the pigs were categorized into high (n = 23) and low (n = 44) yield groups.
Stepwise multivariate linear regression analysis revealed that IE/cm2 of the splenic lobe significantly predicted islet yield. Binary logistic regression analysis indicated that the IE/mm2 of the splenic lobe was the only parameter that significantly correlated with successful pig islet isolations (P = .01; odds ratio 3.605). Variables associated with donor and islet isolation, such as age, gender, ischemic time, or enzyme lot, were not significantly correlated with islet yield.
Our study suggests that islet distribution of splenic lobe biopsies can be a reliable predictor of islet yield from pig pancreata.
PMCID: PMC2922853  PMID: 20692401
7.  Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method 
Transplantation proceedings  2010;42(6):2016-2019.
Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the high dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in lower islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from non–heart-beating donors and preserved by either TLM or persufflation for 24 hours and fixed. Biopsies were collected from several regions of the pancreas, sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries due to gas perfusion and significantly less autolysis/cell death than regions not exposed to persufflation or tissues exposed to TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.
PMCID: PMC2956134  PMID: 20692396
9.  Schwann cell invasion of the conus medullaris: case report 
European Spine Journal  2002;12(3):328-331.
As Schwann cells possess regenerative capabilities there is intense interest concerning their role in central nervous system (CNS) regeneration. We report on a case of an intramedullary schwannoma involving the conus medullaris and spinal cord above it. We discuss the possible origin of these cells and the mechanisms by which these cells may invade the CNS. We offer imaging and discuss experimental studies to support our hypothesis. This case concerns a 48-year-old man, who presented with a 6-month history of bilateral lower extremity weakness. Magnetic resonance imaging (MRI) revealed an intramedullary tumour extending from the conus to T11. At operation, following laminectomy and durotomy, a schwannoma was dissected free from the conus. Total gross resection of tumour was achieved. The patient made an uneventful and full recovery. This case shows that Schwann cells can invade the CNS. Manipulation of the transitional zone astrocytic barrier may offer a potential avenue for Schwann cells to enter the CNS in pathological states.
PMCID: PMC3615487  PMID: 12800008
CNS regeneration Intramedullary schwannoma Transitional zone
10.  Cytotoxic Necrotizing Factor Type 1-Positive Escherichia coli Causes Increased Inflammation and Tissue Damage to the Prostate in a Rat Prostatitis Model 
Infection and Immunity  2001;69(10):6515-6519.
Infection of rat prostates with cytotoxic necrotizing factor type 1 (CNF1)-positive uropathogenic Escherichia coli caused more inflammation-mediated morphological and histological tissue damage than did infection with isogenic CNF1-negative mutants. These striking differences occurred despite the finding that bacterial counts for the strain pairs were indistinguishable. We conclude that CNF1 contributes to E. coli virulence in a model of acute prostatitis. To our knowledge, the results of this study provide the first demonstration of a role for any uropathogenic E. coli virulence factor in acute prostatitis.
PMCID: PMC98788  PMID: 11553597
11.  Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli 
Infection and Immunity  2001;69(4):2066-2074.
Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited cross-reactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.
PMCID: PMC98131  PMID: 11254559
12.  Bacterial toxins: friends or foes? 
Emerging Infectious Diseases  1999;5(2):224-234.
Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus alpha-toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we discuss the information available on its synthesis and structure, mode of action, and contribution to virulence. We also review the role certain toxins have played in unraveling signal pathways in eukaryotic cells and summarize the beneficial uses of toxins and toxoids. Our intent is to illustrate the importance of the analysis of bacterial toxins to both basic and applied sciences.
PMCID: PMC2640701  PMID: 10221874
13.  Coordinate transcription and V(D)J recombination of the kappa immunoglobulin light-chain locus: NF-kappaB-dependent and -independent pathways of activation. 
Molecular and Cellular Biology  1997;17(7):3477-3487.
To further elucidate the potential role of mitogens and cytokines in regulation of the kappa immunoglobulin light-chain locus, we have characterized the activation of transcription factor binding, kappa germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination upon induction of model pre-B-cell lines. We find that both lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) are capable of activating germ line transcription, DNase I hypersensitivity, and recombination of the kappa locus. We also find that transforming growth factor beta is capable of completely inhibiting LPS activation of transcription and recombination but has no apparent effect on activation of transcription factor binding, including activation of NF-kappaB. To address the functional role of NF-kappaB in LPS and IFN-gamma induction of these events, we blocked the nuclear translocation of NF-kappaB by overexpression of a dominant negative mutant of IkappaB-alpha (IkappaB deltaN). Overexpression of the IkappaB deltaN protein results in an inhibition of LPS but not IFN-gamma activation of germ line transcription, DNase I hypersensitivity, and Vkappa-to-Jkappa recombination. Our results demonstrate that activation of NF-kappaB is necessary but not sufficient for LPS activation of transcription and recombination at kappa. These results also suggest that NF-kappaB is not required for IFN-gamma activation of transcription or recombination. These results are important in establishing that there are multiple independent pathways of activation of both transcription and recombination.
PMCID: PMC232201  PMID: 9199283
14.  Simple method for comparing reliability of two serum tumour markers in breast carcinoma. 
Journal of Clinical Pathology  1994;47(2):134-137.
AIMS--To compare the two breast tumour markers, CA15-3 and mucinous-like carcinoma associated antigen (MCA), using Receiver Operating Characteristic (ROC) curve analysis. METHODS--One hundred and ninety six patients "presenting" with breast carcinoma had serum CA15-3 and MCA concentrations measured. RESULTS--Using these markers as indicators of stage IV disease at the recommended laboratory level, true positive rates (TPR) and false positive rates (FPR) were obtained as follows: CA15-3 TPR = 75%, FPR = 7.4%, MCA TPR = 80%, FPR = 59.1%. By increasing the CA15-3 cutoff level to 45 U/ml, a TPR and FPR of 75% and 0.6%, respectively were obtained. By increasing the MCA cutoff level to 23 U/ml, a TPR and FPR of 65% and 2.3%, respectively, were obtained. CONCLUSIONS--Using ROC curve analysis shows that CA15-3 is a superior indicator of metastatic breast disease than MCA at recommended laboratory levels, and by altering the cutoff points, the specificity and sensitivity for both these markers can be improved.
PMCID: PMC501827  PMID: 8132827
15.  Octamer independent activation of transcription from the kappa immunoglobulin germline promoter. 
Nucleic Acids Research  1996;24(23):4805-4811.
Previous analyses of immunoglobulin V region promoters has led to the discovery of a common octamer motif which is functionally important in the tissue-specific and developmentally regulated transcriptional activation of immunoglobulin genes. The germline promoters (Ko) located upstream of the J region gene segments of the kappa locus also contain an octamer motif (containing a single base pair mutation and referred to as the variant octamer) which has been shown previously to bind Oct-1 and Oct-2 transcription factors in vitro. To further elucidate the role of this variant octamer motif in the regulation of germline transcription from the unrearranged kappa locus, we have quantitated the relative binding affinity of Oct-1 and Oct-2 for the variant octamer motif and determined the functional role of this octamer motif in transcriptional activation. We find that, although the variant octamer motif binds Oct-1 and Oct-2 in vitro with 5-fold lower affinity than the consensus octamer motif, mutation of the variant octamer motif to either a consensus octamer or non-octamer motif has no effect on transcriptional activation from the germline promoter. We also find significant differences in activation of germline and V region promoters by kappa enhancers. Our results suggest that the germline promoters and V region promoters differ in their dependence on octamer for activation and respond differently to enhancer activation. These findings have important implications in regulation of germline transcription as well as concomitant activation of the V-J recombination of the kappa light chain locus.
PMCID: PMC146306  PMID: 8972869
16.  Epstein-Barr virus in normal, pre-malignant, and malignant lesions of the uterine cervix. 
Journal of Clinical Pathology  1993;46(10):931-935.
AIM--To detect the presence or absence of Epstein-Barr virus (EBV) in cervical lesions ranging from normality to invasive malignancy. METHODS--Eighteen randomly selected cases of invasive squamous cell carcinomas of the uterine cervix were examined as well as 25 cases each of normal cervices and those showing cervical intra-epithelial neoplasia (CIN) I, II, and III. DNA-DNA in situ hybridisation, using a biotinylated probe to the Bam H1 "W" fragment of EBV, was carried out in addition to the polymerase chain reaction using specific primer sequences that flank a 153 base pair segment of the Bam H1 "W" region of the EBV genome and which do not cross-amplify other DNA herpes viruses. Positive control material included paraffin wax embedded P3 HR1 lymphoblastoid cells (containing high copy numbers of EBV) and two nasopharyngeal carcinomas positive for EBV. RESULTS--Neither normal nor CIN I tissue was positive. Eight per cent of CIN II tissue was positive; 8% of CIN III, and 43% of carcinomas were positive for EBV. CONCLUSION--The study shows that the virus is present in some cases of cervical carcinoma and to a lesser degree in some premalignant lesions of the cervix, but the exact association between it and cervical oncogenesis, be it causative or incidental, remains to be determined.
PMCID: PMC501621  PMID: 8227411
17.  Truncated enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimin (EaeA) fusion proteins promote adherence of EHEC strains to HEp-2 cells. 
Infection and Immunity  1996;64(6):2225-2233.
Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino terminus. The second fusion, RVHdHisEae, consisted of the carboxyl two-thirds of the protein. Purified extracts of either construct enhanced binding of wild-type 86-24 to HEp-2 cells and conferred HEp-2 cell adherence on 86-24eaeDelta10, an eaeA deletion mutant, and B2F1, an EHEC O91:1-121 eaeA mutant strain. When 86-24eaeDelta10 was transformed with either of the plasmids encoding the intimin fusion proteins, the transformant behaved like the wild-type parent strain and displayed localized adherence to HEp-2 cells, with positive fluorescent-actin staining. In addition, polyclonal antisera raised against RIHisEae reacted with both fusion constructs and recognized an outer membrane protein of the same mass as intimin (97 kDa) in EHEC and enteropathogenic E. coli but not E. coli K-12. The intimin-specific antisera also blocked adherence of EHEC to HEp-2 cells. Thus, intimin (i) is a 97-kDa outer membrane protein in EHEC that serves as a requisite adhesin for attachment of the bacteria to epithelial cells, even when the protein is truncated by one-third at its amino terminus and (ii) can be added exogenously to specifically facilitate HEp-2 cell adherence of EHEC but not E. coli K-12.
PMCID: PMC174060  PMID: 8675331
18.  The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin. 
Journal of Bacteriology  1996;178(10):2911-2915.
The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.
PMCID: PMC178028  PMID: 8631681
19.  Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice. 
Infection and Immunity  1996;64(5):1569-1576.
The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from mouse small intestine and colon and that growth in small-intestine mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incubation with human colonic mucus. In contrast, culture supernatants from LB-grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc or SLT-IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxicity activity. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice.
PMCID: PMC173963  PMID: 8613362
20.  Vaccination with genetically modified Shiga-like toxin IIe prevents edema disease in swine. 
Infection and Immunity  1996;64(1):55-60.
Escherichia coli strains producing Shiga-like toxin II variant (SLT-IIe, formerly called SLT-IIv) cause edema disease in weaned pigs. Vaccination of pigs with a genetically modified form of Shiga-like toxin IIe, SLT-IIe(E167Q), has been previously shown to be nontoxic and to induce antibodies to SLT-IIe (V.M. Gordon. S.C. Whipp, H.W. Moon, A.D. O'Brien, and J.E. Samuel, Infect, Immun. 60:485-502, 1992). Fifty micrograms of SLT-IIe(E167Q) toxin was used to vaccinate suckling pigs at 1 and 2 weeks of age. Both vaccinated and nonvaccinated pigs were orally inoculated with an SLT-IIe-producing strain of E. coli after weaning (3 to 4 weeks of age). Pigs fed a low-protein diet that were not vaccinated with SLT-IIe(E167Q) developed subclinical edema disease, histologically evident as vascular necrosis. Pigs fed a high-protein diet that were not vaccinated with SLT-IIe(E167Q) developed clinical edema disease manifested as vascular necrosis, reduced weight gain, ataxia, palpebral edema, lateral recumbency, and death. Pigs vaccinated with SLT-IIe(E167Q) had a reduction in the incidence of subclinical edema disease and never developed clinical edema disease. These data demonstrate that vaccination with a genetically modified form of SLT-IIe prevents edema disease and are consistent with the notion that diet influences susceptibility to edema disease.
PMCID: PMC173727  PMID: 8557374
21.  Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells. 
Infection and Immunity  1995;63(9):3739-3744.
In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O'Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418-1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein.
PMCID: PMC173523  PMID: 7642319
22.  Investigation of enterohemorrhagic Escherichia coli O157:H7 adherence characteristics and invasion potential reveals a new attachment pattern shared by intestinal E. coli. 
Infection and Immunity  1995;63(5):2070-2074.
In this study, the interactions of enterohemorrhagic Escherichia coli (EHEC) O157 strains with human ileocecal (HCT-8) epithelial cells and HEp-2 cells were examined. EHEC adhered to, but did not invade, HCT-8 cells by the localized adherence mechanism and a heretofore unrecognized pattern which we called log jam. The log jam formation was (i) not observed on HEp-2 cells, (ii) independent of the EHEC eaeA gene required for localized adherence, and (iii) shared by pathogenic and nonpathogenic E. coli strains but not K-12 strains. The log jam phenotype may represent a basal means by which E. coli bacteria attach to the human intestine.
PMCID: PMC173266  PMID: 7537254
23.  Detection of Aeromonas salmonicida, causal agent of furunculosis in salmonid fish, from the tank effluent of hatchery-reared Atlantic salmon smolts. 
Applied and Environmental Microbiology  1994;60(10):3874-3877.
The fish pathogen, Aeromonas salmonicida, could be detected only by bacteriological culture from the kidney of dead or moribund fish in one tank in a hatchery rearing Atlantic salmon (Salmo salar L.) smolts. However, by using a DNA probe specific for this species, allied to a PCR assay, the pathogen could be detected in water, feces and effluent samples taken from this fish tank. Also, the presence of the pathogen was found in effluent samples from two fish tanks containing apparently healthy fish. Subsequently, the presence of pathogen in these tanks was confirmed by an increase in the daily mortality rate and by a plate culture from moribund fish.
PMCID: PMC201900  PMID: 7527205
24.  The extent of vitamin K deficiency in patients with cholestatic jaundice: a preliminary communication. 
Eleven patients with cholestatic jaundice had measurements of plasma vitamin K1 performed. Seven of these 11 (64%) had subnormal levels. The prothrombin time (PT) was prolonged in three of 15 patients with cholestasis (20%), the patient with the longest PT had the lowest vitamin K1 level. A single intramuscular (im) dose of 10 mg vitamin K1 lowered the PT in 9/15 patients (includes correcting the three prolonged PTs). The initial mean plasma vitamin K1 level rose 24 h later, to a mean plasma level which was 33 times the upper limit of the normal physiological range. These preliminary results suggest that a majority of patients presenting with cholestatic jaundice have low tissue reserves of vitamin K1, and that guidelines for vitamin K1 therapy in patients with cholestatic jaundice should be revised.
PMCID: PMC1294558  PMID: 8046700
25.  10 year review of invasive aspergillosis detected at necropsy. 
Journal of Clinical Pathology  1991;44(6):452-454.
Between 1980 and 1989, 32 cases of invasive aspergillosis were identified out of 2315 consecutive necropsies, an incidence of 1.4%. The incidence in immunosuppressed "high risk" patients was 10.7%. Twenty out of 32 cases showed spread beyond the lungs, with the brain the most common site. There was an increase in cases in the second half of the decade, attributable to the start of a liver transplantation programme. Liver transplant recipients and patients with haematological malignancies were at significantly greater risk of acquiring aspergillosis than kidney transplant recipients or those with solid malignancies treated with chemotherapy. There was also a greater risk of haematogenous dissemination in liver transplant recipients than in all other groups, and this was significantly associated with the use of high dose steroids as anti-rejection treatment. Aspergillus was isolated during life in only eight cases, which indicates a continuing need for and emphasises the value of necropsy.
PMCID: PMC496822  PMID: 2066421

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