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.

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.

A human monoclonal antibody that protects mice against Pseudomonas-induced pneumonia.

X-linked immunodeficient (xid) (CBA/N female x DBA/2 male) F1 male mice, when treated with cyclophosphamide, were much more susceptible to challenge with aerosolized Pseudomonas aeruginosa serotype 11 than were control F1 female littermates. Mortality of F1 males was decreased significantly after intravenous administration of human P. aeruginosa serotype 11 O-specific monoclonal antibody. Antibody treatment reduced bacterial titers in the lungs as well as the severity of Pseudomonas-induced lung histopathology.

A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA.

BALB/c nude mice were injected intrahepatically with hepatitis B virus (HBV) DNA prepared from recombinant plasmids. Hepatitis B surface antigen appeared in the circulation in 19 of 23 mice (82%) 3 to 20 weeks postinjection and persisted for more than 6 months in most animals. Hepatitis B e antigen appeared transiently in the circulation in 12 of the 23 mice (52%) within a few weeks after the appearance of hepatitis B surface antigen. Antibodies to the core, X, and/or polymerase gene products of HBV have also been observed in 14 (61%) of the mice. Histopathological examination of the livers at 7 months postinjection demonstrated that nearly half had characteristics consistent with chronic hepatitis. HBV DNA appeared to be integrated into host liver DNA. No evidence of viral replication was observed in sera or livers from these mice at 7 months postinjection. These results demonstrate that an HBV chronic carrierlike state can be established in mice and that such a model could be used to study host and virus factors important in the establishment and maintenance of HBV-associated chronic liver disease.

Spectral model of leishmaniasis in congenic strains of mice.

Nineteen congenic, resistant strains of mice on C57BL/10ScSn genetic background were infected with Leishmania donovani and the course of infection quantitated. Early in the infection, parasite burdens in the liver were similar for all strains, indicating that the parasite was able to establish, grow, and reproduce in the liver macrophages of each strain with equal facility. Differences in acquired resistance, indicated by decreases in parasite burden, among the strains were first noted at day 21 and became distinct by day 35 postinfection. The extremes were represented by B10.129(10M) mice in which the parasite burden continued to increase at day 35, and B10.LP-H-3b in which only 10% of the peak parasite population remained at this time. The other strains formed a complete continuum between the two extremes. Differences in hepatic pathology were noted among strains, but the severity was not related directly to the strength of the immune response as indicated by reduction in parasite burden; instead, it was more correlated with spleen-to-body weight ratios. Because of the range of responses observed, congenic strains of mice may be of use not only for immunization and chemotherapy studies of leishmaniasis, but also may yield fundamental information on spectral diseases in general.

Single gene control of resistance to cutaneous leishmaniasis in mice.

A series of inbred, congenic resistant, and hybrid strains of mice were intradermally inoculated with 10(6) promastigotes of Leishmania tropica. These mice were divided into susceptible and resistant groups using the criteria of lesion size, development of metastatic foci and skin-test reactivity. At 16 weeks of infection, resistant strains A/J, DBA/1J, AKR/J, CBA/J, C3H/HeJ, NZB/BINJ, C57BL/6J, C57BL/10Sn, B10.D2, B10.129(10M), and B10.CE(30NX) had completely resolved their lesions, while susceptible SWR/J and BALB/cJ mice demonstrated large, nonhealing cutaneous lesions. In addition, BALB/cJ developed metastatic lesions on the extremities which progressively increased in size. A11 BALB/cJ and SWR/J mice died by 7 1/2 months of infection. The BALB/cJ female x C57BL/6J male F1 hybrid behaved in an intermediate fashion showing a slower expansion of cutaneous ulcers and a delayed development of metastatic foci, however, the infection ultimately proved fatal. The F2 generation could be separated into three distinct groups:resistant, intermediate, and susceptible mice with a lesion size distribution pattern in conformity with a 1:2:1 ratio. Male/female susceptibility differences were not noted. These data indicated that development of acquired resistance may be under the control of a single, autosomal gene. The gene did not appear to be H-2-, Ir-2-, or H-11-linked as is seen with Leishmania donovani infections.

Genetic control of acquired resistance to visceral leishmaniasis in mice.

A series of H-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 10(7) amastigotes of Leishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b x B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity to L. donovani was controlled by a dominant gene at or near the Ir-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at the H-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced, H-2 CR strains with H-2b, H-2a, and H-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to other H-2 CR strains.