Safety of Brucella abortus strain RB51 in Bison

We compared the efficacy of two clinically available drugs with N-methyl-D-aspartate receptor antagonist properties, dextromethorphan and ketamine, in potentiating morphine-induced antinociception. Ketamine alone at 0.3-3 mg/kg had no effect on the hot plate test and at 10 mg/kg caused sedation/motor deficits. The antinociceptive effect of 5 mg/kg morphine was slightly enhanced by 1 mg/kg, but not 0.3 or 3 mg/kg, ketamine. Dextromethorphan alone at 45 mg/kg had no effect, but at 60 mg/kg caused sedation/motor deficit. At 15-45 mg/kg, dextromethorphan significantly and dose-dependently increased the magnitude and duration of morphine-induced antinociception. Dextromethorphan also potentiated morphine at doses that, by themselves, did not cause antinociception (1-2 mg/kg). Implications: Dextromethorphan was more effective than ketamine in potentiating morphine-induced antinociception. Dextromethorphan may thus be the drug of choice for testing the interactions between N-methyl-D-aspartate antagonists and morphine clinically.     

Determination of stability of Brucella abortus RB51 by use of genomic fingerprint, oxidative metabolism, and colonial morphology and differentiation of strain RB51 from B. abortus isolates from bison and elk

Brucella abortus RB51 and isolates from cattle, bison, and elk were characterized by pulsed-field gel electrophoresis and standard techniques for biotyping Brucella species, which included biochemical, morphological, and antigenic techniques, phage susceptibility, and antibiotic resistance. The objectives were to ascertain the stability of RB51 and to differentiate RB51 from other brucellae. Genomic restriction endonuclease patterns produced by pulsed-field gel electrophoresis demonstrated a unique fingerprint for RB51 relative to other brucellae. Comparisons of the oxidative metabolic profiles of RB51 after time in vivo (14 weeks) and in vitro (75 passages) showed no change in characteristic patterns of oxygen uptake on selected amino acid and carbohydrate substrates. Strain RB51 was biotyped as a typical rough B. abortus biovar 1 (not strain 19) after animal passage or a high number of passages in vitro and remained resistant to rifampin or penicillin and susceptible to tetracycline. No reactions with A or M antiserum or with a monoclonal antibody to the O antigen of Brucella lipopolysaccharides were detected; however, RB51 agglutinated with R antiserum. The results indicate that the genomic fingerprint and rough colonial morphology of RB51 are stable characteristics and can be used to differentiate this vaccine strain from Brucella isolates from cattle, bison, and elk.     

Safety and immunogenicity of Brucella abortus strain RB51 vaccine in pregnant cattle

L-Asparaginase derived from Erwinia chrysanthemi which is being investigated as an alternative to E. coli for the treatment of lymphoblastic leukaemia has been found in our laboratory to lose activity upon exposure to consecutive freeze-thaw cycles. An investigation was undertaken using several techniques to characterize fully the physicochemical changes L-Asparaginase is undergoing during freeze-thaw cycling leading to the loss of its activity. A total protein assay suggested that the loss of some enzyme activity was a result of protein precipitation. Circular dichroism (CD) studies showed a decrease of alpha-helical structure with a concomitant increase in beta sheet and random coil content, suggesting alterations in the secondary structure leading to unfolding, the first step of denaturation processes. The elution profiles obtained from size-exclusion chromatography (SEC) studies indicated the formation of several species during the process of freezing and thawing. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed bands corresponding to 1-3 kDa and 32 kDa, suggesting that some of the species are fragments and shortened monomers resulting from cleavage of monomers. The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of polydispersed fragments ranging from 900 Da to 3 kDa and a small fraction of aggregates corresponding to 300 kDa. A scheme was proposed to explain the cascade of events leading to the loss of soluble protein and accompanying loss of enzyme activity. Tetramers of the enzyme dissociate into monomers some of which are cleaved into small fragments. The shortened monomers then aggregate and precipitate.     

Morphometric analysis of CD4+, CD8+, and gamma/delta+ T-lymphocytes in lymph nodes of cattle vaccinated with Brucella abortus strains RB51 and 19

We have often experienced false positive results of the stress Thallium-201 myocardial scintigraphy (TL) for the evaluation of artery bypass graft patency after coronary artery bypass surgery (CABG). The purpose of this study is to clarify the frequency and the clinical significance of this findings. Sixty-two patients undergoing coronary angiography (CAG) after CABG were studied. These patients had undergone at total of 156 bypasses (artery grafts 108, saphenous vein grafts 48, mean bypass grafts number 2.65/cases), and the mean period from CABG to TL was 41.6 +/- 34 days. The territories of stress induced ischemia were divided into 3 territories; left anterior descending (LAD), right coronary artery (RCA), and left circumflex (LCX) territories. Patency of the bypass grafts was estimated on the absence of transient perfusion defect (TPD) on TL images. The incidence of false positive results was higher in Dipyridamole TL (38%) than in Exercise TL (18%) and higher in LAD territories (38%) than in RCA (11%) and LCX (13%) territories. All false positive cases showed no evidence of chest pain and significant ST-T change during stress TL test. High incidence of false positive results of stress TL test was observed for the evaluation of artery bypass graft patency after CABG.     

Experimental infection of pregnant cattle with the vaccine candidate Brucella abortus strain RB51: pathologic, bacteriologic, and serologic findings

To determine the placental tropism and abortigenicity of the vaccine candidate Brucella abortus strain RB51 (SRB51), a rough mutant of the virulent strain 2308, ten Polled Hereford heifers were inoculated intravenously in the 6th month of gestation. Heifers were euthanatized and examined at postinoculation week (PIW) 8 (n = 5) or at full term (n = 5). Four of five infected heifers sampled at PIW 8 and three of four infected heifers at term had placentitis, whereas reproductive tissues of three normal cows used for comparison had no placentitis. Numerous macrophages, immunoreactive for SRB51 antigen, as well as neutrophils, fibrin, and cell debris filled the arcade zone between chorion and maternal septae. Trophoblastic epithelium of the placentomal arcade zone had intracellular bacteria that were immunoreactive for SRB51 antigen. The tips of maternal septa had a lymphoplasmacytic infiltrate with small multifocal erosions and ulcerations of maternal epithelium. SRB51 was cultured from all tissues in which lesions were seen. Placentae of one cow from each group had no placentitis and contained no SRB51. In mammae, interstitial lymphoplasmacytic infiltrates and suppurative infiltrates within alveoli and intralobular ductules were seen in two of five heifers at PIW 8. SRB51 was cultured from liver, spleen, lung, and bronchial lymph nodes in four of five calves at PIW 8 and three of four full-term calves, but no lesions were seen. One near-term heifer had disseminated infection, placentitis, and lymphoplasmacytic endometritis, and delivered a premature weak calf. These results establish that SRB51 is less abortifacient than previously published reports with strain 19, in that only one of four heifers delivered prematurely following intravenous inoculation with SRB51, whereas intravenous inoculation with strain 19 leads to 100% abortion. However, it also shows that SRB51 can infect the bovine placenta, mammary gland, and fetus, can induce placentitis, and, in some cases, can lead to preterm expulsion of the fetus.     

Protection against infection and abortion induced by virulent challenge exposure after oral vaccination of cattle with Brucella abortus strain RB51

A series of non-natural N-acyl derivatives of lactosamine is incubated with recombinant alpha(1-3)galactosyl-transferase and UDP-galactose. The enzyme shows a high promiscuity towards the non-natural acceptors. It selectively transfers a galactose unit onto the 3-OH group of the terminal beta-linked galactose in an alpha-mode to give an array of linear-B trisaccharides.     

Vaccinal strain of Brucella abortus 19-BA in ticks (Argasidae) infected by feeding

Bladder and urethral function was studied in 21 patients with lesions of the cauda equina or conus medullaris using gas cystometry, integrated sphincter electromyography, uroflowmetry and computer assisted measurement of detrusor and urethral innervation. An areflexic cystometrogram, impaired electromyographic sphincter activity and delayed reflex-evoked potentials from stimulation of the detrusor muscle and urethra were the most consistent indicators of lesions of the conus medullaris and/oather than the ice water test is suggested.     

Morphometric and histopathologic analysis of lymphoid depletion in murine spleens following infection with Brucella abortus strains 2308 or RB51 or an htrA deletion mutant

A physician education program altered the consumption of hospital resources by orthopedic surgeons doing total hip replacement. They were presented with verbal and written physician-specific materials during a 7-week period. Xbar and R charts were used to display measurements of resource consumption before and after the educational program. Physicians were provided with surgeon-specific data profiling their own practice and that of their peers. Inferential statistics were used to validate the effects of education. Length of hospital stay for patients receiving total hip replacement dropped from 13.7 days to 9.9 days. Adjusted total charges dropped from $22,103 to $18,607. The variance for length of stay and total charges dropped by one half or more. Physician education programs are effective in reducing consumption of hospital resources. Statistical process control data formatting appears to make the data more usable to these physicians.     

Lymphocyte proliferative responses of goats vaccinated with Brucella melitensis 16M or a delta purE201 strain

The response to a Brucella melitensis purEK deletion mutant, delta purE201 (referred to as strain 201), was compared with the response to its parental strain, 16M, in juvenile goats. Proliferative responses to gamma-irradiated bacteria were detected earlier in strain 201-infected goats. Lymphocytes from strain 16M- or 201-infected goats proliferated in response to one-dimensional polyacrylamide gel electrophoresis-separated proteins of similar mass isolated from strain 16M or Brucella abortus RB51. Data from this study suggest that some antigens stimulating cell-mediated responses are conserved among Brucella species, as 201- and 16M-infected goats recognized similar proteins expressed by RB51 and 16M.     

A study of immunological responses of sheep clinically-affected with paratuberculosis (Johne's disease). The relationship of blood, mesenteric lymph node and intestinal lymphocyte responses to gross and microscopic pathology

Nineteen adult sheep diagnosed as having clinical paratuberculosis (Johne's disease) and 16 unaffected controls were examined in this study. Animals were tested for the presence of circulating antibodies of Mycobacterium avium ssp. paratuberculosis (M. a. paratuberculosis) and lymphocytes derived from the blood, mesenteric lymph nodes and intestines were examined for cell-mediated immune (CMI) responses to Johnin pure protein derivative (Johnin-PPD: J-PPD). Bacteriological examinations were carried out on faeces and tissues and any mycobacterial isolates identified as M. a. paratuberculosis (IS900+) or M. avium ssp. silvaticum (M. a. silvaticum) (IS901+) by polymerase chain reaction (PCR). Full necropsy and histopathological studies were performed and diseased animals were categorised on the basis of having a lepromatous or tuberculoid form of intestinal pathology. Unaffected control sheep were generally antibody-negative and demonstrated varying CMI responses to J-PPD. Clinically-affected animals were almost always antibody-positive with variable CMI responses. A correlation was observed between the histological lesion type in the intestine and the cellular immune response. Tuberculoid-type lesions were associated with strong CMI responses in lymphocytes derived from the peripheral blood, mesenteric lymph node and intestine, whereas lepromatous-type lesions were associated with weak CMI responses in all tissues examined.     

The characteristics of the reproductive capacity of hybrids of the bison (Bison bison L.) with the domestic cow (Bos (Bos) primigenius taurus). The reproductive capacity of hybrid females

Reproduction ability of hybrid females received in various variants of absorbent and variable crossing of bison and domestic cow has been studied. Normal development of genitals, oogenesis, display of sexual activity, as well as fertilization have been found in all females, independently of their blood content. Frequent cases of "relative" sterility in some females of the I-III generations have immunological character and are predominantly caused by the mortality of embryos and fetuses at various stages of pregnancy. Their number decreases gradually as blood portion of one of the initial species increases in each successive generation.     

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)     

-Inguinal irradiation versus no lymph node therapy in small vulvar carcinoma with clinically negative lymph nodes (T1, NO-1)-

OBJECTIVE: The objective of this retrospective study was to determine if groin radiation was superior to no therapy in patients with small vulvar cancer with not palpable or not suspicious inguinal lymph nodes (T1, N0-N1). METHODS: From 1974 to 1990, 135 patients with invasive T1, NO-1 vulvar cancer underwent radical vulvectomy with hot knife, groin nodes were left in situ. In 65 patients vulvectomy was followed by inguinofemoral irradiation: 70 patients had none. There were more cases with clitoris carcinoma (p < 0.04) in the group with groin irradiation but no other significant difference in prognostic factors was found. RESULTS: The actuarial 5-year survival was 93.7% with groin irradiation versus 92.4% without lymph node therapy. Inguinal relapses occurred in only 4.6% of cases with groin irradiation versus 10% without lymph node treatment (n.s.). CONCLUSIONS: Radiation therapy to the groin seems to reduce groin relapses in early vulvar cancer.     

Prospective comparison of 18F-FDG PET with conventional imaging modalities (CT, MRI, US) in lymph node staging of head and neck cancer

The aims of this study were to investigate the detection of cervical lymph node metastases of head and neck cancer by positron emission tomographic (PET) imaging with fluorine-18 fluorodeoxyglucose (FDG) and to perform a prospective comparison with computed tomography (CT), magnetic resonance imaging (MRI), sonographic and histopathological findings. Sixty patients with histologically proven squamous cell carcinoma were studied by PET imaging before surgery. Preoperative endoscopy (including biopsy), CT, MRI and sonography of the cervical region were performed in all patients within 2 weeks preceding 18F-FDG whole-body PET. FDG PET images were analysed visually and quantitatively for objective assessment of regional tracer uptake. Histopathology of the resected neck specimens revealed a total of 1284 lymph nodes, 117 of which showed metastatic involvement. Based on histopathological findings, FDG PET correctly identified lymph node metastases with a sensitivity of 90% and a specificity of 94% (P<10(-6)). CT and MRI visualized histologically proven lymph node metastases with a sensitivity of 82% (specificity 85%) and 80% (specificity 79%), respectively (P<10(-6)). Sonography revealed a sensitivity of 72% (P<10(-6)). The comparison of 18F-FDG PET with conventional imaging modalities demonstrated statistically significant correlations (PET vs CT, P = 0.017; PET vs MRI, P = 0.012; PET vs sonography, P = 0.0001). Quantitative analysis of FDG uptake in lymph node metastases using body weight-based standardized uptake values (SUVBW) showed no significant correlation between FDG uptake (3.7+/-2.0) and histological grading of tumour-involved lymph nodes (P = 0.9). Interestingly, benign lymph nodes had increased FDG uptake as a result of inflammatory reactions (SUVBW-range: 2-15.8). This prospective, histopathologically controlled study confirms FDG PET as the procedure with the highest sensitivity and specificity for detecting lymph node metastases of head and neck cancer and has become a routine method in our University Medical Center. Furthermore, the optimal diagnostic modality may be a fusion image showing the increased metabolism of the tumour and the anatomical localization.     

IgA antibody-forming cell responses in the nasal-associated lymphoid tissue of mice vaccinated by intranasal, intravenous and/or subcutaneous administration

Effects of a single intranasal (i.n.), subcutaneous (s.c.) or intravenous (i.v.) vaccination and their combined vaccination of priming and boosting on a primary and a secondary IgA antibody forming cell (AFC) response were examined in the nasal associated lymphoid tissue (NALT), spleen and popliteal lymph nodes (pLNs) of BALB/c mice. Mice were primed with the vaccine prepared from A/Yamagata/120/86 (H1N1) together with a cholera toxin-adjuvant and boosted with the same vaccine 3 weeks later. Three days after boosting, IgA-AFC responses in each lymphoid tissue were measured as an index of the immunological memory that mediates a secondary IgA-AFC response. Single i.n. vaccination induced a greater primary IgA-AFC response in the NALT not only than that in the spleen or pLNs, but also than that induced by single i.v. or s.c. vaccination. The combination of i.n. priming and i.n. boosting afforded a greater anamnestic IgA-AFC response in the NALT not only than that in the spleen or pLNs, but also than that induced by any other combinations of priming and boosting (i.n.-i.v., i.n.-s.c., s.c.-i.n., s.c.-i.v., and s.c.-s.c.). These results showed that i.n. priming induced a greater primary IgA-AFC response in the NALT and simultaneously induced the immunological memory that mediated a greater secondary-type AFC response following i.n. boosting in the NALT.     

Mucosal or targeted lymph node immunization of macaques with a particulate SIVp27 protein elicits virus-specific CTL in the genito-rectal mucosa and draining lymph nodes

The major routes of HIV transmission are through the rectal and cervico-vaginal mucosa. To prevent dissemination of HIV to the regional lymph nodes (LNs), an effective vaccine may need to stimulate CTL in the rectal or genital tract and the draining LNs. We report that mucosal immunization by the recto-oral and vagino-oral route or s.c. immunization targeting the iliac LNs with a particulate SIVp27:Ty-VLP vaccine elicits SIVgag-specific CTL in the regional LNs as well as in the spleen and PBMC. Targeted LN immunization with this vaccine elicited MHC class I-restricted CD8+ CTL responses, and the highest frequency of CTL was found in the iliac LNs. Moreover, SIVgag-specific CTL activity was detected in short term T cell lines established in mononuclear cells eluted from the rectal and cervico-vaginal mucosa. The relative frequency of CTL in short term cell lines prepared from the rectal mucosa (21/113 or 18.6%) was similar to that obtained from the cervico-vaginal mucosa (16/79 or 20.3%). Examination of the relative frequency of CTL to the T cell epitopes residing within SIVp27 showed a higher frequency in iliac LN cells to peptide aa 41-70 than in that to peptide aa 121-150, and this was significant after both recto-oral (chi-squared 6.500, p < 0.02) and vagino-oral (chi-squared = 10.391, p < 0.01) immunization. In contrast, the relative frequency of CTL in PBMC to peptide aa 41-70 (15.5%) was comparable to that elicited by peptide aa 121-150 (17.6%). This study provides novel evidence that mucosal or targeted LN immunization can generate anti-SIV CTL in the rectal and genital mucosa, in the draining LNs, and in the central lymphoid system.     

Human AP endonuclease 1 (HAP1) protein expression in breast cancer correlates with lymph node status and angiogenesis

Human AP endonuclease (HAP1) plays a major role in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA. We used immunohistochemistry to examine the expression of HAP1 in normal breast and in 102 primary breast carcinomas. In normal breast epithelium, HAP1 had a uniformly nuclear localization. However, in lactating glandular epithelium, the expression of HAP1 was predominantly cytoplasmic. In carcinomas, both nuclear and cytoplasmic (44%), cytoplasmic (28%) or nuclear staining (24%) were observed. In four cases (4%), no HAP1 expression was detected. All patterns of expression for HAP1 were demonstrated for ductal carcinomas in situ (DCIS), although comedo-type DCIS were usually accompanied by mostly cytoplasmic staining. Similarly, the HAP1 expression in regions of invasive tumour necrosis was cytoplasmic. Pure nuclear HAP1 expression was significantly correlated with low angiogenesis (P = 0.007) and negative lymph node status (P = 0.001). In contrast, cases with cytoplasmic as well as nuclear staining were associated with poor prognostic factors, such as high angiogenesis (P = 0.03) and node positivity (P = 0.03). The pure nuclear staining may be related to better differentiation, as in normal breast, and hence better prognostic features, and cytoplasmic staining to a more metabolically active phenotype with high protein synthesis, as in lactating breast.