Isolations of Leclercia adecarboxylata from a patient with a chronically inflamed gallbladder and from a patient with sepsis without focus

Leclercia adecarboxylata was isolated from a patient with a chronically inflamed gallbladder, together with Enterococcus sp. The organism was considered clinically significant and was susceptible to all antibiotics tested. Another strain of L. adecarboxylata was cultured from blood, together with Escherichia hermannii and E. faecalis, from a patient with sepsis.     

Identification and Characterization of a Novel, 37-Kilodalton Leishmania donovani antigen for diagnosis of Indian visceral leishmaniasis

The biggest challenge in the serological diagnosis of visceral leishmaniasis (VL) is to find a biomarker with a high specificity. This study was undertaken to identify novel Leishmania donovani antigens to solve the existing problem. The soluble L. donovani promastigote antigen was separated by SDS-PAGE, and a Western blot was probed with pooled sera of five subjects with confirmed VL before (n = 9 pools) and after (n = 9 pools) treatment and at the 6-month follow-up visit (n = 9 pools), healthy controls not from an area of endemicity (n = 9 pools), and healthy controls from an area of endemicity. The antibody response to the identified partially purified antigen was ascertained by an enzyme-linked immunosorbent assay (ELISA) with 70 sera from patients with parasitologically confirmed VL, 48 sera from healthy controls from an area where the disease is not endemic, 60 sera from healthy controls from an area of endemicity, and 42 sera from patients in different disease groups. The eluted protein was subjected to two-dimensional (2D) gel electrophoresis, Western blotted, and probed with sera from patients with confirmed VL and from healthy controls not from an area of endemicity. The antigenic protein was further characterized by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The identified protein (BHUP2) corresponds to a cytochrome c-like synthesis protein of 37 kDa. ELISA results were 94% sensitive, whereas specificities with sera from healthy controls from an area of endemicity, healthy controls not from an area of endemicity, and disease controls were 98%, 100%, and 97%, respectively. The antigen identified via a proteomics-based approach has a strong potential for further development as a diagnostic tool for VL.     

Reaction of antibody with gamma-glutamyl transpeptidase. I. Isolation of the human enzyme and inhibition of its activity by antiserum.

Use of a four-step method of purification from human kidneys yielded gamma-glutamyl transpeptidase with specific activity 330 times higher than activity in the homogenate. The enzyme from human liver was also purified, and from urine two fractions of it were separated. From rabbits immunized with the enzyme from human kidney, antiserum was obtained which inhibits and precipitates the human-derived enzyme, and in a low degree the enzyme purified from bovine kidneys. This inhibition was independent of incubation time and precipitate formation, but dependent on the amount of antiserum used. A gamma-glutamyl transpeptidase complex with antibody was isolated on a column with CM-cellulose with electrophoretic mobility different from that of the native enzyme.     

Fatal japanese encephalitis virus infection in immunosuppressed spider monkeys

Intracerebral inoculation of twelve spider monkeys with large doses of a virulent strain of Japanese encephalitis virus produced a subclinical encephalomyelitis. When an immunosuppressive dosage schedule of cyclophosphamide was given to a group of four monkeys concurrently with virus, all animals developed prostrating paralysis 12–14 days after infection. Virus was isolated more regularly from the blood and throat swabs of animals treated with cyclophosphamide than from controls. Monkeys inoculated with virus only developed serum antibody, but no antibody was detected in suppressed animals. At time of killing, all spinal cords from suppressed monkeys yielded virus and presented a histological picture resembling that of fatal poliomyelitis, but with a markedly reduced inflammatory response. Virus was isolated from only one of three cords from animals inoculated with virus alone, and histological examination indicated less severe neuronal destruction.     

Histamine modulates mast cell degranulation through an indirect mechanism in a model IgE-mediated reaction

Histamine is released in inflammatory reactions and exerts an immunoregulatory function on cells present in the microenvironment. In this study, we compared the effect of histamine on degranulation of mast cells derived from animals bearing a parasitic infection with those from uninfected animals. Peritoneal mast cells (PMC) were obtained 24 days after infection of Wistar rats with Toxocara canis. The degree of degranulation was assessed either morphologically or by measuring the release of beta-hexosaminidase and TNF-alpha. Non-purified PMC or mast cells immunomagnetically purified with mAb AA4 were used. An increase in degranulation of non-purified mast cells from infected animals was observed after incubation with histamine in vitro or when histamine was injected into the peritoneal cavity. When a purified mast cell population was used, this effect was no longer observed. Supernatants from spleen cells stimulated with histamine induced degranulation of purified mast cells, and again, this was potentiated with PMC from infected animals. However, when supernatants from peritoneal macrophages similarly stimulated were used, a reduction in the degranulation of PMC from infected animals was observed. Our results suggest that histamine may act as a regulator of mast cell degranulation, thus modulating inflammatory responses due to infection with certain parasites.     

Detection of antibodies against the core protein p24 of the bovine leukaemia virus in cattle for confirmatory serological testing

An electrophoretic immunoblotting technique which was developed recently was evaluated for the identification of serum antibodies against the bovine leukaemia virus core protein p24 by using 167 sera from a bovine leukaemia virus-negative herd, and 144 sera from herds naturally infected with the virus. The sensitivity of the immunoblot was 97.4%, relative to sera which were positive in the polymerase chain reaction and in a commercial EBL-ELISA. The specificity of the immunoblot was 99.4%, for the sera from a cattle herd in which all animals were negative by a commercial EBL-ELISA, and it was 96.7% relative to sera which were negative by the polymerase chain reaction and by the agar gel immunodiffusion test from bovine leukaemia virus-infected cattle herds. A p24-specific ELISA was developed, using a monoclonal anti-p24 antibody for coating microtitre plates, a crude antigen preparation, and a monoclonal anti-bovine IgG-horse radish peroxidase conjugate as components. All reagents were commercially available. While the p24-ELISA worked well with sera from serial bleeds from calves infected experimentally with the bovine leukaemia virus and its sensitivity with sera from the naturally-infected cattle was 96.5%, its specificity was relatively low at 85.0 or 53.3%, respectively for the two negative sera groups.     

Thyroid blocking antibodies in thyroiditis

Serum from a woman with a history of Hashimoto's thyroiditis, who had given birth to two children with congenital hypothyroidism, contained potent TSH blocking activity. Immunoglobulin preparation from this serum abolished completely TSH-stimulated cAMP production in human thyroid membranes. The blocking activity was associated with the IgG fraction absorbed to and eluted from a Protein A column. The stimulation of adenylate cyclase by a preparation of thyroid-stimulating antibodies from a patient with Graves' disease was also inhibited by the antibodies. In contrast, no effect was observed upon fluoride-stimulated cAMP production. The data indicate that the antibody activity was directed against the TSH receptor. Immunoglobulin preparations from 22 other patients with Hashimoto's thyroiditis and 16 patients with subacute thyroiditis were examined for the existence of TSH receptor blocking antibodies. A blocking activity was found in two of the 22 Hashimoto patients. No such activity was found in the patients with subacute thyroiditis. It appears that thyroid blocking antibodies sometimes contribute to hypothyroidism associated with Hashimoto's thyroiditis.     

The mechanism of basophil histamine release in patients with periodontal disease.

Histamine release from washed peripheral blood basophils of thirty-three subjects with varying degrees of periodontal disease was studied. Dental plaque, serum and basophil leucocytes were collected from individual patients. There was no histamine release when autologous, washed sonicated plaque was added to leucocytes. However, the incubation of autologous plaque with serum at 37 degrees C for 30 min generated a factor which induced histamine release from basophils. This serum factor was stable to heat (56 degrees C, 30 min), eluted from a Sephadex G-100 column at a volume corresponding to a molecular weight of approximately 16,000 daltons and its action was inhibited by antibody to C5. This factor, therefore, is probably C5a. There was a variation in the degree of histamine release seen with the leucocytes of different donors. This variability was a property of the basophil rather than a function of the serum. Basophils from patients with gingival indices of 0.5 to 1.0 had significantly more histamine release than basophils from patients with gingival indices of less than 0.5 or greater than 1.5 (P less than 0.001). These experiments demonstrate that dental plaque activates serum to form C5a which in turn releases histamine from basophils. However, these experiments do not indicate a role for IgE in this reaction since the direct interaction of plaque with basophils did not cause histamine release. The release of mediators from mast cells could play an important role in the induction of the inflammatory response in periodontal disease.     

Effect of a serum factor on IgE-mediated histamine release from whole blood

IgE-mediated histamine release from whole blood was analyzed in 44 patients with bronchial asthma by observing maximum present release and dose-response curves of histamine release induced by anti-IgE and house dust extract. The maximum histamine release from whole blood induced by anti-IgE correlated with total serum IgE levels. There was a close correlation between allergen-induced release from whole blood and the serum levels of specific IgE antibodies. In the maximum histamine release from whole blood induced by both anti-IgE and allergen, the interaction with a serum factor was not clearly recognized. Effect of a serum factor was shown in the dose-response curves of anti-IgE-induced histamine release, but not in those of allergen-induced histamine release. The dose-response curves caused by anti-IgE showed that basophils from cases with a high serum IgE level require much more anti-IgE to produce maximum histamine release than basophils from cases with a low serum IgE level. The results showed that IgE molecules contained in the serum participate in anti-IgE-induced histamine release from whole blood.     

Detection of antibodies to Mycobacterium tuberculosis plasma membrane antigen by enzyme-linked immunosorbent assay

Antibody activity against Mycobacterium tuberculosis of sera from an area with a high prevalence of tuberculosis was measured by enzyme-linked immunosorbent assay (ELISA) with a plasma-membrane extract from M. tuberculosis strain H37RV. All sera from relapsed tuberculosis patients and 82.5% of sera from new untreated cases gave positive results. The seronegative group of tuberculosis patients gave positive results by direct microscopy and culture. No clear correlation between antibody and delayed hypersensitivity or extent of disease was observed. Chemotherapy was associated with a higher antibody response. Specificity of the test with healthy control subjects from the high prevalence area was 85%. Negative results were obtained with 145 sera from presumed healthy European subjects and with seven sera from BCG-vaccinated subjects.     

Occurrence of mycoplasmas in urinary tracts of patients with acute pyelonephritis

Mycoplasma hominis was isolated from the upper urinary tract in 7 of 80 patients with acute pyelonephritis and from 0 of 60 patients with noninfectious diseases of the urinary tract, a significant difference. In four cases M. hominis was isolated in pure culture, in one it was isolated together with Ureaplasma urealyticum, and in two it was isolated with bacteria. U. urealyticum was isolated from the upper urinary tract of five patients, all with acute pyelonephritis; this was not significantly different from the control group.     

A comparison of the adsorption of three adhesive proteins to biomaterial surfaces.

The adsorption of three cell adhesive proteins with known thrombogenic activity [fibrinogen (FGN), fibronectin (FN), and vitronectin (VN)] was quantified from mono-component protein solutions, from a quaternary-component protein solution, and from plasma and diluted plasma in order to compare their potential for adsorption to polymeric substrates from solutions of varying complexity. The surfaces studied included polyethylene (PE), silicone rubber (SR), Teflon-FEP (FEP), and two polyetherurethanes: one with a poly(tetramethylene oxide) soft segment (PTMO-PU) and one with a poly(ethylene oxide) soft segment (PEO-PU). The adsorption of these proteins from single-component solutions followed the Freundlich isotherm and the adhesive proteins showed similar trends in Freundlich parameters for surfaces of similar surface wettability. Adsorption from a quaternary-component solution composed of physiological molar ratios of the three proteins and human serum albumin (HSA) revealed a significant enrichment of adsorbed vitronectin as determined from ratios of the adsorbed surface fraction of each protein to its respective bulk fraction. The other proteins' adsorption was enriched to a lesser extent in the decreasing order of FGN greater than FN greater than HSA for all surfaces. The relative enrichment of VN from plasma was also high as compared with its bulk concentration, whereas the enrichment of FGN, FN, and HSA was much lower and of approximately the same magnitude. Compared with the three other proteins, VN showed a resistance to displacement from the polymer substrates as either the plasma concentration was increased or the length of contact with plasma and diluted plasma was increased.     

Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils

NAD(P)H oxidase activity was determined in particulate fractions from human neutrophils by measuring the production of hydrogen peroxide. Activity was measured over a wide range of substrate concentrations from 0.0 to 4.0 mM. The activity with NADPH was consistently greater than with NADH. Activity towards both substrates was higher in a particulate fraction derived from cells which had phagocytized opsonized zymosan than in a corresponding fraction from resting cells. This increased activity was apparently due to a decreasedK _m of the enzyme, although no evidence of allosteric kinetics was obtained. The activity was markedly reduced in the presence of superoxide dismutase, indicating the involvement of a superoxide-mediated chain reaction. Particulate fractions derived from cells of a patient with chronic granulomatous disease exhibited decreased activity towards both substrates and an apparent defect in the activation of the enzyme by phagocytosis.     

Demonstration of two disease specific antigens in circulating immune complexes.

A method is described to assess antigenic cross-reactivity between soluble immune complexes precipitated from sera with polyethylene glycol. The precipitated complex from one serum was dissociated in acid and used to coat a plastic cup. Radioiodinated complexes from another serum were dissociated in the cup, neutralized and allowed to reassociate overnight. The binding of the labelled complex was used to measure the cross-reactivity between the complexes. Using this technique, complexes from a group of patients with haematuria and hypertension have been found to share an antigen, and a different antigen was found in patients with bullous pemphigoid. The participation of rheumatoid factors in the cross-reactions is unlikely, and no cross-reactivity of either group was found with sera from patients with rheumatoid arthritis.     

A new serogroup (L) of Neisseria meningitidis

A strain of neisseria meningitidis (LCDC 78189) isolated from the mother of a 3-year-old male with meningococcal meningitis was found to be antigenically distinct from the known serogroups A, B, C, D, H, I, K, X, Y, Z, 29E, and W135; it was designated serogroup L. Anti-78189 serum specifically agglutinated the homologous strain and three other strains which were isolated from the father and two other contacts of the child. Only those strains isolated from the contacts produced immunoprecipitates with the anti-78189 serum by the antiserum-agar method. A structurally unique capsular polysaccharide which was obtained from strain 78189 in a highly purified state was demonstrated to be the antigen responsible for the serological properties of the strain. The polysaccharide formed a precipitin band with the anti-78189 serum but not with the meningococcal grouping sera, and it was also able to absorb both the agglutinating and precipitating activity from the anti-78189 serum.     

Passive hemagglutination test for enteric fever.

A passive hemagglutination (PHA) test for serodiagnosis of enteric fever was developed by sensitizing glutaraldehyde-preserved erythrocytes with lipopolysaccharide from Salmonella serogroups A, B, C, and D singly or simultaneously. The lipopolysaccharide-sensitized erythrocytes were tested with sera from 200 blood donors, 100 patients whose hemoculture was positive for Salmonella species, and 10 patients septicemic for other members of the family Enterobacteriaceae. The PHA test was positive in 90% of 28 acute-phase serum samples from patients with enteric fever from one hospital and in 93% of 72 acute-phase serum samples from another hospital. It was also positive in 100 and 60% of early- and late-convalescent-phase sera, respectively. The PHA test was negative in all patients septicemic for other members of the Enterobacteriaceae. Absorption of sera from patients with enteric fever with lipopolysaccharide from other members of the Enterobacteriaceae did not reduce PHA titers, indicating the specificity of the PHA test. Simultaneous sensitization with lipopolysaccharide from Salmonella serogroups A, B, C, and D was useful as a screening test in a limited trial with 28 acute-phase sera, 10 early-convalescent-phase sera, and 17 late-convalescent-phase sera. The PHA test is indeed a simple, sensitive, specific, and rapid test supplementing hemoculture in laboratory diagnosis of enteric fever.     

Identification of Bacteroides species from adult periodontal disease

Samples from deep (4-7 mm) periodontal pockets were collected from 17 patients with adult-type periodontal disease and one with the juvenile form of the disease. They were streaked immediately on selective and non-selective media and incubated anaerobically for 96 h. There was a heavy growth of Bacteroides spp. from most samples and 10 representative colonies from each sample were sub-cultured for identification. In a total of 149 isolates from patients with adult-type disease, the commonest species were B. oralis (40), B. asaccharolyticus (35), B. intermedius (31), B. fragilis (12) and B. ureolyticus (10); B. gingivalis was not detected. The distribution of species was not distorted by multiple identical isolates from individual patients. There was a heavy growth of a single species, B. ureolyticus, from the patient with juvenile-type disease.     

Tumor necrosis factor and macrophage activation are important in clearance of Nocardia brasiliensis from the livers and spleens of mice.

The roles of tumor necrosis factor (TNF) and macrophage activation in clearance of Nocardia brasiliensis from BALB/c mouse livers and spleens were evaluated. TNF activity was detectable in sera from animals at all stages of infection. Treatment of infected mice with an antiserum against TNF significantly enhanced the experimental infection as judged by enumeration of CFU in the spleens and livers of infected mice. In another set of experiments, a population of activated macrophages from the peritoneal cavities of N. brasiliensis-infected mice was studied by using a cytostatic assay. The observed cytotoxic activity of these activated macrophages against L929 cells was mediated by TNF, since this activity was inhibited by anti-TNF antiserum treatment. The level of TNF activity generated in vitro in the presence of lipopolysaccharide (LPS) by peritoneal macrophages from infected mice was higher than that of adherent peritoneal cells obtained from normal mice after challenge with LPS. When the nocardiacidal activity of peritoneal cells from N. brasiliensis-infected mice was estimated in vitro, a significant decrease in the number of CFU recovered was observed. Moreover, nocardiacidal activity of peritoneal cells obtained from N. brasiliensis-infected mice previously treated with anti-TNF antiserum was significantly reduced compared with the activity of cells obtained from infected mice previously treated with normal rabbit serum and that of cells from uninfected mice. These data suggest a role for TNF in resistance to N. brasiliensis infection.     

The induction of cell-associated and secreted IL-1 by iscoms, matrix or micelles in murine splenic cells.

The kinetics of the expression of membrane-associated IL-1 (mIL-1) and soluble IL-1 (sIL-1) was studied in in vitro stimulated spleen cells from non-primed mice or from mice primed with influenza virus antigens incorporated in the immuno-stimulating complexes (iscoms) or as micelles. Matrix, which is the carrier structure for the antigens in the iscom, was used as a non-antigen stimulus. The IL-1 produced was assayed in an IL-1-dependent cell line and the specificity was demonstrated in a blocking experiment with antiserum to IL-1 alpha. Soluble IL-1 alpha was also quantified in ELISA. Iscoms and matrix induced production of mIL-1 and sIL-1 in cultures from non-treated mice as well as from mice primed 4 days before with iscoms or micelles. Micelles were a less strong stimulus and did not induce production of sIL-1. Micelles induced production of mIL-1 in cultures from non-primed mice or from mice which were recently immunized with micelles. No mIL-1 expression was induced by micelles if the spleen cells originated from mice immunized shortly before with iscoms. Depletion experiments demonstrated that sIL-1 was produced by adherent cells upon stimulation with iscoms or matrix. However, factor(s) from the non-adherent cells seem to be necessary for optimal secretion of sIL-1.