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.

Differential expression of two CYP1A genes in rainbow trout (Oncorhynchys mykiss).

Differential expression of two rainbow trout CYP1A genes was measured in vivo and in vitro in response to treatment with the model CYP1A inducers beta-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), isosafrole (ISF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, only in vitro). Originally described by Berndtson and Chen (Arch. Biochem. Biophys. 310, 187-195, 1994) as CYP1A1 and CYP1A2, these genes were renamed CYP1A3 and CYP1A1, respectively, by the P450 nomenclature committee. A significant, differential, inducer-dependent induction of the two CYP1A mRNAs, as measured by RNase protection assay, was observed in vivo. CYP1A3 and CYP1A1 mRNA levels in liver were significantly induced 50- and 18-fold, respectively, following ip injection with BNF. Conversely, CYP1A3 and CYP1A1 mRNA levels were significantly induced 5- and 66-fold, respectively, following ip injection with 3-MC. Isosafrole had no significant effect on in vivo induction of CYP1A mRNA levels. In primary cultures of hepatocytes, BNF, 3-MC, ISF, as well as TCDD all significantly induced CYP1A3 and CYP1A1 mRNA levels compared to controls. The differential induction of the two CYP1A genes was not as evident in vitro as in vivo. In addition, reanalysis and sequence comparison of the these two trout CYP1A genes with the first trout CYP1A cDNA described by Heilmann et al. (DNA 7, 379-387, 1988) indicate that the Heilmann cDNA is a hybrid of the two trout genes. The 5' portion of the cDNA sequence (212 bp) was determined by sequencing of a genomic clone and is 100% identical to the trout CYP1A3 gene. The majority of the cDNA sequence (2377 bp), however, was sequenced from a partial cDNA clone and is 99.2% identical to trout CYP1A1. Although the nomenclature of these two trout CYP1A genes is undergoing revision, these results demonstrate a differential, inducer-dependent response to model mammalian CYP1A inducers.

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.

Toxicity of sea nettle (Chrysaora quinquecirrha) fishing tentacle nematocyst venom in cultured rat hepatocytes.

Sea nettle (Chrysaora quinquecirrha) venom (CQV) is known to be toxic to the cardiac, respiratory, renal and hepatic systems in animal models. However, the mechanism of toxicity of CQV on hepatocytes is unknown. We utilized isolated rat hepatocytes in culture and measured percentage total lactate dehydrogenase (LDH) release after direct exposure to CQV. Toxicity of CQV in this system produced a linear multiple-dose response curve as well as a linear single-dose kinetics curve. Neither extracellular calcium concentration nor intracellular calcium chelation had a statistically significant effect on the toxicity of CQV in our hepatocyte model. From these results it appears that CQV does not form large membrane channels similar to complement, nor does calcium appear to play a major role in the mechanism of toxicity in hepatocytes. The isolated rat hepatocyye culture and measure of LDH release provided a relatively simple and reproducible model for examining toxicity of CQV.

Uptake of taurocholic acid and cholic acid in isolated hepatocytes from rainbow trout.

The uptake of the bile acids cholate (CHA) and taurocholate (TCHA) was studied in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Both CHA and TCHA were taken up in a concentration- and temperature-dependent manner with optimum temperature at 15 degrees C and a strikingly efficient uptake even at low temperatures (0-5 degrees C). The total uptake was a combination of a saturable [Michaelis-Menten constant (Km) for CHA, 20 microM; Km for TCHA, 19 microM] and a nonsaturable component. The maximal uptake rate of the saturable component was 416 and 805 pmol.mg protein-1.min-1 for CHA and TCHA, respectively. The uptake of both bile acids was shown to be energy dependent, since it was inhibited by the metabolic inhibitors antimycin A, oligomycin and carbonyl cyanide m-chlorophenylhydrazone. The uptake was clearly Na+ independent, since isosmotic replacement of extracellular Na+ by Li+, choline, or K+ did not inhibit the uptake. Furthermore, it seemed to be independent of the presence of extracellular Cl-, since it was not inhibited by replacement of Cl- with sodium gluconate. On the whole, our results show that the hepatocellular uptake of bile acids in rainbow trout is mediated by a Na(+)-independent carrier system, with characteristics resembling the corresponding transport component in mammalian hepatocytes, but with high efficiency even at low temperatures.

Effects of chloroform and bromodichloromethane on DNA synthesis in male F344 rat kidney.

We have been investigating the actions of chloroform (CHCl3) and bromodichloromethane (BDCM) in rat kidney after different routes of exposure. Male F344 rats were exposed by gavage with corn oil or water as the diluting vehicle. All experiments lasted 30 days with gavage exposures 5 days per week for 4 weeks (20 doses). All animals were injected IP with bromodeoxyuridine (BrdU) 3 times over a 6-day period at 50 mg/kg/injection. Kidney tissue was fixed and slides were stained with hematoxylin and eosin for routine viewing and by the PAP (peroxidase-antiperoxidase) technique using anti-BrdU to label cells in DNA synthesis. There were no significant changes in gross parameters evaluated between the control rats and the rats exposed to CHCl3 or BDCM. Rats exposed via corn oil gavage to CHCl3 displayed a segment-specific epithelial cell necrosis (6/6 high dose, 2/6 low dose). The lesions were primarily localized to the second segment of the proximal tubule, although some spread to cells in the first segment was occasionally observed. No histologic lesions were observed in the kidneys of rats exposed to BDCM. Preliminary results indicate a significant increase in DNA synthesis in the CHCl3-treated rats and a slight increase in DNA synthesis in BDCM-treated rats with corn oil as the diluent. The increase in BrdU labeling was primarily in cells of the S2 segment of the proximal tubule and interstitial cells of CHCl3-exposed animals and in cells of the S3 segment of BDCM-exposed animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.

Alteration in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by exposure to gallium, indium, and arsenite.

Patterns of protein synthesis in primary cultures of rat kidney proximal epithelial tubule cells were examined following exposure to gallium (Ga) chloride, indium (In) chloride, and sodium arsenite. After incubation with these chemicals for 20 hr, newly synthesized proteins were labeled with [35S]methionine. 35S-labeled proteins in the cells were separated by SDS/polyacrylamide gel and two-dimensional gel electrophoresis and detected by fluorography. A protein with molecular weight (Mr) of 30,000 was markedly induced by exposure to 300 microM Ga or 10 microM arsenite, and synthesis of proteins with Mr of 85,000, 71,000, 65,000, 51,000, 38,000, and 28,000 was also increased by Ga or arsenite. Arsenite exposure increased synthesis of eight different proteins, which were not induced by Ga. No significant changes in protein synthesis were observed with 300 microM In exposure. Release of lactate dehydrogenase from the cells was not significantly increased by exposure to concentrations of 300 microM Ga and 3 microM arsenite or less. In the absence of overt cell injury, the induction of these proteins may be useful as an early indicator for assessing exposure to Ga.

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes.

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.

Development of newly formed nephrons in the goldfish kidney following hexachlorobutadiene-induced nephrotoxicity.

New nephrons developed in goldfish, Carassius auratus, several weeks following hexachlorobutadiene-induced (HCBD) nephrotoxicity. Basophilic clusters of presumptive nephrogenic cells incorporated 5-bromo, 2'deoxyuridine (BrdU) one week after HCBD injection, indicating initiation of DNA synthesis. These clusters, like renal vesicles in the developing kidney, elongated, fused with collecting ducts and developed into immature nephrons during the next 2 weeks. Stereologic quantification showed the volume percent of the kidney occupied by the developing nephrons was greater in HCBD-treated fish 2, 3, 4, and 10 weeks after injection than in the control fish. The presence of large numbers of developing nephrons may provide a marker for renal injury in fish from contaminated waterways.

Retinoblastoma in a porkfish (Anisotremus virginicus, Linnaeus) and a brown bullhead (Ictalurus nebulosus, Lesueur).

Two cases of retinoblastoma in fish are described. The neoplasms occurred in a porkfish (Anisotremus virginicus, Linnaeus) and in a brown bullhead (Ictalurus nebulosus, Lesueur).

Comparative effects of three carcinogens on human, rat and mouse hepatocytes.

Ultrastructural changes commonly observed in liver cells of rodents exposed to carcinogens in vivo can be induced in hepatocytes exposed to carcinogens in vitro. Human, rat and mouse hepatocytes in primary culture were treated with actinomycin D, aflatoxin B1 (AFB1) and dimethylnitrosamine (DMN). These cultured hepatocytes were examined for ultrastructural alterations following carcinogen exposure for 24 h. Similar to the effects on liver cells in vivo, the most prominent change was a segregation of the nucleolar components. Human, rat and mouse hepatocytes, dosed with 7.9 X 10(-8) M actinomycin D, developed nucleolar segregation in 86%, 98% and 55% of cells, respectively. When incubated with 3.2 X 10(-6) M AFB1, 60% of human and 84% of rat hepatocytes developed nucleolar segregation. However, exposures of mouse hepatocytes less than or equal to 3.2 X 10(-5) M of AFB1 failed to induce segregation of the nucleolus. DMN administered at a dose of 2.0 X 10(-2) M caused segregation in 11% of the rat hepatocytes and in 60% of the mouse hepatocytes. Distinct nucleolar segregation did not occur in human hepatocytes until they were exposed to a concentration of 5.0 X 10(-2) M DMN (31%). Actinomycin D, AFB1, DMN, as well as other compounds that bind to DNA and interfere with template activity cause nucleolar segregation. Morphologic changes observed in cultured rat and mouse hepatocytes correlate well with in vivo experiments with regard to the relative sensitivity of rats and mice to toxicological effects of these carcinogens. Thus, hepatocyte cultures may provide a realistic system to determine the sensitivity of human liver cells to carcinogens.

Histogenesis of dieldrin and DDT-induced hepatocellular carcinoma in Balb/c mice.

Male, Balb/c mice were fed diets containing dieldrin (10 ppm) and DDT (100-175 ppm) for 75 weeks. Control and treated mice were serially killed and their livers analyzed by histological and histochemical procedures after 2, 4, 8, 16, 36, 52 and 75 weeks of exposure. Mice administered both chlorinated hydrocarbons initially responded with centrolobular hepatocytomegaly. The cells were characterized by decreased glucose-6-phosphatase and succinate dehydrogenase activity. At later periods 52 through 75 weeks, foci of phenotypically-altered hepatocytes were noted. The cells of these lesions were basophilic or clear-staining in hematoxylin and eosin-stained sections and displayed increased gamma glutamyl transpeptidase activity. In mice preloaded with iron dextran, cells of foci were negative for iron when the surrounding parenchyma was siderotic. Hepatocellular adenomas (HA) and carcinomas (HPC) were composed of cells with increased gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase and decreased glucose-6-phosphatase and succinate dehydrogenase activity. In iron loaded mice, the cells of HA and HPC did not stain for iron in otherwise siderotic surroundings. Both hepatocellular foci and adenomas may be potential precursors of mouse hepatocellular carcinomas.

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity.

DNA binding levels were determined and compared in cultured hepatocytes from male and female rats as well as other animal species following exposure to aflatoxin B1 (AFB1) or 2-acetylaminofluorene (2-AAF). When human, rat (both male and female) and mouse hepatocytes in primary culture were exposed to 2.0 X 10(-7) M [3H]AFB1 (sp. act. 2.63 microCi/nmol) for 24 h, male rat hepatocytes had the highest degree of [3H]AFB1-DNA binding (203 pmol/mg DNA) and human hepatocytes contained the next highest binding level (42 pmol/mg DNA). Hepatocytes from female rats contained 38 pmol/mg DNA while cultured mouse hepatocytes contained only 1.4 pmol/mg DNA. When the same dose of [3H]AFB1 was administered to the cultured male rat hepatocytes at 24 h, 48 h, 72 h and 1 week after seeding, and incubated for 24 h, the DNA binding levels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallel experiments to the cultured male rat hepatocytes above, the AFB1-DNA binding levels in the cultured female hepatocytes were 42, 41, 37 and 34 pmol/mg DNA respectively. Human, male and female rat hepatocytes in primary culture were exposed to 5.2 X 10(-5) M 2-acetylamino[9-14C]fluorene (sp. act. 0.0094 microCi/nmol) for 24 h. It was determined that male rat hepatocytes contained the highest amount of radioactively labeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), female rat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytes contained 0.29 nmol/mg DNA. Results from our in vitro hepatocyte culture system correlate well with in vivo animal studies dealing with species and sex differences in DNA binding and carcinogenic susceptibility. This indicates that hepatocytes in vitro maintain many of the biological properties necessary for carcinogen response similar to liver cells in vivo. In addition, comparison of genotoxic effect in cultured hepatocytes from animals as well as humans may be useful in evaluating carcinogenic potential of xenobiotics in human liver.

Tropical fish medicine. Necropsy examination of fish.

Necropsy examination of moribund or dead specimens is an essential step in diagnosing fish diseases. This article discusses basic anatomy and necropsy procedures. A large part of the article has been devoted to methods of procuring samples for laboratory analysis and histologic examination. To determine which findings are responsible for the mortalities, the entire case history and gross necropsy findings must be reviewed. Careful observations made during the necropsy examination provide valuable information immediately, as well as later on in the interpretation of laboratory data.

Hyperplastic and neoplastic alterations in the livers of white perch (Morone americana) from the Chesapeake Bay.

White perch (Morone americana) sampled from 15 estuaries of the Chesapeake Bay contained a variety of hyperplastic and neoplastic alterations in their livers. The lesions were derived from bile ductular epithelium and/or hepatocytes. The biliary lesions consisted of hyperplasias and adenomas. The hepatocellular lesions consisted of focal populations of altered cells and neoplasms, of clear-cell and basophilic morphologic appearance. The hepatocellular lesions were conspicuous in the absence of the decreased amount of copper accumulation. In this respect, the cells comprising these lesions are reminiscent of cells from hepatocellular neoplasia in mice and rats resistant to accumulation of iron.