Previous reports indicate that anti-tumor activity arising from systemically injected P. acnes is macrophage-mediated, whereas anti-tumor activity arising from locally injected P. acnes is T cell-mediated. It is possible these P. acnes-induced cytotoxic T cells arise via the Interleukin cascade. Therefore, this study investigated the involvement of Interleukin 2 (IL 2) and Interleukin 3 (IL 3), known components of the Interleukin cascade, in local P. acnes-mediated anti-tumor action. A 500 ug dose of heat-killed stationary phase P. acnes given simultaneously with 10⁴ tumor cells was found to inhibit tumor formation completely, therefore this amount was used as a standard dose throughout the study. Unvaccinated counterparts developed palpable tumors two weeks after tumor cell administration. Lower doses of vaccine protected animals from tumor growth to a lesser degree. A vaccine prepared from logarithmic phase P. acnes exerted a moderate anti-tumor effect in some cases. IL 2 and IL 3 levels were measured in vitro in normal BALB/c mice (N), tumor-bearing mice (TBH), normal vaccinated mice (N+V), and mice receiving both tumor cell and vaccine injection (T+V). IL 2 and IL 3 production was maintained in both N and N+V host splenocyte cultures throughout the study. In a similar fashion, levels of IL 2 and IL 3 in T+V host splenocyte cultures were comparable to those of N+V hosts. However, TBH splenocyte production of IL 2 and IL 3 began to decline when tumors became palpable, at Day 14 after tumor cell inoculation. By Day 28, TBH IL 2 and IL 3 levels were <15% of normal control levels. Causes for this suppression of IL 2 and IL 3 production in TBH were examined. From reports of others it appeared that suppression may be mediated through prostaglandin(s). Addition of the prostaglandin inhibitor indomethacin to splenocyte cultures greatly enhanced IL 2 production by N, N+V and T+V splenocytes, but failed to restore IL 2 production in TBH splenocyte cultures to normal levels. Thus, it appeared prostaglandins were not directly responsible for the majority of suppression seen in TBH. In the non-tumor-burdened host, prostaglandin appeared to play a homeostatic role regarding IL 2 production. Indomethacin-treatment had little effect on IL 3 production. Nylon wool fractionation of N, TBH, N+V and T+V splenocytes suggested a cell removed by nylon wool treatment was largely responsible for the suppression of IL 2 and IL 3 production in TBH. No obvious presence of functional suppressor cells was noted in N, N+V or T+V splenocytes. From these results, it appeared that P. acnes administration maintains and/or restores IL 2 and IL 3 production, thus favoring the production of CTL. In addition, the suppression of IL 2 and IL 3 production seen in TBH may be due to a nylon wool adherent suppressor cell. A model describing the effect of P. acnes administration on local anti-tumor activity was presented. / Master of Science

The fecal flora of healthy bottle or breast-fed infants was examined for the presence of Bifidobacterium. Identification was based on the presence of fructose-6-phosphate phosphoketolase, which is found only in these bacteria. No bifidobacteria were recovered from bottle-fed infants. However, bifidobacteria were readily isolated from 15 day to 3 month old breast-fed infants. Further characterization revealed B. breve and B. longum were the dominant species in feces of breast-fed infants, but atypical strains also were found. A whey-based medium (7% sweet whey, 0.05% cysteine and 0.3% yeast extract, WCY-0.3) was developed to grow Bifidobacterium species without use of anaerobic incubation conditions. Freshly pasteurized WCY-0.3 was inoculated with 0.2% (10⁶ to 10⁷ CFU/ml) of the following active cultures of bifidobacteria: B. bifidum 15696, B. breve 15700, B. longum 15707, B. breve 15698, B. longum L10, B. longum L12, and B. longum 3j. Following incubation for 12 hours, most strains reached cell densities of 10⁹ to 5 x 10⁹ CFU/ml, except B. bifidum 15696 and B. longum 3j. Addition of Oxyrase to the WCY (WC with any level of yeast extract) at 0.03 unit/ml (WCYO) reduced the lag phase of all strains, allowing maximum populations to be reached more quickly. A higher population density (2 to 7 times) could be achieved in the WCOY-0.3 medium with strains 15696, 15700, 15707, and L10 by incorporating 1.9% sodium glycerophosphate or trimagnesium phosphate with incubation for 12 hours at 37°C. Also, viability of these strains was retained throughout a 24-hour incubation period, in contrast to rapid death of cells grown without the neutralizing agents. Inoculation of WCY-0.3 or WCOY-0.3 medium with frozen concentrates (10⁷ to 10⁸ CFU/ml) of bifidobacteria allowed equal growth of all species, except B. bifidum 15696, which grew much better in WCOY-0.3 than in WCY- 0.3. Survival stability of whey-based medium-grown bifidobacteria when resuspended in pasteurized skim milk and refrigerated at 4°C was strain dependent and enhanced by the presence of 0.05% cysteine; generally ATCC strains were more stable than strains freshly isolated from baby feces. In this regard, B. breve 15700, B. longum 15707, and B. breve 15698 did not lose viability in 11% skim milk with 0.05% cysteine within 10 days of storage. Stability of whey-based medium-grown bifidobacteria in WCY with 15% glycerol during six months storage at -40°C was strain dependent. Bifidobacterium bifidum 15696, B. breve 15700, B. longum 15707, B. breve 15698, and B. longum L12 did not lose viability; however B. bifidum L6 lost about 50% viability, while B. longum L10, B. breve T10, and B. breve T2 lost about one log population density. The plasmid profiles of 35 strains of bifidobacteria from human sources were examined. Only one strain, B. breve 15698, harbored a 5.8Kb plasmid. A curing process using UV-light treatment to remove the plasmid was carried out but characterictics of the cured strain were identical to those of the parent strain, indicating the plasmid is cryptic. / Graduation date: 1991

No description available.

No description available.

Includes bibliographical references (leaves 308-327) / xxviii, 356 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 2004?

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 62-68). Also available via the World Wide Web.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Human infections with influenza A virus (IAV) exhibit mild to severe clinical outcomes as a result of differential virus-host interactions. C-type lectin receptors (CLRs) are pattern recognition receptors that may sense carbohydrates, proteins, or lipids derived from infected hosts or the invading microbes including bacteria, viruses, fungi, or parasites. CLR-viral interaction may lead to increased viral entry and spread; furthermore, their interactions have been reported to trigger downstream signaling that further modulates host’s innate immune responses through the induction of pro-inflammatory cytokines. To date, DC-SIGN and DC-SIGNR have been shown to mediate IAV entry; however, the potential interactions between other human transmembrane CLRs with IAV have not yet been systematically investigated. We utilized lentiviral-based pseudoparticles expressing influenza hemagglutinin (HA) to examine the binding potential between HA and a panel of human CLRs expressed in soluble form. CLEC5A was identified as a potential interacting target with the HA proteins derived from a highly pathogenic avian H5N1 virus A/VN/1203/04 (VN1203) or a human seasonal H1N1 virus A/HK/54/98 (HK5498), albeit at different binding intensity. Applying siRNA gene silencing, we confirmed that CLEC5A did not enhance influenza entry in human monocytic U937 cells that constitutively express CLEC5A or in the lentiviral-transduced stable CHO and CHO-Lec2 cells that overly expressed CLEC5A. To investigate downstream signaling upon engagement of CLEC5A to influenza virus, M-CSF or GM-CSF differentiated human macrophages with high expression levels of CLEC5A and DAP12, a known adaptor protein for CLEC5A upon phosphorylation to initiate signal transduction, was subjected to CLEC5A siRNA gene silencing followed by infection with recombinant A/PR/8/34 virus expressing HA and NA derived from either VN1203 (H5N1) or HK5498 (H1N1) viruses. RG-PR8xVN1203HA,NA (H5N1) exhibited a higher infectivity and induced higher levels of pro-inflammatory cytokines (TNF-( and IFN-α) and chemokines (IP-10, MCP-1, MIG and MIP-1α) secretion in M-CSF or GM-CSF differentiated macrophages while compared to that of the RG-PR8xHK5498HA,NA (H1N1) virus. Knocking-down CLEC5A in macrophages led to a universal reduction of cytokines and chemokines secretion after infection with either the RG-PR8xVN1203HA,NA, RG-PR8xHK5498HA,NA, RG-A/VN/1203/04 (H5N1) or A/Shanghai/2/2014 (H7N9) viruses, suggesting that CLEC5A plays a role as cytokine and chemokine amplifier after influenza infection. Since DAP12 phosphorylation is known to activate downstream signaling via Spleen tyrosine kinase (Syk), pre-incubation of M-CSF macrophages with a Syk inhibitor (Bay 61-3606) also lead to a significant reduction of TNF-α and IP-10 in infected macrophages. A higher mortality was observed in CLEC5A-/- mice while compared to the wild-type C57BL/6 mice after challenged with a lethal dose of RG-A/VN/1203/04 (H5N1) influenza virus suggesting that CLEC5A as a host innate response amplifier play a protective role upon influenza infection. In conclusion, we have identified CLEC5A as a novel host factor for influenza pathogenesis by modulating host innate inflammatory response. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

No description available.