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Biomedical subjects

C L Franklin

Publications and source records attributed to C L Franklin.

At least 19 recordsLinked to original sources
PubMed2001

Improved methods for determining the concentration of 6-thioguanine nucleotides and 6-methylmercaptopurine nucleotides in blood.

The conversion of the cytotoxic and immunosuppressive 6-mercaptopurine (6MP) to the active 6-thioguanine nucleotides (6TGN) is necessary for clinical efficacy of 6MP and its prodrug azathioprine. Another metabolite, 6-methylmercaptopurine nucleotide (6MMPN), is formed via a competing pathway by thiopurine methyl transferase. The concentrations of 6TGN and 6MMPN are measured in washed erythrocytes as a surrogate to the intracellular levels of these metabolites in the target tissues. Analysis of 6TGN and 6MMPN in multi-center clinical studies is more complicated because of the requirement to wash erythrocytes. In this investigation, we found no differences in the concentrations of 6TGN and 6MMPN in blood versus washed erythrocytes in samples obtained from patients taking therapeutic doses of oral 6MP or azathioprine for inflammatory bowel disease. We concluded that whole blood could be used for the analysis of these analytes, thus saving sample preparation time. We also found that the erythrocyte 6TGN concentration in blood at ambient temperature declined 2-4% per day, a loss that can be avoided by shipping blood samples frozen. The loss of 6TGN in blood stored at approximately -80 degrees C was 1% after 1 week and 12% after 24 weeks, indicating the analyte was moderately stable. 6MMPN in blood did not significantly change after 24 weeks of storage at approximately -80 degrees C. In addition, the sensitivity of the 6TGN assay was improved by modifying the HPLC conditions, which made the method more suitable for quantifying low levels of 6TGN in human intestinal biopsy samples and blood.

Chromatography, High Pressure Liquid↗
PubMed2001

Posttraumatic stress disorder and major depressive disorder: investigating the role of overlapping symptoms in diagnostic comorbidity.

Studies of posttraumatic stress disorder (PTSD) have found high levels of comorbid major depressive disorder (MDD). One reason suggested for the comorbidity is the symptom overlap (contaminated symptoms) between the disorders. The present study investigated the contribution of contaminated symptoms (anhedonia, concentration, and sleep problems) to the comorbidity of PTSD and MDD. PTSD symptoms were subdivided into two groups: the contaminated symptoms and the 14 unique symptoms. It was speculated that if the contaminated symptoms are responsible for the comorbidity, then they will show less specificity than the unique symptoms, will be less highly correlated with a PTSD symptom total count, and be more frequently endorsed in PTSD patients with than without MDD. These hypotheses were tested in a sample (N = 1300) of psychiatric outpatients, 260 of whom had lifetime PTSD. None of the hypotheses were supported, thereby suggesting that the comorbidity between PTSD and MDD is not an artifact of symptom overlap.

Adolescent↗
PubMed2001

Differential interleukin-10 and gamma interferon mRNA expression in lungs of cilium-associated respiratory bacillus-infected mice.

The cilium-associated respiratory (CAR) bacillus is a gram-negative, extracellular bacterium that causes persistent respiratory tract infections in rodents. We have previously demonstrated that BALB/c mice are more susceptible to CAR bacillus-induced disease than resistant C57BL/6 mice, with elevations in pulmonary gamma interferon (IFN-gamma) and interleukin (IL)-4. IL-10 is a type 2 cytokine that can increase host susceptibility to bacterial diseases through its anti-inflammatory effects, including suppression of macrophage function. The purpose of this study was to further describe the cytokine profiles associated with histologic lesions in CAR bacillus-infected mice and to assess the effects of cytokine depletion on the pathogenesis of disease. Six-week-old female BALB/c and C57BL/6 mice and mice with targeted mutations in IFN-gamma and IL-4 were inoculated intratracheally with 10(5) CAR bacillus organisms, and samples were collected at 6 to 7 weeks postinoculation. Lung samples were collected for histopathologic examination and analysis of cytokine mRNA. IFN-gamma, IL-10, and IL-4 mRNA levels in the lungs of infected mice were semiquantitatively measured using a reverse transcriptase-mediated PCR assay and compared to those in uninfected control animals of each strain. BALB/c mice infected with CAR bacillus had a median lung lesion score of 6 and IL-10 and IL-4 mRNA levels were significantly elevated. The majority of C57BL/6 mice were resistant to disease characterized by lung lesions scores of 2 or less and a dominant IFN-gamma mRNA cytokine profile. A few C57BL/6 mice with lesions scores of 5 or greater had elevations in all three cytokines and were susceptible to disease. C57BL/6 IFN-gamma knockout mice had increased disease with elevations in IL-10 and IL-4 mRNA, while BALB/c IL-4 knockout mice infected with CAR bacillus had a mild decrease in lesion severity and an attenuated IL-10 mRNA expression compared to wild-type BALB/c mice. These data indicate that IL-10 and IL-4 predominate in CAR bacillus-induced histologic lesions in mice, while IFN-gamma may play a role in resistance to disease.

Animals↗
PubMed2001

Helicobacter typhlonius sp. nov., a Novel Murine Urease-Negative Helicobacter Species.

Over the past decade, several Helicobacter species have been isolated from rodents. With the advent of PCR for the diagnosis of infectious agents, it has become clear that several previously uncharacterized Helicobacter species also colonize rodents. In this report, we describe a novel urease-negative helicobacter, Helicobacter typhlonius sp. nov., which was isolated from colonies of laboratory mice independently by two laboratories. Infection of immunodeficient mice by this bacterium resulted in typhlocolitis similar to that observed with other helicobacter infections. H. typhlonius is genetically most closely related to H. hepaticus. Like H. hepaticus, it is a spiral bacterium with bipolar sheathed flagella. However, this novel species contains a large intervening sequence in its 16S rRNA gene and is biochemically distinct from H. hepaticus. Notably, H. typhlonius does not produce urease or H(2)S nor does it hydrolize indoxyl-acetate. Compared to other Helicobacter species that commonly colonize rodents, H. typhlonius was found to be less prevalent than H. hepaticus and H. rodentium but as prevalent as H. bilis. H. typhlonius joins a growing list of helicobacters that colonize mice and are capable of inducing enteric disease in various strains of immunodeficient mice.

Animals↗
PubMed2001

Leucopenia resulting from a drug interaction between azathioprine or 6-mercaptopurine and mesalamine, sulphasalazine, or balsalazide.

AIM: We evaluated the effect of coadministration of sulphasalazine, mesalamine, and balsalazide on the pharmacokinetics and pharmacodynamics of azathioprine and 6-mercaptopurine. METHODS: Thirty four patients with Crohn's disease receiving azathioprine or 6-mercaptopurine were enrolled in an eight week non-randomised parallel group drug interaction study and treated with mesalamine 4 g/day, sulphasalazine 4 g/day, or balsalazide 6.75 g/day. The primary outcome measure was the occurrence of clinically important leucopenia during the study, defined separately as total leucocyte counts < 3.0 x 10(9)/l and < or = 3.5 x 10(9)/l. Whole blood 6-thioguanine nucleotide concentrations were determined. RESULTS: Three patients could not be evaluated for the primary outcome measure. In the remaining 31 patients, the frequency of total leucocyte counts < 3.0 and < or = 3.5 were: 1/10 and 5/10 in the mesalamine group; 1/11 and 6/11 in the sulphasalazine group; and 0/10 and 2/10 in the balsalazide group. There were significant increases in mean whole blood 6-thioguanine nucleotide concentrations from baseline at most time points in the mesalamine and sulphasalazine groups but not in the balsalazide group. CONCLUSIONS: In patients with Crohn's disease receiving azathioprine or 6-mercaptopurine, coadministration of mesalamine, sulphasalazine, and possibly balsalazide results in an increase in whole blood 6-thioguanine nucleotide concentrations and a high frequency of leucopenia.

Adult↗
PubMed2001

Measurement of thiopurine methyltransferase activity and azathioprine metabolites in patients with inflammatory bowel disease.

BACKGROUND: Measurement of 6-thioguanine nucleotide concentrations may be useful for optimising treatment with azathioprine and 6-mercaptopurine. METHODS: We conducted a study of 170 patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine to determine the relationship between 6-thioguanine nucleotide concentrations and both disease activity, as measured by the inflammatory bowel disease questionnaire (active disease < 170, remission > or = 170) and leucopenia. Blood was submitted for whole blood 6-thioguanine nucleotide concentration and leucocyte count. RESULTS: Mean (SD) inflammatory bowel disease questionnaire score was 176 (32). There was no correlation between inflammatory bowel disease questionnaire scores and 6-thioguanine nucleotide concentrations (r(s) = -0.09, p = 0.24). Median 6-thioguanine nucleotide concentrations in 56 patients with active disease and 114 patients in remission were similar (139 v 131 pmol/8 x 10(8) red blood cells; p = 0.26). There was no correlation between 6-thioguanine nucleotide concentrations and leucocyte counts. CONCLUSIONS: In patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine, 6-thioguanine nucleotide concentrations did not correlate with disease activity, as measured by the inflammatory bowel disease questionnaire, or leucocyte count. These findings are discrepant with most previous studies, possibly due to selection of responding patients who tolerated the medications. A prospective, randomised, dose optimisation trial using 6-thioguanine nucleotide concentrations is warranted.

Adolescent↗
PubMed2001

Hamster polyomavirus infection in a pet Syrian hamster (Mesocricetus auratus).

An approximately 8-week-old pet Syrian hamster (Mesocricetus auratus) with a 1-week history of dyspnea, hyporexia, and ataxia was submitted for necropsy. On gross examination, the hamster had multiple abdominal adhesions and enlargement of the mesenteric lymph node. Histologic evaluation revealed multicentric lymphoma of the liver, jejunum, mesenteric lymph node, testicular fat pad, and epididymis. Based on the hamster's age and the type and distribution of the lymphoma, a presumptive diagnosis of hamster polyomavirus-induced lymphoma was made. A specific polymerase chain reaction (PCR) was developed, which confirmed the diagnosis. An in situ PCR demonstrated hamster polyomavirus DNA within lymphocytes of the multicentric lymphoma and renal tubular epithelial cells and within clusters of enterocytes in the jejunum. These data are consistent with environmental dissemination of hamster polyomavirus virions through the renal tubular epithelium and into the urine and with fecal shedding of hamster polyomavirus virions; however, additional studies will be needed to confirm these observations.

Animals↗
PubMed2001

Diagnostic polymerase chain reaction assays for identification of murine polyomaviruses in biological samples.

PURPOSE: Mouse polyoma virus and K virus are murine polyomaviruses frequently used in carcinogenicity and cellular biology studies in mice. These viruses can cause persistent infections, which increase the likelihood of transmission through transplantation of cells from infected mice. To identify polyomavirus-infected biological samples, several diagnostic polymerase chain reaction (PCR) assays were developed. METHODS: Polyomavirus-family and virus-specific PCR assays were designed and optimized for specificity and sensitivity. The generic (polyomavirus-family) PCR assay and mouse polyoma virus-specific assays were compared with the mouse bioassay for diagnosis of infected cellular samples. RESULTS: Specificity of the PCR assays was confirmed by testing a battery of other murine viruses. The mouse polyoma virus PCR test was the most sensitive assay, detecting as few as 2,000 copies of homologous virus. The K virus PCR assay was about eightfold less sensitive, and the generic PCR test was the least sensitive. Mouse polyoma virus and generic PCR assays amplified mouse polyoma virus in the inoculum and tissues from experimentally infected mice, and performed better than did the mouse bioassay. CONCLUSIONS: Results of this study confirm that PCR is a specific and sensitive method for detection of murine polyomaviruses in biological samples.

Animals↗
PubMed2001

Coxiella burnetii infection in C.B-17 scid-bg mice xenotransplanted with fetal bovine tissue.

Two from a group of approximately 50 C.B-17 scid-bg mice were examined because of lethargy, dehydration, and rough coat. Three months prior to development of clinical signs of disease, mice of this study had been surgically implanted with fetal bovine liver, thymus, and lymph node. At necropsy, marked splenomegaly and mild hepatomegaly were observed in both animals. Large areas of necrosis and inflammation, with associated intracytoplasmic granular basophilic inclusions, were observed in histologic sections of multiple organs. Aerobic and anaerobic culturing of the liver yielded negative results. Six months after the initial case, four more reconstituted scid-bg mice from a different fetal donor had identical clinical, gross, and histologic signs of disease. To determine whether the basophilic inclusions represented an infective agent, 4-month-old immune-naive C.B-17 scid-bg mice were inoculated intraperitoneally with a liver and spleen homogenate from an affected mouse. Two weeks after inoculation, mice developed clinical signs of disease and lesions identical to those seen in the signal mice. On ultrastructural examination of the liver, pleomorphic bacteria were found in large cytoplasmic vacuoles of hepatocytes. Bacterial DNA was amplified from the liver, using primers that amplify a segment of the 16S rRNA gene from many bacterial species. Sequencing of the polymerase chain reaction (PCR) product revealed gene sequence identical to that of Coxiella burnetii, the agent of Q-fever. These results highlight the need to consider infective agents of the donor species when working with xenografted animals.

Abdomen↗
PubMed2000

A novel class of zinc-binding inhibitors for the phosphatidylcholine-preferring phospholipase C from Bacillus cereus.

The phospholipase C (PLC) isozymes catalyze the hydrolysis of phospholipids to provide diacylglycerol (DAG) and a phosphorylated headgroup. Because DAG has been implicated in cellular signal transduction cascades in mammalian systems, there has been considerable interest in the development of inhibitors of these enzymes. Toward this end, we have discovered that the cyclic N,N'-dihydroxyureas 6-10 inhibit the phosphatidylcholine preferring PLC from Bacillus cereus (PLCBc). This class of inhibitors is believed to function by the bidentate chelation of the N,N'-dihydroxyurea array to one or more of the zinc ions at the active site of the enzyme. Because the affinities of these compounds correlate with the pKaS of the N-OH hydroxyl groups, it is apparent that one or both of the hydroxyl groups must be ionized for effective coordination to the zinc ions. It is also apparent that there may be rather strict steric requirements for these inhibitors.

Bacillus cereus↗
PubMed2000

Antibody and cytokine responses to the cilium-associated respiratory bacillus in BALB/c and C57BL/6 mice.

The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response. To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 10(5) CAR bacillus organisms. CAR bacillus-specific serum immunoglobulins (immunoglobulin M [IgM], IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], and interleukin-4 [IL-4]) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days. BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time. Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-alpha, IFN-gamma, and IL-4. C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA. Cytokine perturbations were not detected in C57BL/6 mice. The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-gamma and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease. Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.

Animals↗
PubMed2000

Helicobacter mesocricetorum sp. nov., A novel Helicobacter isolated from the feces of Syrian hamsters.

A spiral-shaped bacterium with bipolar, single, nonsheathed flagella was isolated from the feces of Syrian hamsters. The bacterium grew as a thin spreading film at 37 degrees C under microaerobic conditions, did not hydrolyze urea, was positive for catalase and alkaline phosphatase, reduced nitrate to nitrite, did not hydrolyze hippurate, and was sensitive to nalidixic acid but resistant to cephalothin. Sequence analysis of the 16S rRNA gene and biochemical and phenotypic criteria indicate that the novel bacterium is a helicobacter. The novel bacterium is most closely related to the recently described mouse enteric helicobacter, Helicobacter rodentium. This is the first urease-negative Helicobacter species with nonsheathed flagella isolated from feces of asymptomatic Syrian hamsters. We propose to name this novel helicobacter Helicobacter mesocricetorum. The type strain is MU 97-1514 (GenBank accession number AF072471).

Alkaline Phosphatase↗
PubMed1999

Acanthamoeba castellanii: characterization of an adhesin molecule.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein.

Acanthamoeba↗
PubMed1999

Cloning and expression of an immunogenic membrane-associated protein of Helicobacter hepaticus for use in an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a bacterial pathogen that causes chronic active hepatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hepaticus was constructed and was screened with sera from H. hepaticus-infected mice. A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified. The gene had high identity with genes coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusion protein was detected on Western blots probed with sera from H. hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice free of Helicobacter infection. The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H. hepaticus antibodies in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bilis, or H. muridarum and sera from mice free of Helicobacter infection were evaluated. Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%). These data indicate that H. hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.

Amino Acid Sequence↗
PubMed1999

Isolation of cilia-associated respiratory (CAR) bacillus from pigs and calves and experimental infection of gnotobiotic pigs and rodents.

Filamentous, gram-negative bacteria morphologically similar to cilia-associated respiratory (CAR) bacillus of rodents and rabbits were isolated from the tracheas of 5 pigs and 4 calves. All pigs but none of the calves had histologic lesions of chronic tracheitis. In silver-stained histologic sections, CAR bacilli were adhered to the tracheal epithelium of each pig but were not found in the calves. Like CAR bacillus of rats, the bacteria displayed gliding motility and grew only in cell culture or cell culture medium supplemented with fetal serum. Initially, all isolates were contaminated by Mycoplasma spp. This contamination was eliminated from 4 pig isolates by limiting dilutions, and mycoplasma-free isolates were used to intranasally inoculate gnotobiotic pigs and CAR bacillus-free mice and rats and to immunize guinea pigs. The gnotobiotic pigs remained healthy, and when they were necropsied 4 and 7 weeks after infection no macroscopic or microscopic lesions were found in the respiratory tract. However, CAR bacillus was isolated at both times from the nasal cavities and tracheas of inoculated pigs, and the ciliated tracheal epithelium of infected pigs necropsied 7 weeks after infection was colonized by low numbers of CAR bacillus-like bacteria. The rats and mice remained healthy through week 12 postinoculation, and evidence of short- or long-term colonization was not detected by histologic examination or culture. When used as primary antibody for immunohistochemical staining, sera from guinea pigs immunized with pig CAR bacillus specifically stained CAR bacilli colonizing the respiratory epithelium of naturally infected pigs, whereas sera collected prior to immunization failed to react with the bacteria. These results indicate that CAR bacilli are unlikely to be primary pathogens of pigs or cattle and that rodents do not act as reservoirs.

Animals↗