Troy Auffenberg

A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor α (TNFα) and abrogates endotoxin-induced lethality

Excessive tumor necrosis factor α (TNFα) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNFα production rely on monoclonal antibodies or immunoadhesins that bind TNFα and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNFα processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 μg/mL endotoxin, GM-6001 at concentrations <5 μg/mL blocked release of TNFα, but did not affect the release of either IL-1β or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNFα. To confirm the role of secreted TNFα in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 μg/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNFα response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1β and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNFα processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNFα both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

A role for tumor necrosis factor-alpha in the increased mortality associated with Vibrio vulnificus infection in the presence of hepatic dysfunction.

OBJECTIVE The present study was designed to evaluate whether pre-existing hepatic dysfunction (cirrhosis) leads to increased morbidity and mortality, in part through an inappropriate in vivo tumor necrosis factor-alpha response. SUMMARY BACKGROUND DATA Vibrio vulnificus is the most commonly isolated member of the noncholera Vibrio sp., responsible for fulminant and frequently fatal septicemia. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from Vibrio sp. infection. However, the underlying mechanism behind this association has not been fully delineated. METHODS Cirrhosis was induced in C57BL/6 (15 to 20 g) mice using thrice-weekly injections of carbon tetrachloride (CCl4) for 7 weeks. Either a 7.0 to 9.5 X 10(7) (low dose) or a 0.8 to 1.2 X 10(9) colony-forming unit (high dose) of V. vulnificus was administered through a mini-laparotomy incision via transgastric puncture into both cirrhotic and control animals. RESULTS Mortality in cirrhotic mice to low- and high-dose Vibrio infection was 88% (7/8) and 100% (8/8), respectively, whereas mortality in control animals was 0% (0/8) and 12% (1/8), respectively (p<0.01). Tumor necrosis factor-alpha mRNA could be detected by reverse transcriptase polymerase chain reaction in livers and lungs from infected animals 2 and 4 hours after Vibrio administration in both control and cirrhotic animals. Lung and liver tumor necrosis factor-alpha bioactivity, however, was significantly lower in cirrhotic animals infected with Vibrio when compared with controls. Serum tumor necrosis factor-alpha was only sporadically detected in both groups of Vibrio-infected animals. When cirrhotic mice challenged with a low dose of Vibrio sp. were pretreated with 1.0 mg/kg body weight of a novel tumor necrosis factor-alpha receptor immunoadhesin, the increased mortality was completely prevented. CONCLUSIONS Cirrhotic mice show increased mortality to Vibrio infection, and this increased mortality is dependent on an in vivo tumor necrosis factor-alpha response.

Leptin produces anorexia and weight loss without inducing an acute phase response or protein wasting

The ob gene product leptin is known to produce anorexia and loss of body fat when chronically administered to both lean and genetically obese mice. The current study was undertaken to examine whether administration of recombinant leptin in quantities sufficient to produce decreases in food intake and body weight and alterations in body composition would elicit either an hepatic acute phase protein response or preferential loss of carcass lean tissue. Mice were administered increasing quantities of recombinant human leptin or human tumor necrosis factor-α as a positive control. Although leptin (at 10 mg/kg body wt) produced significant anorexia and weight loss (both P < 0.05), human leptin administration did not appear to induce an hepatic acute phase protein response in either lean or genetically obese mice, as determined by protein synthetic rates in the liver or changes in the plasma concentration of the murine acute phase protein reactants, amyloid A, amyloid P, or seromucoid (α1-acid glycoprotein). In addition, human leptin administration did not induce a loss of fat-free dry mass (protein) in lean or obese animals. The findings suggest that at doses adequate to alter food intake and body weight leptin is not a significant inducer of the hepatic acute phase response nor does leptin promote the preferential loss of somatic protein characteristic of a chronic inflammatory process.The ob gene product leptin is known to produce anorexia and loss of body fat when chronically administered to both lean and genetically obese mice. The current study was undertaken to examine whether administration of recombinant leptin in quantities sufficient to produce decreases in food intake and body weight and alterations in body composition would elicit either an hepatic acute phase protein response or preferential loss of carcass lean tissue. Mice were administered increasing quantities of recombinant human leptin or human tumor necrosis factor-alpha as a positive control. Although leptin (at 10 mg/kg body wt) produced significant anorexia and weight loss (both P < 0.05), human leptin administration did not appear to induce an hepatic acute phase protein response in either lean or genetically obese mice, as determined by protein synthetic rates in the liver or changes in the plasma concentration of the murine acute phase protein reactants, amyloid A, amyloid P, or seromucoid (alpha1-acid glycoprotein). In addition, human leptin administration did not induce a loss of fat-free dry mass (protein) in lean or obese animals. The findings suggest that at doses adequate to alter food intake and body weight leptin is not a significant inducer of the hepatic acute phase response nor does leptin promote the preferential loss of somatic protein characteristic of a chronic inflammatory process.

Transfection of the Type Ii Tgf-β Receptor Into Colon Cancer Cells Increases Receptor Expression, Inhibits Cell Growth, and Reduces the Malignant Phenotype

OBJECTIVE To determine if transfection of SW48 colon cancer cells with the type II transforming growth factor-beta (TGF-beta) receptor restores growth inhibition and reverses the in vitro and in vivo malignant phenotype. SUMMARY BACKGROUND DATA The authors have previously shown that SW48 colon cancer cells that are replication error positive in both alleles lack functional cell surface TGF-beta type I (RI) and type II (RII) receptors and are insensitive to TGF-beta1-induced growth inhibition. METHODS SW48 cells were stably transfected with the cDNA for the normal type II TGF-beta receptor (RII). Once transfected, the cells were evaluated for in vitro phenotypic changes and in vivo changes in tumor growth. RESULTS Denaturing sequencing gel electrophoresis of the reverse transcriptase-polymerase chain reaction product from SW48 cells revealed that the RII coding sequence contained a single base deletion mutation. When these cells were transfected with normal RII cDNA, Northern and Western blot analyses revealed increased levels of RII mRNA and protein. Affinity labeling techniques revealed that RII-transfected SW48 cells produced functional RI and RII protein. Transfection of SW48 cells also led to changes in cell phenotype, as shown by inhibition of both in vitro growth rate and incorporation of [3H]-thymidine. SW48 cells expressing normal RII also exhibited reduced cloning efficiency in semisolid medium and reduced growth as a xenograft in NOD/LtSz-scid/J mice. CONCLUSIONS The results confirm that RII is a tumor-suppressor protein that is required for TGF-beta-induced growth inhibition in SW48 colon cancer cells.

Inhibition of transforming growth factor alpha stimulation of human squamous cell carcinoma of the head and neck with anti-TGF-α antibodies and tyrphostin

AbstractBackground: Transforming growth factor alpha (TGF-α) and its receptor (EGF-R) may regulate normal and malignant epithelial cell growth by an autocrine mechanism. We investigated the role of TGF-α in regulating head and neck SCC tumor growth. Methods: TGF-α and EGF-R levels were measured in 7 SCC cell lines and 14 SCC biopsies by RIA, Scatchard, and Western analysis. TGF-α autocrine stimulation of DNA synthesis in SCC cell lines was assessed by incubation with TGF-α neutralizing antibodies and tyrphostin AG 1478, a selective and potent inhibitor of EGF-R kinase. Results: All SCC cell lines synthesized TGF-α and expressed elevated EGF-R levels compared to normal keratinocytes. Twelve of the 14 SCC biopsies contained TGF-α protein and 8 had specific EGF-R. Exogenous TGF-α or EGF significantly increased DNA synthesis in 4 of 5 SCC cell lines. TGF-α neutralizing antibodies or tyrphostin AG 1478 reduced DNA synthesis in the two SCC cell lines (FaDu and SCC9) tested. Conclusions: These results indicate that SCC cell lines and tumors usually synthesize TGF-α, have elevated levels of EGF-R, and are mitogenically stimulated by a TGF-α autocrine system. Selective inhibition of the TGF-α system by EGF-R kinase inhibitors or TGF-α neutralizing antibodies may be useful strategies for treating SCC that overexpress TGF-α and its receptor.

Amblyomma helvolum (Acarina:Ixodidae) as a parasite of varanid and scincid reptiles in the Philippines

Abstract Infestation levels and attachment sites of male and female Amblyomma helvolum on hosts Varanus salvator and V. grayi and on 14 other lizard species, representing the families Scincidae and Agamidae, were recorded for 19 months in the Philippines. The frequency of A. helvolum varies with season of the year, and the highest tick levels occur during the first monsoon in June and July following 4 months with little rain. Both varanids are more frequently infested with A. helvolum than other lizard species. Female ticks attach anteriorly on all hosts, and on both varanid hosts their attachment sites are similar, most commonly between the toes of the front feet and in the axillary area. Male ticks, however, attach to different sites on each varanid host. On V. sulvator male ticks primarily attach on the ventral surface, while on V. grayi they are largely restricted to the base of the claws.