Subcutaneous Implants of a Cholesterol-Triglyceride-Buprenorphine Suspension in Rats.

A Target Animal Safety protocol was used to examine adverse events in male and female Fischer F344/NTac rats treated with increasing doses of a subcutaneous implant of a lipid suspension of buprenorphine. A single injection of 0.65 mg/kg afforded clinically significant blood levels of drug for 3 days. Chemistry, hematology, coagulation, and urinalysis values with 2- to 10-fold excess doses of the drug-lipid suspension were within normal limits. Histopathology findings were unremarkable. The skin and underlying tissue surrounding the drug injection were unremarkable. Approximately 25% of a cohort of rats given the excess doses of 1.3, 3.9, and 6.5 mg/kg displayed nausea-related behavior consisting of intermittent and limited excess grooming and self-gnawing. These results confirm the safety of cholesterol-triglyceride carrier systems for subcutaneous drug delivery of buprenorphine in laboratory animals and further demonstrate the utility of lipid-based carriers as scaffolds for subcutaneous, long-acting drug therapy.

Subcutaneous Implants of Buprenorphine-Cholesterol-Triglyceride Powder in Mice.

Subcutaneous drug implants are convenient systems for the long-term delivery of drugs in animals. Lipid carriers are logical tools because they generally allow for higher doses and low toxicity. The present study used an US Food and Drug Administration Target Animal Safety test system to evaluate the safety of a subcutaneous implant of a cholesterol-triglyceride-buprenorphine powder in 120 BALB/c mice. Mice were evaluated in 4- and 12-day trials with 1- and 5-fold doses of the intended 3 mg/kg dose of drug. One male mouse treated with three 3 mg/kg doses and surgery on days 0, 4, and 8 died on day 9. The cause of death was not determined. In the surviving 119 mice there was no evidence of skin reaction at the site of the implant. Compared to control animals treated with saline, weight measurements, clinical pathology, histopathology, and clinical observations were unremarkable. These results demonstrate that the lipid carrier is substantially safe. Cholesterol-triglyceride-drug powders may provide a valuable research tool for studies of analgesic and inflammatory drug implants in veterinary medicine.

Neuroimmune semaphorin 4A as a drug and drug target for asthma.

Neuroimmune semaphorin 4A (Sema4A) has been shown to play an important costimulatory role in T cell activation and regulation of Th1-mediated diseases such as multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), and experimental autoimmune myocarditis (EAM). Sema4A has three functional receptors, Tim-2 expressed on CD4+ T cells, Th2 cells in particular, and Plexin B1 and D1 predominantly expressed on epithelial and endothelial cells, correspondingly. We recently showed that Sema4A has a complex expression pattern in lung tissue in a mouse model of asthma. We and others have shown that corresponding Plexin expression can be found on immune cells as well. Moreover, we demonstrated that Sema4A-deficient mice displayed significantly higher lung local and systemic allergic responses pointing to its critical regulatory role in the disease. To determine the utility of Sema4A as a novel immunotherapeutic, we introduced recombinant Sema4A protein to the allergen-sensitized WT and Sema4A(-/-) mice before allergen challenge. We observed significant reductions in the allergic inflammatory lung response in Sema4A-treated mice as judged by tissue inflammation including eosinophilia and mucus production. Furthermore, we demonstrated that in vivo administration of anti-Tim2 Ab led to a substantial upregulation of allergic inflammation in WT mouse lungs. These data highlight the potential to develop Sema4A as a new therapeutic for allergic airway disease.

Neuroimmune semaphorin 4D is necessary for optimal lung allergic inflammation.

Neuroimmune semaphorin 4D (Sema4D) was found to be expressed and function in the nervous and immune systems. In the immune system, Sema4D is constitutively expressed on T cells and regulates T cell priming. In addition, it displays a stimulatory function on macrophages, DC, NK cells, and neutrophils. As all these cells are deeply involved in asthma pathology, we hypothesized that Sema4D plays a critical non-redundant regulatory role in allergic airway response. To test our hypothesis, we exposed Sema4D(-/-) and WT mice to OVA injections and challenges in the well-defined mouse model of OVA-induced experimental asthma. We observed a significant decrease in eosinophilic airway infiltration in allergen-treated Sema4D(-/-) mice relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL IL-5, IL-13, TGFß1, IL-6, and IL-17A levels. In addition, T cell proliferation in OVA323₋339-restimulated Sema4D(-/-) cell cultures was downregulated. We also found increased Treg numbers in spleens of Sema4D(-/-) mice. However, airway hyperreactivity (AHR) to methacholine challenges was not affected by Sema4D deficiency in either acute or chronic experimental disease setting. Surprisingly, lung DC number and activation were not affected by Sema4D deficiency. These data provide a new insight into Sema4D biology and define Sema4D as an important regulator of Th2-driven lung pathophysiology and as a potential target for a combinatory disease immunotherapy.

Neuroimmune semaphorin 4A downregulates the severity of allergic response.

To define the role of semaphorin 4A (Sema4A) in allergic response, we employed Sema4A⁻/⁻ and wild-type (WT) mice in the experimental model of ovalbumin (OVA)-induced allergic airway inflammation. We observed a selective increase in eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A⁻/⁻ mice relative to WT mice. This enhanced inflammatory response was associated with a selective increase in bronchoalveolar lavage (BAL) interleukin 13 (IL-13) content, augmented airway hyperreactivity, and lower regulatory T cell (Treg) numbers. In vivo allergen-primed Sema4A⁻/⁻ CD4+ T cells were more effective in transferring T helper type 2 (Th2) response to naive mice as compared with WT CD4+ T cells. T-cell proliferation and IL-13 productions in OVA323₋339-restimulated Sema4A⁻/⁻ cell cultures were upregulated. Generated bone marrow chimeras showed an equal importance of both lung-resident cell and inflammatory cell Sema4A expression in optimal disease regulation. These data provide a new insight into Sema4A biology and define Sema4A as an important regulator of Th2-driven lung pathophysiology.

Exposure to febrile temperature upregulates expression of pyrogenic cytokines in endotoxin-challenged mice.

Fever is a phylogenetically ancient response that is associated with improved survival in acute infections. In endothermic animals, fever is induced by a set of pyrogenic cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1, and IL-6] that are also essential for survival in acute infections. We studied the influence of core temperature on cytokine expression using an anesthetized mouse model in which core temperature was adjusted by immersion in water baths. We showed that raising core temperature from basal (36.5-37.5 degrees C) to febrile (39.5-40 degrees C) levels increased peak plasma TNF-alpha and IL-6 levels by 4.1- and 2. 7-fold, respectively, and changed the kinetics of IL-1beta expression in response to lipopolysaccharide challenge. TNF-alpha levels were increased predominantly in liver, IL-1beta levels were higher in lung, and IL-6 levels were widely increased in multiple organs in the warmer mice. This demonstrates that the thermal component of fever may directly contribute to shaping the host response by regulating the timing, magnitude, and tissue distribution of cytokine generation during the acute-phase response.

Febrile-range temperature modifies early systemic tumor necrosis factor alpha expression in mice challenged with bacterial endotoxin.

Fever improves survival in acute infections, but the effects of increased core temperature on host defenses are poorly understood. Tumor necrosis factor alpha (TNF-alpha) is an early activator of host defenses and a major endogenous pyrogen. TNF-alpha expression is essential for survival in bacterial infections but, if disregulated, can cause tissue injury. In this study, we show that passively increasing core temperature in mice from the basal (36.5 to 37.5 degrees C) to the febrile (39.5 to 40 degrees C) range modifies systemic TNF-alpha expression in response to bacterial endotoxin (lipopolysaccharide). The early TNF-alpha secretion rate is enhanced, but the duration of maximal TNF-alpha production is shortened. We identified Kupffer cells as the predominant source of the excess TNF-alpha production in the warmer animals. The enhanced early TNF-alpha production observed at the higher temperature in vivo could not be demonstrated in isolated Kupffer cells or in precision-cut liver slices in vitro, indicating the participation of indirect pathways. Therefore, expression of the endogenous pyrogen TNF-alpha is regulated by increments in core temperature during fever, generating an enhanced early, self-limited TNF-alpha pulse.

Production of enterotoxin by Yersinia bercovieri, a recently identified Yersinia enterocolitica-like species.

Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii) Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

Parenteral gene therapy with p53 inhibits human breast tumors in vivo through a bystander mechanism without evidence of toxicity.

Mutations of the p53 tumor suppressor gene are the most frequently observed genetic lesion in human cancer. Previously, we found that multiple intravenous injections of a liposome:p53 complex inhibited the growth of a malignant human breast cancer cell line that was implanted into nude mice. In the present study, we evaluated the toxicity of the liposome:p53 complex and the mechanism of this in vivo treatment in reducing tumor growth. Intravenously delivered liposome:p53 complex at dosages sufficient to inhibit human breast cancer in nude mice showed no evidence of toxicity. Clinical chemistries, complete blood counts, and histopathologic examination of various organs from the p53-treated groups did not demonstrate any difference from the control groups. To elucidate the mechanism by which the liposome:p53 complex inhibits cancer, the transfection efficiency of a liposome:chloramphenicol acetyltransferase (CAT) complex into the tumor was determined. Interestingly, less than 5% of the tumor was transfected with a liposome:CAT complex. A mechanism that could account for p53 reduction of tumor size and a low transfection efficiency is inhibition of angiogenesis. After one treatment, we found that the liposome:p53 complex reduced the number of blood vessels in the p53-treated group by approximately 60% compared to the control group (p < 0.001). The close correlation between the antitumor effect of p53 and the reduction of blood vessel density in the tumor suggests that p53 effects are mediated, at least in part, by an antiangiogenesis mechanism.

Subdiaphragmatic vagotomy inhibits intra-abdominal interleukin-1 beta stimulation of adrenocorticotropin secretion.

Although interleukin (IL)-1 beta activates the hypothalamic-pituitary-adrenal (HPA) axis, the mechanisms by which peripheral IL-1 beta acutely stimulates adrenocorticotropin (ACTH) secretion are not clear. Recently, the vagus has been implicated in mediating peripheral cytokine signalling of the brain. To investigate a possible central mechanism for peripheral cytokine stimulation of the HPA axis, we tested the hypothesis that the vagus mediates IL-1 beta activation of the HPA axis by an intra-abdominal stimulus. We studied the effect of subdiaphragmatic vagotomy on plasma ACTH stimulation in rats by intraperitoneal (i.p.) IL-1 beta. Adult male Sprague-Dawley rats underwent subdiaphragmatic vagotomy or sham surgery 1 week prior to study. Rats were killed 1 and 2 h after i.p. saline (control) and low- (4 micrograms/kg) and high-dose (20 micrograms/kg) IL-1 beta. Vagotomy markedly attenuated plasma ACTH secretion at 2 h after high-dose IL-1 beta stimulation and abolished plasma ACTH secretion at 2 h after low-dose IL-1 beta stimulation. At 1 h after low-dose IL-1 beta, stimulation of plasma ACTH in vagotomized animals was also markedly diminished compared to sham animals. However, vagotomy did not alter stimulation of plasma corticosterone at 1 or 2 h after low-dose IL-1 beta or at 2 h after high-dose IL-1 beta. In addition, vagotomy did not alter stimulation of plasma ACTH or corticosterone secretion by insulin-induced hypoglycemia. We conclude that: (1) the vagus plays an important role in stimulation of ACTH secretion by intra-abdominal (i.p.) IL-1 beta; (2) stimulation of corticosterone secretion by i.p. IL-1 beta is not altered by vagotomy; and (3) the inhibitory effect of vagotomy on activation of the HPA axis appears to be specific for immune stimulation by cytokines.

Toxicity of the novel animal-derived anticancer agent, VRCTC-310: acute and subchronic studies in beagle dogs.

Acute and subchronic toxicities of VRCTC-310, a combination product of crotoxin (CT) and cardiotoxin (CD), which has shown antitumor activity in vivo, have been studied in Beagle dogs. Single i.m. doses of 0.25, 0.5 and 1.0 mg/kg resulted in dose-dependent local muscular toxicity consisting of myofiber atrophy, interstitial edema and macrophage infiltration. Also, AST, ALT and LDH levels increased on day 2, returning to normal values on days 6-8. Local lesions were absent after recovery on day 45. At 2.0 mg/kg, signs of neurotoxicity (ataxia) appeared, in addition to vomitus, salivation, hematuria and myotoxicity in tongue and diaphragm on day 8. Local lesions healed with fibrosis at the site of injection on day 45. Administration of fixed (0.025 and 0.05 mg/kg) or escalating (0.025-0.1 mg/kg) daily doses for 30 days also produced local muscular damage, which was absent at day 75. The increases in AST, ALT and LDH serum activities on days 2-4 were independent of dosing schedule and sharply decreased on day 8, despite continuation of treatment. An escalating dose schedule of 0.025-2.0 mg/kg showed local muscle damage at the site of injection on day 31, however, there were no lesions of myotoxicity in the tongue or diaphragm and no clinical signs of neurotoxicity were observed. Animals tolerated the subchronic treatment better than the acute. The resolution of serum enzymes to normal values during treatment may be attributed to a decrease of sensitivity to VRCTC-310-mediated myotoxic effects.

Serologic response to Helicobacter pylori among children and teenagers in northern Chile.

The serologic response to Helicobacter pylori was determined in 388 children and teenagers living in Iquique, Chile by using an IgG enzyme-linked immunosorbent assay. Serum antibody levels, as measured by optical density, correlated strongly with age. Increases in the mean antibody level were seen primarily after age five, with rates of seropositivity increasing to > or = 70% among teenagers. The reasons for this age-related pattern of acquisition of infection remain to be determined.

Early colonic damage and invasion of Campylobacter jejuni in experimentally challenged infant Macaca mulatta.

Experimental challenge studies with Campylobacter jejuni were conducted in 3.5-month-old infant Macaca mulatta. One infant monkey (92-1) was challenged with 2.7 x 10(10) cfu of strain 78-37. A second infant was infected intentionally by natural transmission. The infants developed diarrhea 32 h after challenge of infant 92-1. Electron microscopic observations indicate that cell invasion is the primary mechanism of colon damage and diarrheal disease caused by C. jejuni. Intracellular C. jejuni were located in membrane-bound vacuoles and were free in the cytoplasm. Damaged epithelial cells exhibited premature apoptosis and were exfoliated into the lumen of the colon. C. jejuni were also located extracellularly in the mucosa and submucosa. Some cells had dilated endoplasmic reticulum, indicating possible alteration in ion and water transport.

Human immune response to Campylobacter jejuni proteins expressed in vivo.

Campylobacter jejuni 81-176 grown in vivo in rabbit ileal loops expresses novel proteins that are not expressed under standard laboratory culture conditions. A new protein with a molecular mass of ca. 180 kDa is expressed at 14, 24, and 48 h of infection. Three other proteins, with molecular masses of ca. 66, 43, and 35 kDa, are overexpressed during different phases of infection. Expression of these proteins stops immediately during the first passage in laboratory media, and they do not elicit a human immune response. Two other proteins, with molecular masses of ca. 84 and 47 kDa, expressed 48 h after infection can be identified by using convalescent sera from human volunteers who were immune to C. jejuni infection upon rechallenge; these proteins were not visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by Coomassie blue staining or silver staining. Antibodies to the 84- and 47-kDa proteins are of the immunoglobulin G class. Both preinfection and convalescent human sera react strongly to the C. jejuni flagellin (a 58-kDa protein), suggesting the presence of cross-reactive antibodies to this protein in healthy humans. Major outer membrane protein and flagella may play a role in providing protection against C. jejuni disease, but our data suggest that there are other proteins expressed only during in vivo growth of the organism that elicit a strong immune response in human C. jejuni infections.

Uncommon Campylobacter species in infant Macaca nemestrina monkeys housed in a nursery.

Studies were conducted to characterize 18 isolates of Campylobacter spp. that could not be identified as either Campylobacter jejuni or C. coli. The isolates were cultured from specimens from 13 of 18 infant nonhuman primates during a prospective epidemiologic study reported previously. Phenotypic tests, DNA hybridization, and analysis of DNA coding for rRNA identified the isolates as C. butzleri (seven isolates), C. hyointestinalis (seven isolates), and C. fetus subsp. fetus or C. fetus subsp. fetus-like organisms (four isolates). Ribotype and polyacrylamide gel electrophoresis patterns indicated that there was heterogeneity among the isolates of C. butzleri and C. fetus subsp. fetus-like organisms.

Mouse model for exoerythrocytic stages of Plasmodium falciparum malaria parasite.

Research on the exoerythrocytic (EE) stages of human malaria parasites has been hindered because of the lack of an easily available suitable animal model. We report here an approach to produce mature EE-stage Plasmodium falciparum parasites by using severe combined immunodeficient (scid) mice with transplanted human hepatocytes. Transplantation of human hepatocytes into scid mice (scid hu-hep), their subsequent intravenous infection with P. falciparum sporozoites, and the development of mature liver-stage merozoites was achieved. Immunofluorescent staining of scid hu-hep kidney tissue sections demonstrated the presence of circumsporozoite protein (early during infection), merozoite surface antigen 1, and liver schizont antigen 1. The scid hu-hep model can serve as a source of human malaria liver-stage parasites, decreasing the need for nonhuman primates. Use of this model will facilitate characterization of EE-stage antigens and the assessment of stage-specific chemotherapeutic agents and candidate vaccines.

Development of an in vitro model for study of non-O1 Vibrio cholerae virulence using Caco-2 cells.

Non-O1 Vibrio cholerae strains have been reported as a causative agent of diarrhea throughout the world. We recently reported that non-O1 V. cholerae strains cause diarrhea in human volunteers. In this study we evaluated the virulence of three strains of non-O1 V. cholerae in a Caco-2 cell adherence assay by light and electron microscopy. A-5 is an environmental isolate which failed to colonized volunteers and did not cause diarrhea. It exhibited low numbers of organisms adherent to Caco-2 cells, leaving the microvilli intact. Strain 2076-79, isolated from a patient with diarrhea, colonized human volunteers without producing disease. It adhered to Caco-2 cells in moderate numbers without producing any damage to the microvilli. Strain NRT36S, a clinical isolate, colonized human volunteers and produced significant diarrhea disease. This strain adhered in very large numbers to Caco-2 cells and caused damage to the brush borders. Membrane-bound bacteria were also seen within the cytoplasm of these cells. Scanning electron microscopy confirmed the generalized adherence of NRT36S to the microvilli of Caco-2 cells. The three strains did not appear to compete with each other for binding sites on Caco-2 cells and were not adherent when assays were conducted at 4 degrees C. Our results with strains A-5, 2076-79, and NRT36S correlate well with observations in human volunteer studies, suggesting that Caco-2 cells provide an appropriate in vitro system for further investigation of the pathogenesis of non-O1 V. cholerae gastroenteritis.