Enhancement of Bacteroides intermedius growth by Fusobacterium necrophorum.

Previous work from this laboratory has demonstrated the persistence of Bacteroides intermedius in the livers of mice receiving an intraperitoneal inoculum of B. intermedius and Fusobacterium necrophorum. This study was undertaken to determine whether F. necrophorum enhanced the in vitro growth of B. intermedius. Tryptose phosphate broth did not support the growth of B. intermedius alone, but the bacterium did survive in a tryptose phosphate broth culture of F. necrophorum. B. intermedius cultured in F. necrophorum-conditioned tryptose phosphate broth grew impressively, reaching maximal absorbance at 24 h after inoculation. The growth of B. intermedius in F. necrophorum-conditioned tryptose phosphate broth was proportional to the amount of conditioned medium present. The B. intermedius growth-stimulating factor was detectable in conditioned medium 8 h after inoculation with F. necrophorum and could be detected throughout the 96-h incubation period. Growth-factor-active fractions eluted from a Sephadex G-100 column did not absorb at 280 nm and were retained on the column until 4 column volumes were eluted. The growth factor was nondialyzable and stable to boiling, lyophilization, extraction with hot aqueous phenol, and trypsin digestion. The factor was inactivated by exposure to pH 2.0 in the pepsin digestion protocol. Significant amounts of hexose, methyl pentose, and 2-keto-3-deoxyoctonate were detected in pooled growth-factor-active fractions eluted from the Sephadex column. This pool was also active in the Limulus lysate endotoxin assay. These results suggest that the B. intermedius growth-stimulating factor produced by F. necrophorum is a lipopolysaccharide.     

Antigen provocation to the skin, nose and lung, in children with asthma; immediate and dual hypersensitivity reactions.

Most (18/21) children with perennial asthma gave dual (immediate and late) responses to bronchial provocation with two of Dermatophagoides pteronyssinus, and either timothy grass pollen or cat fur. Most (19/21) also showed dual responses to skin prick tests, only half (11/21) gave dual responses in the nose, mainly with timothy grass pollen, and these were associated with allergic rhinitis. Only two children gave dual responses in lung, skin and nose to both antigens, and only two gave immediate reactions without late reactions in all positive tests; most showed different patterns of response according to the organ tested or the antigen used for provocation. Our results suggest that local factors may be important in determining the pattern of allergic response in a 'target' organ, and that dual responses are strongly associated with the patient's symptoms.     

Isolation of a variant of Candida albicans.

During the course of Candida albicans antigen production, a variant of this organism was encountered which did not produce hyphae at 37 degrees C. Presented here are some of the characteristics of this variant. It produces hyphae at 25 degrees C on cornmeal agar and synthetic medium plus N-acetylglucosamine and Tween 80. At 37 degrees C, it does not produce hyphae on these media, although C. albicans normally does produce hyphae under these circumstances. In liquid synthetic medium, this variant does not produce hyphae at 37 degrees C. The variant strain was analyzed for DNA, RNA, protein content, and particle size. After 50 to 70 h in balanced exponential-phase growth, particle size distribution was narrow, and there were no differences in the DNA, RNA, or protein content per particle in the two strains. When balanced exponential-phase cultures were brought into stationary phase, both strains contained the same amount of DNA per cell.     

Molecular identification of pathogenetic IdLNF+1 autoantibody idiotypes derived from the NZBxSWR F1 model for systemic lupus erythematosus

The acceleration of nephritis in SNF(1) mice by CD4(+) T-cell clones reactive with a nephritogenic idiotype, Id(LN)F(1) [1], as well as the ability of anti-Id(LN)F(1) antisera to down-regulate the production of Id(LN)F(+)(1) immunoglobulin (Ig) in vivo and delay nephritis [2], suggests that dysregulation of this idiotype may contribute to the development of SNF(1) nephritis. Herein, we show that a monoclonal Id(LN)F(1)-expressing antibody, 540, significantly (P< or = 0.01) stimulated Id(LN)F(1)-reactive T-cell clones B6 and D2 to proliferate, while other Id(LN)F+1 antibodies did not. Further, injection of 540-producing hybridoma cells into nonautoimmune (SWRxBalb/c)F(1) mice resulted in the deposition of Id(LN)F(+)(1) Ig in the kidneys, in a pattern indicative of early nephritis. To identify the pathogenetic Id(LN)F(1) epitope(s) at the molecular level, we compared the deduced amino acid sequences of the heavy and light chain variable regions of pathogenetic and non-pathogenetic Id(LN)F(1)-expressing Igs 540, 317, and 533. Two overlapping peptides derived from the V(H) sequence of 540 (aa 54-66 and 62-73), which both contain the triple basic amino acid motif K(X)K(X)K, stimulated SNF(1) T cells and T-cell clones B6 and D2. These results further support the involvement of a subset of Id(LN)F(1)-expressing Ig in SNF(1) nephritis.     

Rickettsia-macrophage interactions: host cell responses to Rickettsia akari and Rickettsia typhi

The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-beta and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-gamma), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari- and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.     

Molecular characterization of the Clostridium difficile toxin A gene

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.     

Who should give lifestyle advice in general practice and what factors influence attendance at health promotion clinics? Survey of patients' views.

BACKGROUND: Health promotion activity in general practice has increased greatly since 1990. A large proportion of this work is undertaken by practice nurses. Little is known about patients' views about the providers of health promotion or their views about general practice health promotion clinics. AIM: A study was carried out in 1992 to determine patients' views about the provision of health promotion advice by general practitioners and practice nurses and their views about attending health promotion clinics. METHOD: A postal questionnaire was sent to a random sample of 1750 patients aged 16 years and over from five general practices in south Tyneside. The questionnaire explored patients' preferences regarding health promotion advice from the general practitioner or practice nurse in relation to four areas of lifestyle advice and factors that might encourage patients to attend a health promotion clinic. RESULTS: A response rate of 75% was obtained from 1639 eligible patients. Receiving health promotion advice from either the general practitioner or the practice nurse was the most commonly preferred option expressed by patients overall. The ability of health promotion clinic staff to deal with patients' concerns about their illness and short waiting times were more likely to influence patients' attendance at health promotion clinics than the presence of a general practitioner or practice nurse. CONCLUSION: In the present study, many patients found health advice received from practice nurses and general practitioners equally acceptable. However, it was the ability of health professionals to respond to patients' health concerns in the health promotion clinic rather than the type of health professional running the clinic that was important for patients.     

Responses against complex antigens in various models of CD4 T-cell deficiency

The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antibody (Ab), CD4 knockout (CD4^−/−) and major histocompatibility complex (MHC) class II knockout (class II^−/−) mice. We recently described the anti-CD4 Ab transgenic mouse (GK) as an improved CD4 cell-deficient model. This review compares this new GK mouse model with the widely available class II^−/− and CD4^−/− mice, when exposed to complex antigens (foreign grafts and during bacterial or viral infection). We highlight here the cytometric and functional differences (including Ab isotype, viral or bacterial clearance, and graft survival) among these CD4 cell-deficient models. For example, whereas grafts are generally rejected in class II^−/− and CD4^−/− mice as quickly as in wild-type mice, they survive longer in GK mice. Also, CD4^−/− mice produce IgG against both simple model and complex antigens, but class II^−/− and GK mice produce small amounts of IgG2a against complex antigens but not simple model antigens. These differences harbinger the caveats in the use of these various mice.