Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo.

In a series of in vivo and in vitro experiments, the concept has been established that breast cancer cells that express LH/CG or LHRH receptors can be targeted and destroyed by constructs consisting of a lytic peptide moiety and a 15-amino acid segment of the beta-chain of CG or by an LHRH lytic peptide conjugate. Data obtained in vitro established the validity of this concept, showed the specificities of the Hecate-betaCG, and Phor14 and Phor21-betaCG conjugates in killing cells that express functional LH/CG receptors and proved that the LH/CG receptor capacity is directly related to the compound's specificity. In in vivo experiments, Hecate-betaCG, Phor14-betaCG, and Phor21-betaCG(ala) each caused highly significant reductions of tumor volume and tumor burden in nude mice bearing breast cancer xenografts; Hecate and Phor21 alone or conjugated with non-specific peptides were not effective. Most importantly, the lytic peptide conjugates were all highly effective in targeting and destroying disseminated breast cancer metastases in lymph nodes, bones, lungs and other organs.

Conjugates of lytic peptides and LHRH or betaCG target and cause necrosis of prostate cancers and metastases.

In a series of in vivo and in vitro experiments, it was shown that membrane disrupting lytic peptides (Hecate, Phor14, or Phor21) conjugated to a 15 amino acid segment of the beta chain of CG or to LHRH were able to target and destroy hormone dependent and independent human prostate cancer xenografts in nude mice. In vitro sensitivity of the cells to the drugs was directly related to LH/CG receptor expression, and pretreatment in vitro or in vivo with estrogens or FSH to enhance LH/CG receptor expression capacity and increased sensitivity to the drugs. Administration of unconjugated Hecate and LHRH was ineffective. Most importantly, all of the lytic peptide-betaCG conjugates tested were highly effective in destroying prostate cancer metastatic cells in lymph nodes, bones and lungs.

Spiroplasma spp. from transmissible spongiform encephalopathy brains or ticks induce spongiform encephalopathy in ruminants.

Spiroplasma, small motile wall-less bacteria, are linked by molecular and serological studies to the transmissible spongiform encephalopathies (TSEs), which include scrapie in sheep, chronic wasting disease (CWD) in deer and Creutzfeldt-Jakob disease in humans. In this study, two experiments were undertaken to determine the role of spiroplasma in the pathogenesis of TSE. In experiment 1, Spiroplasma mirum, a rabbit tick isolate that had previously been shown to experimentally induce spongiform encephalopathy in rodents, was inoculated intracranially (IC) into ruminants. S. mirum-inoculated deer manifested clinical signs of TSE after 1.5 to 5.5 months incubation. The deer, as well as sheep and goats, inoculated with S. mirum developed spongiform encephalopathy in a dose-dependent manner. In experiment 2, spiroplasma closely related to S. mirum were isolated from TSE-affected brains via passage in embryonated eggs, and propagated in cell-free M1D media. Spiroplasma spp. isolates from scrapie-affected sheep brain and from CWD-affected deer brain inoculated IC into sheep and goats induced spongiform encephalopathy closely resembling natural TSE in these animals. These data show spiroplasma to be consistently associated with TSE, and able experimentally to cause TSE in ruminant animal models, therein questioning the validity of studies that have concluded the prion, a miss-folded protease-resistant protein that builds up in TSE brains during the course of the disease, to be the sole causal agent. The spiroplasma infection models reported here will be important for investigating factors involved in the pathogenesis of TSE since ruminants are the natural hosts.

Brucella abortus strain RB51 vaccination in elk. I. Efficacy of reduced dosage.

Bovine brucellosis is a serious zoonotic disease affecting some populations of Rocky Mountain elk (Cervus elaphus nelsoni) and bison (Bison bison) in the Greater Yellowstone Area, USA. The fear that elk and/or bison may spread Brucella abortus to livestock has prompted efforts to reduce or eliminate the disease in wildlife. Brucella abortus strain RB51 (RB51) vaccine has recently been approved for use in cattle. Unlike strain 19 vaccine, RB51 does not cause false positive reactions on standard brucellosis serologic tests. If effective, it may become the vaccine of choice for wildlife. In February 1995, 45 serologically negative female elk calves were trapped and taken to the Sybille Wildlife Research and Conservation Education Unit near Wheatland, Wyoming, USA. In May 1995, 16 of these elk calves were hand-vaccinated with 1 x 10(9) colony forming units (CFU) of RB51, 16 were vaccinated with 1 x 10(8) CFU RB51 by biobullet, and 13 were given a saline placebo. The elk were bred in fall of 1996 and they were challenged with 1 x 10(7) CFU of B. abortus strain 2308 by intraconjunctival inoculation in March 1997. Thirteen (100%) control elk aborted, 14 (88%) hand-vaccinated elk aborted, and 12 (75%) biobullet vaccinated elk aborted or produced nonviable calves. These results suggest that a single dose of 1 x 10(8) to 1 x 10(9) CFU RB51 does not provide significant protection against B. abortus induced abortion in elk. However, the vaccine appears to be safe at this dose and additional study may reveal a more effective RB51 vaccine regimen for elk.

Resistant starch, fermented resistant starch, and short-chain fatty acids reduce intestinal fat deposition in Caenorhabditis elegans.

Obesity is a growing global public health dilemma. The objective of this project is to develop and validate a screening mechanism for bioactive compounds that may reduce body fat and promote health. Resistant starch (RS) reduces body fat in rodents. Amylose starch that has a high content of RS, endogenous compounds obtained from the ceca of amylose starch fed mice (fermented RS), and individual short-chain fatty acids (SCFA) were tested. The Caenorhabditis elegans model and Nile red staining were selected to determine the intestinal fat deposition response to bioactive components. The fluorescence intensity of Nile red was reduced to 76.5% (amylose starch), 78.8% (fermented RS), 63.6% (butyrate), or 28-80% (SCFAs) of controls, respectively (P < 0.001). The reduced intestinal fat deposition suggests reduced food intake or increased energy expenditure. C. elegans is a practical animal model to screen for bioactive compounds that may prevent or treat obesity.