Isolation of Corynebacterium pseudotuberculosis from the cervical canal of clinically normal sows

Two isolates from the cervical canal of clinically normal sows were indistinguishable from Corynebacterium pseudotuberculosis in their biochemical properties and genetic homology by DNA-DNA hybridisation. Furthermore, three sheep inoculated with one of the isolates developed typical lesions of caseous lymphadenitis and gave antibody responses specifically to C pseudotuberculosis.     

Serological studies on Corynebacterium pseudotuberculosis infections in goats using enzyme-linked immunosorbent assay

This study investigated matrix formation of rabbit meniscal fibrochondrocytes synthesized in vitro and the viability of fibrochondrocytes encapsulated in fibrin gel after transplantation into rabbit meniscal lesion sites in vivo. The matrix of newly formed meniscal fibrochondrocytes encapsulated in fibrin gel was evaluated using immunohistochemical methods. The meniscal cells in the three-dimensional encapsulating fibrin gel proliferated well for a 2-month period. Examination of the in vivo transplantation confirmed that the newly formed cartilage produced S-100 protein. These observations suggest that meniscal fibrochondrocytes encapsulated in fibrin gel are suitable for cell division and matrix production, and the use of fibrin gel as a scaffolding delivery substance may be beneficial for the transplantation of viable meniscal fibrochondrocytes in resurfacing meniscoplasty.     

Application and evaluation of a mailed questionnaire for an epidemiologic study of Corynebacterium pseudotuberculosis infection in horses

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood gammadelta T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 10(7)) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN-alpha and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood gammadelta T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 10(9) BRM-activated killer (BAK) cells composed of CD56+ gammadelta T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90-100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated gammadelta T cells producing IFN-gamma were assayed as an indication marker of BAK therapy. The normal range of IFN-gamma producing gammadelta T cells comprised 6.9 +/- 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN-gamma producing gammadelta T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood gammadelta T cells of Patients Nos. 1-7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN-gamma producing gammadelta T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial gammadelta T cell counts, a low frequency of these cells may contraindicate BAK therapy.     

Reevaluation of the virulence phenotype of the inv yadA double mutants of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis and Yersinia enterocolitica are closely related human pathogens causing gastroenteritis. Invasin and YadA are two of the most extensively studied virulence factors of the Yersinia genus. Invasin is the primary invasion factor encoded by the inv gene on the chromosome and is required for the penetration of the epithelial cells. YadA is encoded by the yadA gene on the 70-kb virulence plasmid and has multiple functions. Previous studies indicate that an inv yadA double mutant of Y. enterocolitica is avirulent while an inv yadA mutant of Y. pseudotuberculosis is hypervirulent. In this study, we investigated this unexpected difference. New constructs of the inv yadA mutants of Y. pseudotuberculosis were made and tested in mice. These new constructs were not hypervirulent; rather, they maintained the same virulence as the wild-type strain. Further examination of the inv mutant used for the previous study revealed that it carries an aberrant inv phenotype and has an altered outer membrane profile and an altered colony morphology. Therefore, the mutants used previously were not isogenic to the parental wild-type strain, which may in part account for the difference in the results obtained.     

Lectin-bearing polymerized liposomes as potential oral vaccine carriers

PURPOSE: The potential of using lectin-modified polymerized liposomes as Peyer's patch targeted oral delivery vehicles was examined. METHODS: Two types of lectins, Ulex Europaeus Agglutinin I (UEA I) and Wheat Germ Agglutinin (WGA), were modified with a hydrophobic anchor N-glutaryl-phosphotidylethanolamine (NGPE). The modified lectins were incorporated into liposome bilayers and the liposomes were subsequently stabilized through polymerization. The presence of the lectins on the liposome surfaces was first confirmed with X-ray photoelectron spectroscopy. Surface-immobilized lectins were then shown to retain their carbohydrate binding activities as well as specificities based on an in vitro aggregation assay. Finally, delivery efficiencies of lectin-bearing liposomes were determined in mice. RESULTS: About 10.5% UEA I liposomes and 5.8% WGA liposomes were taken up from the gastrointestinal tract. These numbers are significantly higher than the 3.2% observed in the case of lectin-free liposomes. At the same time, UEA I liposomes exhibited the most effective Peyer's patch targeting among the three, which directly correlated with the highest delivery efficiency observed. CONCLUSIONS: This establishes that lectin modification of liposomes can promote binding to Peyer's patches, which will give improved efficiency for Peyer's patch targeted delivery. All these point to the potential for these lectin-modified liposomes as novel vehicles for oral vaccination.     

The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison

A large number of drug trials for prevention of restenosis have been conducted with many showing little or conflicting benefit. Antiplatelets such as aspirin, ticlopidine and thromboxane A2 receptor inhibitors have not shown a clear benefit. Similarly, antithrombotics, either acting indirectly such as heparin, or as direct thrombin inhibitors such as hirudin and hirulog, do not prevent restenosis. Trials with ACE inhibitors, HMG-CoA reductase inhibitors and fish-oil supplements have yielded inconclusive results. The antiproliferatives, angiopeptin, trapidil and tranilast have shown some benefit in small-scale studies. Other drug classes of potential benefit include the glycoprotein IIb/IIIa receptor antagonists, inhibitors of the early coagulation cascade, calcium channel blockers and nitric oxide donors. Drug research into restenosis prevention has been hampered by problems with the definition of restenosis and the applicability in humans of animal models. Although no single drug has conclusively proven effective yet, the promise of a number of agents, together with other nonpharmacological strategies will likely result in further reductions in the incidence of restenosis.     

Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants

There is no effective vaccine for Marburg virus (MBGV) or any other filovirus, nor enough pertinent information to expedite rational vaccine development. To ascertain some of the minimal requirements for a MBGV vaccine, we determined whether whole inactivated MBGV, or a baculovirus-expressed virion subunit, could be used to immunize guinea pigs against a lethal infection. Baculovirus recombinants were made to express the MBGV glycoprotein (GP) either as a full-length, cell-associated molecule or a slightly truncated (5.4%) product secreted into medium; the latter, for its far greater ease in manipulation, was tested for its vaccine potential. Like MBGV GP, both the full-length and truncated GP expressed by baculovirus recombinants were abundantly glycosylated with both N- and O-linked glycans; differences in glycosylation were detectable, but these could not be shown to affect antigenicity with respect to available antibodies. The recombinant truncated glycoprotein elicited protection against lethal challenge with the MBGV isolate from which it was constructed and less effectively against an antigenically disparate MBGV isolate. Killed (irradiated) MBGV antigen was protective, in a reciprocal fashion, against both MBGV types. In a preliminary assessment of possible protective mechanisms, serum antibodies from immune animals were shown to be sufficient for protecting naive guinea pigs from lethal MBGV infections     

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.     

The potential of various HIV-1 mutants to inhibit the replication of wild-type virus

Transluminal placement of a stent graft in patients with an abdominal aortic aneurysm is a new endovascular technique that offers a potentially less invasive and less risky alternative to open surgery. Complications after stent graft placement are not infrequent, but in most cases secondary endovascular intervention is successful. We describe a late major leak in the aneurysmal sac caused by a distal migration of the iliac limb of a bifurcated graft. This late complication was successfully treated by covered stent placement, excluding and thrombosing completely the reformed aneurysm.     

In-vitro activity of psychiatric drugs against Corynebacterium urealyticum (Corynebacterium group D2)

We tested the in-vitro activity of amoxycillin, amoxycillin/clavulanic acid, cefotaxime, gentamicin, trimethoprim-sulphamethoxazole, tetracycline, norfloxacin, ciprofloxacin, vancomycin, teicoplanin, clindamycin and five psychiatric drugs (chlorpromazine, sertraline, fluoxetine, paroxetine and risperidone) against 32 strains of Corynebacterium urealyticum. Resistance rates exceeded 90% for all antibiotics except glycopeptides, quinolones and tetracycline. Sertraline was the most active psychiatric drug. We tested the influence of sertraline on the activity of amoxycillin, amoxycillin/clavulanic acid, cefotaxime, gentamicin, trimethoprim-sulphamethoxazole, tetracycline and ciprofloxacin. We did not observe antagonism in any case. Sertraline enhanced the activity of ciprofloxacin and tetracycline against all strains (MIC decrease: 4-64-fold for ciprofloxacin, 2-32-fold for tetracycline).     

The transferrin binding protein B of Moraxella catarrhalis elicits bactericidal antibodies and is a potential vaccine antigen

The transferrin binding protein genes (tbpA and tbpB) from two strains of Moraxella catarrhalis have been cloned and sequenced. The genomic organization of the M. catarrhalis transferrin binding protein genes is unique among known bacteria in that tbpA precedes tbpB and there is a third gene located between them. The deduced sequences of the M. catarrhalis TbpA proteins from two strains were 98% identical, while those of the TbpB proteins from the same strains were 63% identical and 70% similar. The third gene, tentatively called orf3, encodes a protein of approximately 58 kDa that is 98% identical between the two strains. The tbpB genes from four additional strains of M. catarrhalis were cloned and sequenced, and two potential families of TbpB proteins were identified based on sequence similarities. Recombinant TbpA (rTbpA), rTbpB, and rORF3 proteins were expressed in Escherichia coli and purified. rTbpB was shown to retain its ability to bind human transferrin after transfer to a membrane, but neither rTbpA nor rORF3 did. Monospecific anti-rTbpA and anti-rTbpB antibodies were generated and used for immunoblot analysis, which demonstrated that epitopes of M. catarrhalis TbpA and TbpB were antigenically conserved and that there was constitutive expression of the tbp genes. In the absence of an appropriate animal model, anti-rTbpA and anti-rTbpB antibodies were tested for their bactericidal activities. The anti-rTbpA antiserum was not bactericidal, but anti-rTbpB antisera were found to kill heterologous strains within the same family. Thus, if bactericidal ability is clinically relevant, a vaccine comprising multiple rTbpB antigens may protect against M. catarrhalis disease.     

Serodiagnosis of caseous lymphadenitis (pseudotuberculosis) in sheep

Random postmortal isolation of Corynebacterium pseudotuberculosis from the sheep used in a research project and the consequently determined serological positivity of these animals indicated the serological examination of the flock from which the sheep came. No symptoms of caseous lymphadenitis were ever observed in the flock in question and no data on the occurrence of this disease were available in the flock history. The sera of 228 head of sheep were used for examinations, while this number of sheep represented half the flock head. Agar-gel immunodiffusion (AID) and neutralization of the toxin C. pseudotuberculosis (NTX) were applied as serological assays. The antigen in both tests was an isolated form of toxin i.e. phospholipase D (PLD). To determine the effect of PLD, the intensity of its synergic hemolytic effect with the equi factor Rhodococcus equi was found out and it was expressed in activity units (AU). Undiluted serum with PLD containing 5,000 AU/ml was examined in AID. Positive precipitation was observed in 79 examined sera, out of which 71 were also positive in NTX. NTX demonstrated the inhibition of synergic hemolytic activity of PLD with the equi factor, which was used to sensibilize sheep erythrocytes in agar medium. PLD with the terminal content of 10 AU/ml was used, as well as twofold dilution series of sera. The first evaluated dilution series was 1:10. In this dilution series, the highest number of sera, 22, had the positive reaction. The highest positivity at a dilution series 1:5,120 was found out in one serum. A total of 71 sera had positive reactions in NTX and those sera were also positive in AID. The determined 34.7% positivity substantiated the importance of serodiagnosis for the intravital diagnosis of caseous lymphadenitis of sheep, particularly of its chronic form. AID and NTX are usable tests for serodiagnosis of this disease.     

Site-directed mutants of pseudoazurin: explanation of increased redox potentials from X-ray structures and from calculation of redox potential differences

In order to understand the origins of differences in redox potentials among cupredoxins (small blue type I copper-containing proteins that reversibly change oxidation state and interact with redox partners), we have determined the structures of the native and two mutants (P80A and P80I) of pseudoazurin from Alcaligenes faecalis S-6 in oxidized and reduced forms at resolutions of 2.2 A in the worst case and 1.6 A in the best case. The P80A mutation creates a surface pocket filled by a new water molecule, whereas the P80I mutant excludes this water. Distinct patterns of change occur in response to reduction for all three molecules: the copper position shifts, Met 7 and Pro 35 move, and the relative orientations of residues 81 to 16, 18 to the amide planes of 77 and 86, all change. Systematic changes in the weak electrostatic interactions seen in the structures of different oxidation states can explain the Met 7/Pro 35 structural differences as well as some fluctuating solvent positions. Overall displacement parameters increase reversibly upon reduction. The reduced forms are slightly expanded over the oxidized forms. The geometries of the mutants become more trigonal in their reduced forms, consistent with higher redox potentials (+409 mV for P80A and +450 mV for P80I). Calculations of the differences in redox potentials, using POLARIS, reveal that a water unique to the P80A mutant is required (with correctly oriented hydrogens) to approximate the observed difference in redox potential. The POLARIS calculations suggest that the reduced forms are additionally stabilized through changes in the solvation of the copper center, specifically via the amides of residues 16, 39, 41, 79, and 80 which interact with either Phe 18, Met 86, or Cys 78. The redox potential of P80A is increased largely due to solvation effects, whereas the redox potential of P80I is increased largely due to geometrical effects.     

Rain Loads and Flow Attenuation on Roofs

Basic relationships are derived for calculating the flow attenuation and depth of storm water on roofs. Hyetographs and corresponding hydrographs are specialized to the case of roof drainage and incorporated in methods which are presented for analysis and design of roof drains. Roof storage and outflow are characterized for various roof geometries and outflow devices. The methods are applied to examples found in the literature, including those in standards of Factory Mutual Insurance Company, ASCE, and others. Assumptions implicit in those standards are tested and their limitations demonstrated. A critical duration at which the greatest water depth and outflow occur must be found on a case-by-case basis. The critical durations can differ significantly from the fixed durations specified in standards. The corresponding maximum water depths and peak outflows can also differ accordingly. For determination of roof loads, these results will have their greatest importance in warmer climates in which the rain loads govern over snow loads. An example of design specifically for flow attenuation is also presented.