Age-related defects in Th1 and Th2 cytokine production by human T cells can be dissociated from altered frequencies of CD45RA+ and CD45RO+ T cell subsets.

The present study investigated whether age-related changes in the production of Th1 and Th2 cytokines by human T cells might be linked to altered frequencies of naive (CD45RA+) and memory (CD45RO+) T cell subsets. T cells from healthy elderly humans (n = 32) stimulated with anti-CD3epsilon monoclonal antibody OKT3 plus PMA produced significantly lower levels of IL-2 and IFNgamma (Th1 type) and of IL-4 (Th2 type) cytokines compared with T cells from young subjects. Although considerable heterogeneity was observed in the levels of cytokines produced by activated T cells from elderly individuals, linear regression analysis failed to demonstrate any significant shift in Th1 to Th2 type cytokine profiles of human T cells during aging. Sufficient T cells were available from eighteen elderly subjects to quantitate the levels of cytokine production in parallel with flow cytometry analysis of the frequencies of CD45RA+ naive and CD45RO+ memory T cells. Compared with the group of young subjects, the elderly group exhibited significant decreases in the frequencies of naive T cells with reciprocal increases in memory T cells. However, defects in Th1 and Th2 cytokine production were not significantly correlated with altered frequencies of naive/memory T cells among elderly individuals. In addition, those elderly individuals with normal frequencies of naive/memory T cells exhibited decreases in cytokine production comparable to the reductions observed for elderly donors with alterations in the frequencies of naive/memory T cells. These findings suggest that age-related defects in Th1 and Th2 cytokine production cannot be attributed entirely to alterations in the frequencies of naive/memory T cell subsets and point toward intrinsic aberrancies within human T cell cytokine networks during aging.

Downregulation of class II antigen expression by FGF-heparin complexes is due primarily to heparin effect.

Fibroblast growth factors are thought to play a role in the pathogenesis of autoimmune inflammation. The evidence linking these growth factors to autoimmunity stems in part from their presence in mononuclear cells from inflammatory sites during disease processes. We sought to further dissect the mechanisms through which fibroblast growth factors might affect the inflammatory response. Peritoneal macrophages from autoimmune MRL 1pr/1pr mice and congenic wild-type MRL +/+ mice were cultured for 72 hours in the presence of either IFN-gamma, heparin, FGF-1, FGF-2, FGF-1 plus heparin, FGF-2 plus heparin or medium alone. Expression of MHC class II (I-Ak and I-Ek) antigens were analyzed using direct immunofluorescence and flow cytometry. As expected, at baseline there were higher numbers of I-Ak bearing cells in elicited peritoneal cells from 1pr mice relative to +/+ cells (70.8 +/- 14.9 versus 43.4 +/- 19.7, p = 0.046). Expression of I-Ak and I-Ek and percentage of I-E bearing cells were essentially the same between strains. Cells from 1pr and +/+ mice displayed reductions in the percentage of I-Ak expressing cells following culture with FGF-1 plus heparin and FGF-2 plus heparin. Similarly, cells from both 1pr and +/+ mice displayed significant reductions from baseline I-Ak expression following culture with FGF-1 and FGF-2 in the presence of heparin. Similar reductions were seen in cells from both strains cultured with heparin alone. No change from baseline was discernible when cells were cultured in the presence of FGF-1 or FGF-2 alone. Titration studies showed a maximum heparin effect at 5 units/ml culture. However, reduced amounts of heparin in the cell culture were directly proportional to decreased levels of I-Ak expression. These results suggest that cells from autoimmune MRL 1pr/1pr mice and wild type MRL +/+ mice respond similarly with a general reduction of I-Ak expression and a decrease in the percentage of I-Ak bearing cells in response to heparin with little discernible effect from addition of either FGF-1 or FGF-2. This change in class II expression suggests that the heparin-heparan component in FGF-heparin complexes may serve to downregulate class II expression during inflammation.

Presence of NK1 receptors on a mucosal-like mast cell line, RBL-2H3 cells.

Reverse transcription--polymerase chain reaction of mRNA from rat RBL-2H3 cells yielded a 316 base pair band consistent with that predicted for the neurokinin-1 (NK1) receptor. Saturation and competition binding with 125I-labeled Bolton-Hunter substance P, substance P fragments, and a series of selective tachykinin receptor agonists and antagonists demonstrated that RBL-2H3 cells express high affinity binding sites for substance P on their surfaces with the kinetic and pharmacological properties of NK1 receptors. The pharmacology of these 125I-labeled substance P binding sites was (from most to least potent) [Sar9,Met(O2)11]substance P > substance P 4-11 >> GR82334 << MEN 10,376. However, substance P 1-4, substance P 8-11, substance P 9-11, and [Trp7, beta-Ala8]neurokinin A 4-10 failed to compete for binding. The metabolically stable NK1 receptor agonist, [Sar9,Met(O2)11] substance P, caused a 49% increase in 5-hydroxytryptamine release above basal levels. The results demonstrate the presence of functional NK1 receptors on RBL-2H3 cells, a mucosal-like mast cell line.

Phosphorylation and coupling of zeta-chains to activated T-cell receptor (TCR)/CD3 complexes from peripheral blood T-cells of elderly humans.

Aging is often accompanied by altered T-cell signaling and functions. Signals mediated through the T-cell receptor (TCR)/CD3 complex are associated with tyrosine phosphorylations of zeta-chains by the regulated activities of protein tyrosine kinases p56(lck) and p59(fyn) as well as protein tyrosine phosphatases. In the present investigation, the coupling and phosphorylation of zeta-chains to TCR/CD3 immunocomplexes were examined in peripheral blood T-cells from 13 elderly and young humans stimulated by ligation of the TCR/CD3 with cross-linked anti-CD3epsilon monoclonal antibody OKT3. Western blots analyzing the non-covalent coupling of zeta-chains to TCR/CD3 immunocomplexes from Brij-96 detergent lysates of anti-CD3 ligated T-cells showed that the levels of zeta-chains within TCR/CD3 immunocomplexes from T-cells of elderly and young subjects did not significantly differ. By contrast, the levels of phosphorylated zeta-chains generated during in vitro phosphorylations of TCR/CD3 immunocomplexes from elderly subjects were significantly reduced and averaged 44% of those observed for anti-CD3epsilon ligated T-cells from young subjects. Analyses of the levels of zeta-chain coupling and phosphorylations in T-cells from each of the 13 elderly individuals also showed that the reductions in zeta-chain phosphorylations were heterogeneous and unrelated to modest reductions in coupling. Furthermore, the age-related decreases in zeta-chain phosphorylations were not due to diminished frequencies of CD3epsilon+ cells or densities of CD3epsilon surface receptors and could be observed without reductions in epsilon-chain phosphorylations. These results suggest that aberrancies of zeta-chain phosphorylations can occur in T-cells of elderly humans independent from any uncoupling of zeta-chains to activated TCR/CD3 complexes.

IFN-gamma-stimulated enhancement of MHC class II antigen expression by the human mast cell line HMC-1.

The expression of MHC class II molecules by human mast cells has been reported in immunohistochemical surveys of inflammatory conditions, such as in tuberculin hypersensitivity. While these data suggest that human mast cells may act as antigen-presenting cells under inflammatory conditions, the induction of class II antigens on human mast cells has not been examined. In this study, we determined the effects of the inflammatory cytokines IFN-gamma and IL-4 on the expression of class II antigens HLA-DR, -DP, and -DQ by the human mast cell line HMC-1. HMC-1 cells were incubated with or without 1000 U/ml recombinant human IFN-gamma (rhIFN-gamma) and IL-4 (rhIL-4) for 72 hr and analyzed for expression of MHC class II antigens by direct immunofluorescence and flow cytometry. HMC-1 cells expressed significant levels of HLA-DR and moderate levels of HLA-DP and -DQ at baseline and when cultured without exogenous cytokines. Stimulation by rhIFN-gamma for 72 hr significantly increased the levels of HLA-DR and -DP expression but did not affect levels of HLA-DQ. Stimulation by rhIL-4 for 72 hr had minimal effect on expression of class II molecules, but induced a significant difference in levels of ICAM-1 (CD54) expression, indicating that this cytokine is involved instead in the control of certain accessory molecules. Our data showing constitutive expression of MHC class II molecules on HMC-1 cells and upregulation of that expression by rhIFN-gamma suggest that human mast cells function as antigen-presenting cells at sites where inflammatory cytokines are present.

Rat peritoneal mast cells present antigen to a PPD-specific T cell line.

Rat peritoneal mast cells were examined to determine whether mast cells can stimulate T cell proliferation through antigen presentation. Mast cells were obtained by peritoneal lavage and purified to 98% using density gradient centrifugation. Purified peritoneal mast cells expressed MHC class II molecules as determined by flow cytometry using monoclonal antibody OX6 specific for common determinants of rat class II. The intensity of class II expression by mast cells was not significantly increased upon incubation with recombinant rat IFN-gamma. Peritoneal mast cells also were found to express the accessory molecules ICAM-1 (CD54) and LFA-1 beta (CD18) but not LFA-1 alpha (CD11a). In the presence of antigen, purified mast cells stimulated proliferation of an autologous CD4+, PPD-specific T cell line. This stimulation was blocked by OX6 antibody, confirming that the proliferation was class II dependent. T cell proliferation was similarly induced by purified mast cell populations that were completely monocyte and macrophages depleted. These results demonstrate that mast cells, through their expression of MHC class II and accessory molecules, are capable of antigen presentation.

Intestinal mast cell responses in idiopathic inflammatory bowel disease. Histamine release from human intestinal mast cells in response to gut epithelial proteins.

Mucosal and submucosal mast cell hyperplasia is a feature of the chronic inflammatory bowel diseases--ulcerative colitis and Crohn's disease. The mast cells are often seen to be degranulated in areas of active disease, suggesting that the inflammatory mediators released from these cells contribute to the pathophysiology of these disorders. We examined the hypothesis that epithelial cell-derived proteins, intestinal epithelial cell-associated components (ECAC), interact with the mast cells of patients with chronic inflammatory bowel disease to trigger the local release of mast cell mediators. Aliquots of human intestinal mucosal mast cell suspensions obtained from surgically resected specimens of colon or small intestine (ulcerative colitis, 12; Crohn's disease, 3; histologically normal controls, 8) were incubated with 1-100 micrograms/ml of colon or small bowel-derived murine ECAC or control kidney protein, or 1 microgram/ml goat anti-human IgE positive control for 30 min at 37 degrees C. Supernatants were analyzed in duplicate for histamine content by fluorometric assay. The median percent total histamine released by chronic inflammatory bowel disease mast cell suspensions to colonic epithelium-derived protein (ECAC-C) was 4% histamine (range 0-20%), such that the distribution of histamine release values in inflammatory bowel disease specimens was significantly different from the distribution of values in mast cells taken from normal mucosa (median 0%, P < 0.05). The median histamine release by all chronic inflammatory bowel disease specimens was also increased in response to the ECAC preparations derived from small bowel epithelium in that a third of the inflammatory bowel disease specimens showed greater than 10% histamine release to ECAC.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of rat methylbenzylnitrosamine metabolism by dietary zinc and zinc in vitro.

Methylbenzylnitrosamine is an esophageal-specific carcinogen in the rat, and the incidence of methylbenzylnitrosamine-induced esophageal carcinoma is increased by dietary zinc deficiency. Methylbenzylnitrosamine requires activation by cytochrome P-450 to be mutagenic; the present study examined the role of dietary zinc deficiency and the in vitro addition of zinc on the cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. Dietary zinc deficiency significantly increased the cytochrome P-450-dependent esophageal and hepatic microsomal metabolism of methylbenzylnitrosamine. These changes occurred without alteration in the specific content of total microsomal cytochrome P-450 of the esophagus or liver. The addition of zinc in vitro, at concentrations found in normal tissues, irreversibly inhibited the esophageal and hepatic cytochrome P-450-dependent microsomal metabolism of methylbenzylnitrosamine. These results suggest that physiological levels of zinc may be an endogenous inhibitor of methylbenzylnitrosamine metabolism. Dietary zinc deficiency appears to reduce this inhibition of cytochrome P-450 activity, resulting in an increase in carcinogen activation.

Modulation of mediator release from human intestinal mast cells by sulfasalazine and 5-aminosalicylic acid.

Intestinal mast cells are thought to contribute to the mucosal inflammation in ulcerative colitis and Crohn's disease through release of inflammatory mediators. Since sulfasalazine and its metabolite 5-aminosalicylic acid are effective therapeutic agents in inflammatory bowel disease and have been shown to inhibit generation of inflammatory products in other cells, we examined the effect of these agents in vitro on human intestinal mast cell mediator release. Sulfasalazine (5 x 10(-4)-10(-3) M) was found to significantly enhance goat anti-human IgE-induced histamine release from intestinal mast cells, which is the same response as seen in human blood basophils, whereas its metabolite 5-aminosalicylic acid was an effective inhibitor of stimulated histamine release in both mast cells and basophils. 5-Aminosalicylic acid also inhibited production of prostaglandin D2 by the stimulated intestinal mast cells. Sulfasalazine alone, without immunologic stimulation, did not induce histamine release from mast cells or basophils, but the enhancement of ongoing mast cell activation by sulfasalazine may explain some cases of adverse reactions to the drug. The inhibition of mast cell histamine release and prostaglandin generation by 5-aminosalicylic acid demonstrates a potential therapeutic modality of this agent.

Enhancement of human intestinal mast cell mediator release in active ulcerative colitis.

To further define the role of mast cells in the idiopathic inflammatory bowel diseases, mediator release from intestinal mast cells derived from actively inflamed and relatively quiescent areas of ulcerative colitis was studied. It was hypothesized that mast cells in the actively diseased segments would indicate involvement in the disease process by releasing a different profile of mediators than cells in uninflammed tissue. Mast cell-containing suspensions derived from matched segments of 12 ulcerative colitis specimens were compared for responsiveness to the mast cell stimulus goat anti-human immunoglobulin E. Supernatants from challenged cells were analyzed for levels of three mast cell mediators, histamine, prostaglandin D2, and the sulfidopeptide leukotriene C. Mast cells from the actively involved areas released significantly greater amounts of histamine, prostaglandin D2, and sulfidopeptide leukotriene. The difference in histamine release was not a result of greater stores of histamine in the active tissue cells, because the total histamine content of the mast cells from the active areas was not significantly greater. The enhanced release of both preformed and newly generated mediators indicates activation of those cells in the course of the disease and points to the mast cell contribution to the inflammatory process in these disorders.

Dexamethasone does not inhibit the release of mediators from human mast cells residing in airway, intestine, or skin.

Glucocorticoids are potent anti-inflammatory drugs that are widely used in the treatment of allergic disorders. Their actions are often species specific or cell-type specific. Previous studies have demonstrated that glucocorticoids inhibit mediator release from mast cells derived from the peritoneum of mouse or rat and from guinea pig lung, but not those residing in human lung parenchymal tissue. In the present study, we have analyzed the effect of overnight culture with dexamethasone (10(-6) to 10(-7)M) on the subsequent IgE-dependent release of mediators from human mast cells derived from airway tissue, intestine, and skin. Airway tissue was passively sensitized with antigen-specific, IgE-rich serum during the culture period and subsequently challenged with ragweed antigen E. Skin and intestinal mast cells were challenged with anti-IgE. Histamine and immunoreactive LTC4 and PGD2 release was monitored in all experiments. Prostaglandin E release was quantitated in the experiments using airway tissue. Dexamethasone treatment failed to inhibit the release of mast cell mediators from all three tissues, but it inhibited the antigen-induced release of immunoreactive PGE from other cells residing in airway tissue. These results confirm earlier studies of the effects of glucocorticoids on human lung parenchymal mast cells, but contrast with the inhibitory effects of steroids observed in murine mast cells and human basophils.

Dietary ellagic acid reduces the esophageal microsomal metabolism of methylbenzylnitrosamine.

Dietary ellagic acid has been shown to reduce the incidence of methylbenzylnitrosamine-induced esophageal carcinoma in the rat. Methylbenzylnitrosamine (MBN) is a naturally occurring carcinogen which requires cytochrome P-450 dependent activation to be mutagenic. We examined whether the reduction in tumor incidence observed with dietary ellagic acid was associated with alterations in the cytochrome P-450 dependent microsomal metabolism of MBN. Dietary ellagic acid was shown to significantly reduce total esophageal and hepatic microsomal cytochrome P-450 (P less than 0.05) and significantly reduce the esophageal microsomal metabolism of MBN (P less than 0.05). The addition of ellagic acid in vitro also resulted in a significant inhibition (P less than 0.05) of the esophageal microsomal metabolism of MBN. In contrast, dietary ellagic acid and the addition of ellagic acid in vitro did not alter the hepatic microsomal metabolism of MBN. The reduced rate of MBN metabolism by the esophageal microsomes from the ellagic acid fed rats may contribute to the decreased incidence of esophageal carcinoma observed in these animals.

Selective inhibition of methylbenzylnitrosamine-induced formation of esophageal O6-methylguanine by dietary ellagic acid in rats.

Ellagic acid is a naturally occurring plant phenol which has been shown to reduce the incidence of a number of carcinogen-induced tumors including methylbenzylnitrosamine (MBN)-induced esophageal carcinoma in the rat. The postulated mechanism of MBN-induced esophageal carcinogenesis is through oxidation of MBN to form benzaldehyde and an activated metabolite which methylates DNA forming a variety of methylated DNA adducts including O6-methylguanine (O6-mGua) and 7-methylguanine (m7Gua). O6-mGua adducts have been shown to induce DNA mutations which can lead to cancer, while m7Gua adducts do not appear to be related to tumor induction. In this study, we examined whether the decreased incidence of MBN-induced esophageal carcinoma observed with dietary ellagic acid was associated with a decrease in the in vivo and in vitro formation of MBN-induced DNA adducts and whether this reduction was specific to O6-mGua or due to a reduction in total methylation. Weanling male Sprague-Dawley rats were fed a nutritionally complete diet with and without the addition of 0.4 g of ellagic acid per kg of diet. This dose of dietary ellagic acid has previously been shown to reduce the incidence of MBN-induced esophageal carcinoma by 30 to 50%. After 3 wk on the diets, rats were given injections of a single dose of MBN (2.0 mg/kg of body weight i.p.) and sacrificed 1 h after injection. Dietary ellagic acid significantly reduced the MBN-induced in vivo formation of esophageal O6-mGua, without significantly reducing the formation of esophageal m7Gua. Examination of this effect in an in vitro methylation assay demonstrated that dietary ellagic acid did not reduce the ability of esophageal microsomes to methylate purified calf thymus DNA; however, pretreatment of the calf thymus DNA with ellagic acid selectively reduced the MBN-induced formation of O6-mGua by microsomes from both ellagic acid-fed and control animals without altering the in vitro formation of m7Gua. These results suggest that ellagic acid bound to DNA selectively blocks methylation of the O6-position of guanine without inhibiting the activation of MBN or the ability of MBN to methylate DNA.

Role of cecal pH in intestinal oxalate absorption in the rat.

Hyperoxaluria occurs with many gastrointestinal disorders complicated by malabsorption. This hyperoxaluria is known to be the result of increased colonic absorption of dietary oxalate. Proposed mechanisms for this effect include alterations in fecal fatty acids, alterations in fecal bile acids, and acidification of colonic pH. Using an animal model of lactulose-induced chronic colonic acidification, we examined the effect of pH on oxalate absorption in vivo. Rats were fed a diet containing 6.77 mg oxalate per day with and without lactulose. Cecal pH of the animals receiving lactulose was significantly lower than controls (4.90 +/- 0.42 vs 7.17 +/- 0.38; p less than 0.001). Urinary excretion of oxalate was significantly greater in animals receiving the lactulose diet than in controls (0.975 +/- 0.144 vs 0.844 +/- 0.172 mg oxalate per day; p less than 0.001). These results demonstrate that acidification of the colon results in a significant increase in urinary oxalate excretion. Thus, acidification of the colon may be an important factor in the pathogenesis of enteric hyperoxaluria.

Effect of zinc deficiency on hepatic enzymes regulating vitamin A status.

The elevation of hepatic vitamin A (VA) in zinc deficiency (ZD) is thought to be due, in part, to a reduced synthesis of plasma retinol-binding protein with a subsequent decrease in the release of retinol into the circulation. We hypothesized that the hepatic VA elevation may also be secondary to a change in the activity of enzymes that either regulate retinol degradation or affect the synthesis or hydrolysis of retinyl esters. To examine this question, 20 rats were divided into two groups and pair-fed for 3 wk. ZD rats received a ZD diet (zinc 2.3 mg/kg diet) and controls were fed a zinc-sufficient diet (zinc 50 mg/kg diet). The enzymes studied were retinyl ester hydrolase (REH) and microsomal acyl coenzyme A:retinol acyl transferase (ARAT), the principal enzymes regulating retinyl ester hydrolysis and synthesis. The activities of retinol (alcohol) dehydrogenase (ADH) and retinal oxidase (RO), enzymes regulating retinol degradation to polar metabolites, were also studied. ZD caused an increase in both total hepatic VA concentration and total hepatic VA content, but did not alter the ratio of retinol to retinyl esters. The specific activities of REH and ARAT were not affected by ZD. However, ZD caused a significant reduction in the activity of ADH, the enzyme that catalyzes the first step in retinol oxidation. In contrast, the activity of RO, the enzyme that regulates the irreversible oxidation of retinal to retinoic acid, was significantly increased in ZD rats. These findings indicate that the elevation in hepatic levels of VA in ZD rats may be, in part, secondary to decreased retinol degradation.

Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells.

The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [35S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. We here demonstrate that human lung mast cells of 96% purity incorporate [35S] sulfate into separate heparin and chondroitin sulfate proteoglycans in an approximately equal to 2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [35S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [35S]sulfate E proteoglycans and the [35S]heparin proteoglycans were exocytosed from the [35S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

Comparison of human lung and intestinal mast cells.

Since much of the data on heterogeneity in the rodent systems are based on differential effects of drugs, we compared responses of human lung mast cell and intestinal mucosal mast cell populations to agents that have revealed mast cell heterogeneity in rat models, as well as to other agents that are effective in both rat peritoneal and intestinal mast cell preparations. Disodium cromoglycate and theophylline inhibit histamine release (HR) from rat peritoneal, but not intestinal, mast cells. We found disodium cromoglycate (10(-4) to 10(-8) mol/L) to be ineffective in both human lung and human intestinal mast cell preparations, whereas theophylline (10(-3) to 10(-6) mol/L) inhibited both lung and intestinal mast cell HR. Quercetin (10(-4) to 10(-8) mol/L) and doxantrazole (3 X 10(-4) to 10(-7) mol/L), which inhibit both rat mast cell types, also inhibit HR in both human mast cell preparations. We also examined the actions of indomethacin and exogenous arachidonic acid and found that neither the lung nor the intestinal mast cells demonstrated the enhancement of HR stimulated by these agents in the basophil. In summary, we have found no pharmacologic differences between the human lung and intestinal mucosal mast cell such as exist in the rodent models. This underscores the limitations of applying the data from animal mast cells to the human mast cells, and at the same time demonstrates that human basophils and mast cells are different.

Dietary zinc deficiency increases the methylbenzylnitrosamine-induced formation of O6-methylguanine in the esophageal DNA of the rat.

Dietary zinc deficiency in combination with environmental exposure to methylbenzylnitrosamine (MBN) is associated with an increased incidence of esophageal carcinoma in man. The proposed mechanism of MBN-induced esophageal carcinoma is through metabolic activation of MBN to form benzaldehyde, and a carbonium ion which methylates DNA. MBN is known to methylate DNA forming O6-methylguanine (O6-MeG) adducts. These adducts can induce guanine to adenine point mutations and such mutations are responsible for certain carcinogen-induced tumors. Rats maintained on a zinc-deficient diet exhibit an increased incidence of MBN-induced esophageal carcinoma when compared with ad libitum and pair-fed controls. The caloric restriction of the pair-fed controls was associated with a lower incidence of MBN-induced esophageal carcinoma than was observed in the ad libitum controls. These differences in tumor incidence were associated with alterations in the formation and clearance of MBN-induced esophageal O6-MeG. Weanling male Sprague-Dawley rats were raised on egg protein diets containing 2.3 p.p.m. zinc (low zinc) or 50 p.p.m. zinc (control). One group of control animals was fed the control diet ad libitum and a second group pair-fed the control diet to match the intake of the zinc-deficient group. After 3 weeks on the diets the animals were injected with a single dose of MBN (2.0 mg/kg b.w.) and levels of esophageal O6-MeG were determined after 1, 3, 6 and 24 h. O6-MeG was significantly higher in the zinc-deficient animals than in controls, with the pair-fed controls demonstrating O6-MeG levels lower than the ad libitum controls. Thus, dietary zinc deficiency results in significantly increased levels of MBN-induced esophageal O6-MeG, and caloric restriction results in decreased levels of MBN-induced esophageal O6-MeG. These changes in esophageal O6-MeG may in part explain the increased incidence of MBN-induced esophageal carcinoma observed with dietary zinc deficiency.

Evaluation of anaerobic culture and effect of culture medium supplementation with factor V on colonial morphology and efficacy of isolation of Streptococcus pneumoniae from sputum.

The use of anaerobic incubation for the culture of Streptococcus pneumoniae from sputum was compared with incubation in carbon dioxide in air. A coagglutination test for pneumococcal antigen was used as an index of the number of specimens containing pneumococci. A total of 334 specimens were examined. There was evidence of pneumococcal colonisation by culture or coagglutination, or both, in 48 (14.37%), of which 41 (12.27%) yielded S pneumoniae on culture. Anaerobic incubation was better than incubation in carbon dioxide in air for the primary culture of S pneumoniae from sputum. Primary isolation of S pneumoniae was achieved in 11 of the 41 strains (26.82%) by anaerobic incubation alone, by incubation only in carbon dioxide in air in one strain (2.43%), and by both anaerobic incubation and incubation in carbon dioxide in air in 29 strains (70.73%). Anaerobic incubation gave large moist or mucoid colonies that were easy to recognise, but it suppressed the typical draughtsman colony of S pneumoniae. The factor V supplement routinely used in our medium also inhibited the formation of draughtsman colonies. It is suggested that draughtsman colonies occur because of a relative lack of the coenzyme nicotinamide adenine dinucleotide (factor V), which is required as a reducing agent in aspartate and glutamate metabolism. This nutritional deficiency may lead to bacterial cell wall defect and hence to the autolysis which gives the typical draughtsman colony.