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Biomedical subjects

B E Anderson

Publications and source records attributed to B E Anderson.

At least 19 recordsLinked to original sources
PubMed2001

Molecular cloning, sequence and characterization of cjsT, a putative protease from Rickettsia rickettsii.

The cloning and sequencing of a gene from Rickettsia rickettsii which confers haemolytic activity on Escherichia coli strain TB1 is described. The open reading frame of the haemolysis-promoting gene, cjsT, is 1041 bp and encodes a putative protein with a molecular mass of 33 825 Da. CjsT has high sequence similarity to several bacterial proteases, particularly type IV signal peptidases. Cell lysates from an E. coli clone containing cjsT in pUC19 (pJON1) exhibited greater protease activity in functional assays than found in E. coli containing pUC19 alone. Disruption of the cjsT gene by insertional inactivation with a kanamycin cassette reduced both the protease and haemolytic activities conferred by cjsT. The protease inhibitors antipain and diisopropylfluorophosphate (DFP) both reduced the proteolytic activity of pJON1. The mechanism by which the R. rickettsii cjsT promotes haemolysis in E. coli remains unclear.

Amino Acid Sequence↗
PubMed2001

Host pathogenesis in urinary tract infections.

Urinary tract infections (UTIs) are the result of an interaction between bacterial virulence and host defense factors that compete to invade or protect the host, respectively. Research over the past 30 years has demonstrated that vaginal colonization with uropathogens precedes most UTIs. Receptivity of the vaginal mucosa for uropathogens is an essential initial step in vaginal mucosa colonization. When vaginal and buccal epithelial cells were collected from patients susceptible to reinfection and compared with such cells obtained from controls resistant to UTIs, the strains that caused cystitis adhered much more avidly to the epithelial cells from susceptible women. These genotypic traits for epithelial cell receptivity may be a major susceptibility factor in UTIs. The presence or absence of blood group determinants on the surface of uroepithelial cells may influence an individual's susceptibility to UTIs. The protective effect in women with the secretor phenotype may be due to fucosylated structures at the cell surface which decrease the availability of putative receptors for Escherichia coli. Susceptibility among women who do not secrete blood group antigens may be due to specific E. coli-binding glycolipids that are absent in women who secrete blood group antigens. Recent studies have shown that the vaginal fluid, which forms an interface between uropathogens and epithelial cells, also influences vaginal colonizations.

Bacterial Adhesion↗
PubMed2000

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival.

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.

Animals↗
PubMed2000

Isolation, sequencing and expression of Bartonella henselae omp43 and predicted membrane topology of the deduced protein.

The infection of and interaction of human endothelial cells with Bartonella henselae is one of the most interesting aspects of Bartonella -associated disease. The gene encoding the 43 kDa B. henselae outer membrane protein (Omp43) that binds endothelial cells was cloned and sequenced. Sequence analysis revealed an open reading frame of 1206 nucleotides coding for a protein of 402 amino acids. Analysis of the deduced amino acid sequence shows 38% identity over the entire sequence to the Brucella spp. In addition to this Omp2b porin also shows a signal sequence and peptidase cleavage site. Cleavage of the signal peptide results in a mature 380 amino acid polypeptide with a predicted molecular weight of 42 kDa. Omp43 was expressed in Escherichia coli as a fusion protein. Purified recombinant Omp43 at concentrations of 11 and 2.75 microg/ml bound to intact human umbilical vein endothelial cells. Membrane topology analysis predicts that Omp43 exists as a 16 stranded beta barrel protein, similar to that predicted for the Omp2b Brucella abortus porin. Characterization and expression of the gene encoding Omp43 should provide a tool for further investigation of the role of adherence to endothelial cells in the pathogenesis of B. henselae.

Amino Acid Sequence↗
PubMed2000

Distribution of, and immune response to, chicken anti-alpha Gal immunoglobulin Y antibodies in wild-type and alpha Gal knockout mice.

Chicken antibodies (immunoglobulin Y; IgY) to the alpha Gal epitope (galactose alpha-1,3-galactose) bind to alpha Gal antigens of mouse and porcine tissues and endothelial cells in vitro and block human anti-alpha Gal antibody binding, complement activation and antibody-dependent cell-mediated lysis mechanisms. The activities and toxicity of anti-alpha Gal IgY have not been tested in vivo. In this study, we tested the effects of multiple injections of affinity-purified anti-alpha Gal IgY (AP-IgY) in both wild-type (WT) and alpha-1,3-galactosyltransferase knockout (Gal KO) mice. WT and Gal KO mice were injected once, twice, three, or four times intravenously (i.v.) with AP-IgY and killed at 1 hr or 24 hr. Mice displayed no toxicity to four injections of AP-IgY. Heart, lung, liver, kidney, spleen and pancreatic tissue were evaluated using immunohistochemical techniques for the presence of the alpha Gal epitope using the GSI-B4 lectin, and for bound IgY, as well as mouse IgM and IgG. The binding of AP-IgY antibodies to the endothelium of WT mouse tissues was essentially identical to the pattern of binding of the GSI-B4 lectin after injection of WT mice and death at 1 hr. WT mice killed 24 hr after i.v. injection of AP-IgY showed little remaining bound IgY in their endothelia, indicating that IgY is cleared over that time period. We also evaluated the blood drawn at the time of death for the presence of anti-alpha Gal IgY, anti-IgY IgM and anti-IgY IgG by enzyme-linked immunosorbent assay. Anti-alpha Gal IgY was almost undetectable in WT mouse sera at all injection and killing times. In contrast, Gal KO mouse sera showed increasing anti-alpha Gal IgY levels until 24 hr after the fourth injection, when anti-alpha Gal IgY levels were almost undetectable. Anti-IgY IgM and IgG levels in WT and Gal KO mouse sera showed a typical increase in anti-IgY IgM 24 hr after the second injection (3 days after the first injection) and an increase in anti-IgY IgG 24 hr after the third injection (5 days after the first injection). These results show that IgY binds to alpha Gal epitopes in the WT mice and is cleared sometime over a 24-hr time period and that IgY is an expected immunogen in mice eliciting a rather typical anti-IgY IgM and IgG response.

Animals↗
PubMed2000

Transcriptional activation of the htrA (High-temperature requirement A) gene from Bartonella henselae.

Bacterial htrA genes are typically activated as part of the periplasmic stress response and are dependent on the extracytoplasmic sigma factor rpoE. A putative promoter region, P1, of the sigma(E)-type heat-inducible promoters has previously been identified upstream of the htrA gene of Bartonella henselae. Further analysis of the htrA mRNA by primer extension demonstrated that transcription initiates from P1 and a second region downstream of P1. This second promoter region, termed P2, had no sequence identity to sigma(E)-type heat-inducible promoters. Promoter regions were cloned individually and in tandem into pANT3 upstream of a promoterless version of the green fluorescent protein (GFP) gene (gfpmut3) and transformed into B. henselae by electroporation. The contiguous promoter region containing both P1 and P2 were necessary for the optimal transcriptional activation of the htrA gene. Promoter activity at 37 degrees C was distinctively higher than at 27 degrees C. However, thermal induction at 47 degrees C did not increase expression of gfpmut3. Invasion of human microvascular endothelial cells (HMEC-1) by B. henselae resulted in the formation of well-defined vacuoles containing clusters of bacteria exhibiting marked expression of gfpmut3 transcribed from the P1-P2 region. In addition, a moderate yet significant increase in the ratio of bacterial GFP to DNA was detected for intracellular bacteria compared to extracellular bacteria, indicating upregulation of htrA upon invasion of HMEC-1. The activation of specific genes in the intracellular environment may help us better understand the novel pathogenic mechanisms used by this bacterium.

Angiomatosis, Bacillary↗
PubMed2000

Nutritional therapy for peripheral arterial disease: a double-blind, placebo-controlled, randomized trial of HeartBar.

We investigated the clinical effects of a food bar enriched with L-arginine and a combination of other nutrients known to enhance the activity of endothelium-derived nitric oxide (EDNO) in individuals with claudication from atherosclerotic peripheral arterial disease. The study was a 2-week, double-blind, placebo-controlled trial of subjects randomized to three groups (two active bars, one active and one placebo bar, and two placebo bars per day) followed by an 8-week open-label period. Subjects (n=41) were outpatient volunteers with intermittent claudication. Pain-free and total walking distances were measured by variable-grade, treadmill exercise testing. Quality of life was assessed using the Medical Outcome Survey (SF-36). After 2 weeks of treatment, the pain-free walking distance increased 66% while the total walking distance increased 23% in the group taking two active bars/day. The general and emotional/social functioning components of the SF-36 also improved. These effects were not observed in the one active bar/day and placebo groups. The effects were maintained after 10 weeks and, in addition, an improvement in walking distance was observed in the group taking one active bar. These findings reveal that use of a nutrient bar designed to enhance EDNO activity improves pain-free and total walking distance as well as quality of life in individuals with intermittent claudication.

Aged↗
PubMed2000

Characterization of an immortalized human vaginal epithelial cell line.

PURPOSE: Adherence of type 1 piliated Escherichia coli to vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections (UTIs) in women. Progress in understanding the mechanism of adherence to the vaginal surface could be enhanced by the utilization of well-characterized vaginal epithelial cells. The objective of this study was to immortalize vaginal epithelial cells and study their bacterial adherence properties. MATERIALS AND METHODS: Primary vaginal cells were obtained from a normal post-menopausal woman, immortalized by infection with E6/E7 genes from human papillomavirus 16 (HPV 16) and cultured in serum free keratinocyte growth factor medium. RESULTS: Positive immunostaining with a pool of antibodies to cytokeratins 1, 5, 10 and 14 (K1, K5, K10 and K14) and to K13 confirmed the epithelial origin of these cells. The immortalized cells showed binding of type 1 piliated E. coli in a pili specific and mannose sensitive manner. CONCLUSION: This model system should facilitate studies on the interaction of pathogens with vaginal mucosal cells, an essential step in the progression of ascending UTIs in women.

Bacterial Adhesion↗
PubMed1999

V2 regions of 16S ribosomal RNA used as a molecular marker for the species identification of streptococci in peripheral blood and synovial fluid from patients with psoriatic arthritis.

OBJECTIVE: To detect the 16S ribosomal RNA (rRNA) of 3 streptococcal species in the peripheral blood and synovial fluid of patients with psoriatic arthritis (PsA). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) detection targets bacterial 16S rRNA, which is present in bacteria at high copy numbers. The 3 species-specific primers for group A streptococci (GAS; Streptococcus pyogenes), group B streptococci (GBS; Streptococcus agalactiae), and Streptococcus pneumoniae were designed from the fragments of highly variable V2 regions of 16S rRNA. Total RNA was prepared from whole peripheral blood and joint fluid obtained from patients with PsA and rheumatoid arthritis (RA). All positive PCR reactions were then sequenced with a Pharmacia ALF DNA sequencing system. RESULTS: Our data in 19 PsA patients showed that 7 peripheral blood samples were positive for GAS (P = 0.006 versus GAS-positive RA patients [n = 0], by Fisher's exact test), and 2 were also positive for GBS. One synovial fluid sample from a PsA patient was positive for GAS. S pneumoniae was absent from all specimens. Seventeen patients with RA were PCR negative for the 3 streptococcal species. Peripheral blood from a patient with inflammatory bowel disease was positive for GAS. CONCLUSION: The presence of GAS 16S rRNA in the peripheral blood and synovial fluid of patients with PsA supports the concept that PsA is a reactive arthritis to certain streptococci.

Arthritis, Psoriatic↗
PubMed1999

Roles of glycoproteins and oligosaccharides found in human vaginal fluid in bacterial adherence.

Adherence of type 1-piliated Escherichia coli to carbohydrate structures of vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections in women. Colonization of the vaginal introitus is influenced by interactions between pathogens, vaginal fluid, and vaginal epithelium. In this study, the type and amount of carbohydrates and glycoproteins present in vaginal fluid were determined. Free and protein-bound oligosaccharides in vaginal fluid specimens were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pressure liquid chromatography (HPLC). Two-dimensional electrophoretic separations of vaginal fluid glycoproteins were performed together with bacterial overlay assays. The results of FACE showed that the majority of the oligosaccharides are in the free state and the bound oligosaccharides are undetectable. HPLC analysis of free sugars revealed glucose as the major sugar (3.3 +/- 0.3 mM), and the concentrations of mannose and glucosamine were 0.065 +/- 0.04 and 0.02 +/- 0.001 mM, respectively. Radiolabeled E. coli bound three vaginal fluid glycoproteins with the following molecular masses and pIs: 82 kDa and pI 5.5, 55 kDa and pI 4.5, and 55 kDa and pI 6.5. The binding was inhibited by mannose and by deglycosylation of the proteins prior to the overlay assay. One of these putative receptors was identified to be the heavy chain of secretory IgA (S-IgA). These data suggest that the free mannose in the fluid is less than that required to affect E. coli-epithelial cell binding interactions and that S-IgA may bind E. coli in the vaginal introitus.

Adult↗
PubMed1999

A holistic approach to environmental health research.

One of the most formidable questions facing the environmental health research community today is how to translate basic fundamental research into a product (e.g., disease outcome) that meets the needs of its stakeholders--the medical community, regulatory agencies, and ultimately, the citizens of our nation. Historically, a successful research program could be defined as one that received continuous funding, produced high-quality publications, and was respected by scientists in related fields. However, it is now apparent that this is not sufficient for attaining improved public health--the ultimate goal of these research efforts. Research results must be transferred in a more active way to the communities and professionals who have need of this information. The link must be recognized, and the roles of the stakeholders in the entire research process must be acknowledged to ensure full impact of the research endeavors.

Environmental Health↗
PubMed1998

Outer membrane proteins of Bartonella henselae and their interaction with human endothelial cells.

Members of the genus Bartonella are unique in that they are bacteria which cause proliferation of microvascular endothelial cells and neovascularization (angiogenesis). The mechanisms by which Bartonella henselae causes these processes are unknown. Given the importance of surface-exposed determinants in the pathogenesis of many organisms, outer membrane proteins (OMPs) of B. henselae were identified. Enrichment of the outer membrane fraction of B. henselae by sarkosyl treatment of total membranes, together with radioiodination and biotinylation of intact organisms, suggest that at least nine proteins, with molecular weights of 28, 30, 35, 43, 58, 61, 79, 92 and 171 kDa, are located in the outer membrane. Triton X-100-extracted biotinylated human umbilical vein endothelial cell (HUVEC) surface proteins bound to the 43 kDa B. henselae OMP after B. henselae whole-cell lysates and sarkosyl-fractionated OMPs were separated by SDS-PAGE and transferred onto nylon. Biotinylated B. henselae surface proteins of 28, 32, 43, 52 and 58 kDa were shown to bind intact HUVEC, with the 43 kDa protein being the major adhesin. Preincubation of HUVEC with an increasing concentration (20 microg/ml to 4 mg/ml) of sarkosyl-fractionated unlabelled B. henselae outer membrane proteins inhibited the attachment of all identified HUVEC binding proteins. The identification of B. henselae OMPs, as well as adhesins, should provide a basis for further investigation of the role of adherence in the pathogenesis of B. henselae.

Adhesins, Bacterial↗
PubMed1998

Conversion of native oligomeric to a modified monomeric form of human C-reactive protein.

C-reactive protein (CRP) is a pentameric oligoprotein composed of identical 23 kD subunits which can be modified by urea-chelation treatment to a form resembling the free subunit termed modified CRP (mCRP). mCRP has distinct physicochemical, antigenic, and biologic activities compared to CRP. The conditions under which CRP is converted to mCRP, and the molecular forms in the transition, are important to better understand the distinct properties of mCRP and to determine if the subunit form can convert back to the pentameric native CRP form. This study characterized the antigenic and conformational changes associated with the interconversion of CRP and mCRP. The rate of dissociation of CRP protomers into individual subunits by treatment in 8 M urea-10 mM EDTA solution was rapid and complete in 2 min as assayed by an enzyme-linked immunofiltration assay using monoclonal antibodies specific to the mCRP. Attempts to reconstitute pentameric CRP from mCRP under renaturation conditions were unsuccessful, resulting in a protein retaining exclusively mCRP characteristics. Using two-dimensional urea gradient gel electrophoresis, partial rapid unfolding of the pentamer occurred above 3 M urea, a subunit dissociation at 6 M urea, and further subunit unfolding at 6-8 M urea concentrations. The urea gradient electrophoresis results suggest that there are only two predominant conformational states occurring at each urea transition concentration. Using the same urea gradient electrophoresis conditions mCRP migrated as a single molecular form at all urea concentrations showing no evidence for reassociation to pentameric CRP or other aggregate form. The results of this study show a molecular conversion for an oligomeric protein (CRP) to monomeric subunits (mCRP) having rapid forward transition kinetics in 8 M urea plus chelator with negligible reversibility.

C-Reactive Protein↗
PubMed1997

Bartonella spp. as emerging human pathogens.

Members of the genus Bartonella (formerly Rochalimaea) were virtually unknown to modern-day clinicians and microbiologists until they were associated with opportunistic infections in AIDS patients about 6 years ago. Since that time, Bartonella species have been associated with cat scratch disease, bacillary angiomatosis, and a variety of other disease syndromes. Clinical presentation of infection with Bartonella ranges from a relatively mild lymphadenopathy with few other symptoms, seen in cat scratch disease, to life-threatening systemic disease in the immunocompromised patient. In some individuals, infection manifests as lesions that exhibit proliferation of endothelial cells and neovascularization, a pathogenic process unique to this genus of bacteria. As the spectrum of disease attributed to Bartonella is further defined, the need for reliable laboratory methods to diagnose infections caused by these unique organisms also increases. A brief summary of the clinical presentations associated with Bartonella infections is presented, and the current status of laboratory diagnosis and identification of these organisms is reviewed.

Animals↗
PubMed1996

[Human ehrlichiosis: report of the 1st case in Venezuela].

Human ehrlichiosis is a zoonotic disease, caused by a rickettsia that infects leukocytes. It was described for the first time in the United States of America in 1986. More than 300 cases have been reported in that country. One case has been reported in Portugal, two in France and one more in a tourist coming from Mali (Africa). In Venezuela, a tropical country, where ehrlichiosis is endemic in dogs and horses, the first case of human ehrlichiosis is reported in a seventeen month old girl. She initially had symptoms compatible with a viral illness. Then she developed a rash, acute respiratory failure, hepato-splenomegaly, neurologic abnormalities, renal failure and hematologic alterations including pancytopenia and disseminated intravascular coagulation (DIC). Different diagnoses were given before it was concluded that it was a case of ehrlichiosis. She was treated with tetracycline and very soon recovered. The initial diagnosis was made using buffy coat blood smears stained with Diff Quick Stain. Indirect Immunofluorescence (IFA) test was used to detect antibodies against Ehrlichia chaffeensis (1:126) but not to other rickettsias. Also the presence of platelets with ehrlichia bodies similar to E. platys from dogs, it was demonstrated, in peripheral blood from the child. The presence of these bodies in human platelets has not been previously reported. In this case, it could not be proved that the disease was transmitted by a tick bite. The presence of ehrlichia bodies in platelets from the girl's peripheral blood is also described.

Adolescent↗