The in vivo immunomodulatory effect of recombinant tumour necrosis factor-alpha in guinea pigs vaccinated with Mycobacterium bovis bacille Calmette-Guérin.

Previous studies from our laboratory demonstrated that treatment in vitro with recombinant guinea pig tumour necrosis factor TNF (rgpTNF)-α-enhanced T cell and macrophage functions. Similarly, injection of Mycobacterium tuberculosis-infected guinea pigs with anti-TNF-α altered splenic granuloma organization and caused inflammatory changes and reduced the cell-associated mycobacteria in the tuberculous pluritis model. In this study, rgpTNF-α was injected into bacille Calmette-Guérin (BCG)-vaccinated guinea pigs to modulate immune functions in vivo. Guinea pigs were vaccinated intradermally with BCG, 2 × 10(3) colony-forming units (CFU) and injected intraperitoneally with either rgpTNF-α (25 µg/animal) or 1% bovine serum albumin (BSA) for a total of 12 injections given every other day. Treatment with rgpTNF-α significantly enhanced the skin test response to purified protein derivative (PPD), reduced the number of CFUs and increased the PPD-induced proliferation in the lymph nodes at 6 weeks after vaccination. The levels of interleukin (IL)-12 mRNA were increased in the lymph node and spleen cells stimulated with PPD. TNF-α treatment induced a decrease in TNF-α, IL-12p40 and IL-10 mRNA levels in peritoneal cells following PPD stimulation while live M. tuberculosis caused an increase in TNF-α mRNA and a decrease in the IL-10 mRNA expression. TNF-α injection also induced an increase in the infiltration of mononuclear cells and in the proportions of CD3(+) T cells in the lymph nodes. These results indicate that rgpTNF-α enhances some aspects of T cell immunity and promotes control of mycobacteria in the tissues. Future studies will address the role of TNF-α in BCG-vaccinated guinea pigs following low-dose pulmonary challenge with virulent M. tuberculosis.

Coxiella burnetii isolates cause genogroup-specific virulence in mouse and guinea pig models of acute Q fever.

Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.

Experimental exposure of cattle to a precise aerosolised challenge of Mycobacterium bovis: a novel model to study bovine tuberculosis.

Non-aerosol models of bovine tuberculosis are limited in reproducibility and relevance to natural cases seen in farmed animals. Therefore, there is a need for aerosol models of infection in cattle that can reproduce bovine tuberculosis as seen in natural cases of the disease. This manuscript describes a cattle tuberculosis model based on the inhalation of a precisely defined dose of Mycobacterium bovis in aerosol form, and defines those sites of M. bovis deposition following aerosol inhalation. The dissemination of bacilli and the resultant pathological change following infection is also described. Cattle aged 4-5 months, were infected with approximately 10(4) colony forming units (CFU), using a Madison chamber that had been modified to deliver aerosols to calves. In Experiment 1, calves were examined for gross pathology at post mortem (PM) examination at 93 and 132 days post-infection (PI), respectively. In Experiment 2, pairs of calves were examined for gross pathology at PM examination at 1 day PI and 7 days PI, respectively. At PM examination, samples were taken for bacteriology. Retrospective counts showed that the calves inhaled between 3 x 10(4) and 8 x 10(4)CFU of M. bovis. In Experiment 1, pathology indicative of tuberculosis and detection of M. bovis by qualitative bacteriology was found throughout the lower respiratory tract (LRT). In Experiment 2, pathology was only observed in a single site of one calf at day 7 PI. Samples positive for M. bovis by bacteriology were predominantly in the LRT. The numbers of M. bovis CFU recovered and the distributions of positive sites were greater at day 7 PI than day 1 PI. This study describes an aerosol exposure method that can deliver a defined dose of M. bovis almost exclusively to the LRT. The distribution of M. bovis and lesions indicative of tuberculosis suggests this aerosol method replicates the primary mode of tuberculosis transmission in cattle.

Tuberculosis vaccine development: recent progress.

Recent years have seen a renewed effort to develop new vaccines against tuberculosis. As a result, several promising avenues of research have developed, including the production of recombinant vaccines, auxotrophic vaccines, DNA vaccines and subunit vaccines. In this article we briefly review this work, as well as consider the pros and cons of the animal models needed to test these new vaccines. Screening to date has been carried out in mouse and guinea pig models, which have been used to obtain basic information such as the effect of the vaccine on bacterial load, and whether the vaccine can prevent or reduce lung pathology. The results to date lead us to be optimistic that new candidate vaccines could soon be considered for evaluation in clinical trials.

Purified dietary n-3 polyunsaturated fatty acids alter diacylglycerol mass and molecular species composition in concanavalin A-stimulated murine splenocytes.

A low-dose, short-term dietary supplementation with highly purified (n-3) fatty acid ethyl esters was studied in mice to determine the effect on splenic cell membrane diacylglycerol mass and composition. Mice were fed diets containing either 3% safflower oil (SAF) ethyl esters, 2% SAF plus 1% eicosapentaenoic acid ethyl ester (EPA), or 2% SAF plus 1% docosahexaenoic acid ethyl ester (DHA). Following a 10-day feeding period, pathogen-free mice were sacrificed and splenic cells isolated and stimulated with concanavalin A (Con A) at 10 micrograms/ml. After 0 min (basal), 5 min, and 180 min, 1,2-diacyl, 1-O-alkyl-2-acyl, and 1-O-alkenyl-2-acyl-sn-glycerol subclasses were isolated and quantitated by HPLC. Diacylglycerol (DAG) was found to be the major diradylglycerol (DG) component in murine splenocytes. DHA-fed mice had significantly (P < 0.05) higher levels of DAG at all stimulation time points relative to EPA and SAF animals. Significant effects (P < 0.05) of diet, time, and a diet x time interaction (P < 0.05) were noted for various DAG molecular species. In general, a significantly higher (n-3) polyunsaturated fatty acid (PUFA) content in the EPA and DHA groups, and a significantly higher (n-6) PUFA content in the SAF group was noted. 18:0-22:5(n-3), 18:1-22:5(n-3) and 16:1-20:5(n-3) species were present only in EPA and DHA-DAG, confirming the incorporation of (n-3) fatty acids into splenocyte DAG. The data indicate that the molecular species composition of murine splenocyte DAG is significantly modulated by low-dose, short-term EPA and DHA feeding. In addition, substitution of SAF with DHA results in an increase in DAG mass. These alterations could potentially influence signal transduction pathways regulating lymphocyte function.

Vaccine-induced cytokine responses in a guinea pig model of pulmonary tuberculosis.

Guinea pigs exposed to very small numbers of virulent tubercle bacilli by the respiratory route develop a disease which mimics many of the important features of the pathogenesis of human tuberculosis (TB), including the expression of strong protective immunity following vaccination with BCG. In order to elucidate the precise immunological mechanisms of vaccine-induced resistance in this model, both mRNA and protein assays for several guinea pig cytokines and chemokines have been developed. The coordinated expression of cytokine and chemokine mRNA and protein was examined in various leukocyte populations and in inflammatory cells and fluid collected following the induction of tuberculous pleurisy in BCG-vaccinated guinea pigs. Real-time RT-PCR assays revealed that the mRNA levels for IFNgamma, TNFalpha, and IL-8 rose over the first few days of TB pleuritis and then declined over the 9 days of the study. Injection of anti-TGFbeta on day 8 following pleurisy induction resulted in significant changes in cytokine mRNA levels and PPD-induced proliferation in pleural effusion lymphocytes taken 24h later. BCG vaccination induced significantly higher levels of bioactive TNFalpha protein in the supernatants of alveolar, peritoneal and splenic cells from BCG-vaccinated guinea pigs cultured in the presence of attenuated or virulent mycobacteria. In sharp contrast, following virulent challenge, all three cell types from BCG-vaccinated guinea pigs produced significantly less TNFalpha. Thus, BCG vaccination appears to modulate the potentially harmful effects of TNFalpha in this model of pulmonary TB. Levels of mRNA for IL-12p40 were upregulated by exposure of infected and uninfected macrophages to recombinant guinea pig (rgp)TNFalpha. The intracellular survival of mycobacteria was enhanced when endogeous TNFalpha activity was neutralized with anti-rgpTNFalpha antiserum. rgp RANTES (CCL5) upregulated mRNA levels for TNFalpha, IL-1beta, MCP-1 (CCL2), and IL-8 (CXCL8) in alveolar and peritoneal macrophages. These results illustrate the profound effects of prior vaccination with BCG on the cytokine and chemokine responses of distinct cell populations in the guinea pig following exposure to attenuated and virulent strains of M. tuberculosis.

Disease model: pulmonary tuberculosis.

In spite of a massive effort to apply the tools currently available for tuberculosis (TB) control, both in this country and abroad, it is clear that complicating factors [for example, HIV co-infection, drug resistance, lack of patient compliance with chemotherapy, variable efficacy of Bacille Calmette-Guerin (BCG) vaccine] will prevent disease control unless new drugs, vaccines and diagnostic tests are developed (1). The publication of the complete genome sequence of Mycobacterium tuberculosis in 1998 (2) has facilitated a directed search for virulence genes, new drug targets, and vaccine antigens. This research effort has been made possible by the availability of highly biologically relevant animal models of pulmonary TB ((3)).

Obesity promotes colonic stem cell expansion during cancer initiation.

There is an urgent need to elucidate the mechanistic links between obesity and colon cancer. Convincing evidence for the role of Lgr5(+) stem cells in colon tumorigenesis has been established; however, the influence of obesity on stem cell maintenance is unknown. We assessed the effects of high fat (HF) feeding on colonic stem cell maintenance during cancer initiation (AOM induced) and the responsiveness of stem cells to adipokine signaling pathways. The number of colonic GFP(+) stem cells was significantly higher in the AOM-injected HF group compared to the LF group. The Lgr5(+) stem cells of the HF fed mice exhibited statistically significant increases in cell proliferation and decreases in apoptosis in response to AOM injection compared to the LF group. Colonic organoid cultures from lean mice treated with an adiponectin receptor agonist exhibited a reduction in Lgr5-GPF(+) stem cell number and an increase in apoptosis; however, this response was diminished in the organoid cultures from obese mice. These results suggest that the responsiveness of colonic stem cells to adiponectin in diet-induced obesity is impaired and may contribute to the stem cell accumulation observed in obesity.

Effect of renutrition on humoral and cell-mediated immunity in severely malnourished children.

Forty-three Colombian children suffering from either kwashiorkor (21), combined protein-calorie malnutrition (11), or maramus (11) were hospitalized and provided a high protein, high calorie diet for 4 to 5 wk. Improvement in clinical and nutritional status was accompanied by significant increases in levels of serum immunoglobulins G and M and C3 complement and by significant decreases in serum immunoglobulin A concentrations, especially in infants with kwashiorkor. Skin test reactions to purified protein derivative and candidin improved during renutrition. Lymphocyte blastogenesis after stimulation in vitro with phytohemagglutinin and pokeweed mitogen increased rapidly during hospitalization. After 1 yr posttreatment, cell-mediated immune responses, both in vivo and in vitro, had diminished. These results indicate that some aspects of the immune response are affected to a different degree in kwashiorkor, maramus, and combined malnutrition. Short-term nutritional rehabilitation has a differential effect on the long-term restoration of various aspects of immunity.

Effect of age, malnutrition and renutrition on free secretory component and IgA in secretions.

The development of free secretory component (FSC) was studied in the tears of normal infants, children and adults. The level of FSC in tears was higher in older adults than in children. Free secretory component was also measured in the tears of normal, moderately and severely malnourished Colombian children. Children suffering from kwashiorkor, combined protein-calorie malnutrition or marasmus were studied before and after renutrition. No change was detected in the concentration of FSC in tears of moderately malnourished (Grade I and II) children. There was a significant difference between normal and severely malnourished children which improved with renutrition. The levels of tear IgA were decreased in the moderately malnourished children. These results indicate that reduction in secretory IgA levels in moderate malnutrition may not be explained by a lack of available free secretory component in tears, but that severe malnutrition may impair the S-IgA system by significantly reducing the availability of free secretory component.

Effect of moderate malnutrition on concentrations of immunoglobulins and enzymes in tears and saliva of young Colombian children.

The influence of moderate malnutrition on immunoglobulins and enzymes in the sera and secretions of 71 Colombian children was studied. Concentrations of immunoglobulin A, immunoglobulin G, lysozyme, albumin, and aminopeptidase were measured in the sera, tears, and saliva of 27 normal, 32 grade I, 9 grade II, and 3 grade III malnourished children. The most severely malnourished children, grades II and III, had markedly reduced immunoglobulin A concentrations and elevated immunoglobulin G concentrations in tears. Immunoglobulin A levels in whole saliva were also reduced in these malnourished children. In contrast, the concentration of immunoglobulin A in the sera of these children was significantly elevated. There was no influence of malnutrition on levels of lysozyme, albumin, total protein, and aminopeptidase in tears or saliva. These results indicate that secretory immunity may be impaired in moderately malnourished children due to decreased levels of immunoglobulin A in secretions.

Effect of maternal nutritional status on immunological substances in human colostrum and milk.

Substances in colostrum and breast milk confer significant disease resistance to the breast-fed infant. The influence of maternal nutritional status on both immunological and nonimmunological milk factors was studied in a group of 23 Colombian women during the first 2 months of lactation. Maternal malnutrition was characterized by significantly lower weight/height ratio, creatinine/height index, total serum proteins, serum albumin, and serum IgG and IgA. The colostrum of malnourished mothers contained only one-third the normal concentration of immunoglobulin G and less than half the normal level of albumin. Significant reductions in colostrum levels of IgA and the fourth component of complement (C4) were also observed in the malnourished group. No differences were observed in colostral concentrations of lysozyme, C3 complement, or IgM. Titers of antibody in milk directed against respiratory syncytial virus were not influenced by maternal nutritional status. The differences noted above tended to disappear in mature milk, concomitant with improvement in the nutritional status of malnourished mothers during the first several weeks postpartum. We conclude that the protective qualities of colostrum and milk may be significantly influenced by maternal nutritional status.

Development of impaired cell-mediated immunity in mild and moderate malnutrition.

The development of moderate malnutrition and cell-mediated immune function was studied in 71 Colombian infants from birth through 2 yr of age. Based upon weight-for-age criteria 31 remained normal, 33 were classified as grade I, and seven were grade II malnourished at the end of their 2nd yr of life. Delayed hypersensitivity reactions to purified protein derivative were significantly reduced in all malnourished children 8 wk after Bacille Calmette-Guerin vaccination at birth, and also at 2 yr in the Grade II group. Nearly half of the latter group could not be sensitized to dinitrochlorobenzene at 2 yr of age. A 50% reduction in the blastogenic response of peripheral blood lymphocytes to phytohemagglutinin in vitro was detected in grade II children. Both mildly and moderately malnourished infants exhibited a significant reduction in tonsil size at 2 yr of age. These results indicate that a majority of newborns in this poor, urban setting will develop measurable malnutrition associated with impaired cell-mediated immune function before their 2nd birthday.

Pancreatic and salivary amylase activity in undernourished Colombian children.

Amylase activities were quantitated in secretions of marginally and severely malnourished Colombian children. In young children with a mean age of 21 months, the relative pancreatic and salivary amylase isozyme activities of urine were significantly changed in marginally malnourished children compared to normal children. There was a relative increase in salivary and decrease in pancreatic amylase activity in the undernourished children and total amylase activity was somewhat decreased. Amylase activity in saliva and tears was significantly lower in these malnourished children. Older children who were more severely malnourished had significantly lower amylase activity in their sera and tears. Thus marginal and severe malnutrition affects the production of amylase by the pancreas and salivary glands of young children distinctly. It significantly suppresses amylase activity in tears, saliva, and serum.

Nutritional modulation of host responses to mycobacteria.

Nutritional status determines the generation and functioning of cellular and molecular components of the immune system which are responsible for host resistance to various infectious diseases, including tuberculosis. Studies carried out by us and others have demonstrated that malnutrition exerts detrimental effects on many aspects of host immune responses against mycobacterial infection. First, dietary deficiencies of single nutrients, such as protein and zinc, cause thymic atrophy and impair the generation and maturation of T lymphocytes in animal models of tuberculosis, resulting in reduced number of immunocompetent T cells in lymphoid compartments including the blood. Second, deficiencies of protein, zinc and vitamin D impair T-cell functions, including decreased production of the Th1 cytokines IL-2 and IFN-gamma, and depressed dermal tuberculin reactions and PPD-induced lymphoproliferation in guinea pigs and mice infected with virulent Mycobacterium tuberculosis. Third, protein malnutrition causes trapping or sequestration of reactive T lymphocytes and loss of tuberculosis resistance following BCG vaccination. Finally, protein malnutrition potentiates M. tuberculosis H37Rv-infected monocyte-macrophages to produce higher levels of TGF-beta1 a cytokine which has been implicated as a likely mediator of immunosuppression and immunopathogenesis in tuberculosis.

Isolation and characterization of a gene associated with a virulent strain of Babesia microti.

Babesia microti genomic DNA was purified from parasitized murine erythrocytes, digested with mung bean nuclease and used to construct an expression library in lambda gt11. Polyspecific antisera from mice infected with virulent B. microti organisms (ATCC30221) were used to screen the genomic library for genes encoding major immunogens. High titer antisera selected a recombinant phage, Bm13, containing 3.3 kb of B. microti DNA. Hybridization analysis confirmed the parasite origin of the clone; affinity-purified antibody revealed a native molecular weight of 54,000 for the B. microti protein encoded by the recombinant. Only genomic DNA isolated from the virulent strain of B. microti contained sequences which hybridized to Bm13. Genomic DNA prepared from the Peabody attenuated strain of B. microti or from Babesia bovis DNA did not contain any complementary sequences. These data suggest a possible role for the gene in the virulence of the organism.

A coordinated strategy for evaluating new vaccines for human and animal tuberculosis.

There is a remarkable convergence in the current efforts to develop and evaluate new tuberculosis (TB) vaccine candidates for use in humans, domestic animals, and wild animal reservoirs. It is quite likely that similar vaccination strategies will prove useful in these diverse host species. Many TB vaccine candidates are being screened for protective efficacy in conventional laboratory animals (e.g. mouse, guinea pig), in captive wild species under laboratory conditions (e.g. brushtail possum), and in the target hosts (e.g. cattle, deer). These systems share some important features, e.g. direct challenge infection of the lung by intratracheal or aerosol exposure, and the use of bacterial enumeration, and gross and microscopic histopathology, as the readouts. Some TB vaccine candidates have been tested in many models, yielding important insights into common mechanisms of resistance to Mycobacterium tuberculosis and M. bovis, and providing evidence of the vaccine's ability to induce protection under widely different circumstances. Coordination of this global search for better TB vaccines, irrespective of target species, would facilitate the rapid application of new technologies and maximize the sharing of materials and experiences between human and veterinary TB researchers. The creation of liaisons between TB vaccine research efforts of government-sponsored medical and agricultural research programs, international bodies such as the World Health Organization (WHO) and the European Community (EC), private foundations and the vaccine industry, will yield a high return.

Immune responsiveness of splenocytes after chronic daily melatonin administration in male Syrian hamsters.

The interrelationships between the immune system and the pineal hormone, melatonin, have been explored recently. The present studies investigated the effects of daily melatonin injections on reproductive and spleen function in male Syrian hamsters. Testes weights and serum testosterone levels were depressed after 8-10 weeks of daily melatonin injections. Melatonin-treated hamsters exhibited increased splenic lymphoproliferative responses to a polyclonal T-cell mitogen (concanavalin A (Con-A)), but decreased proliferation following stimulation with a polyclonal B-cell mitogen (lipopolysaccharide). It appears that daily melatonin injections in male hamsters increase the T-cell-mediated immune capacity while reducing the antibody-mediated immune potential. These data suggest that chronic, daily melatonin alters immune system responsiveness in hamsters by shifting the balance of cellular and humoral reactivity.

Diacylglycerol and ceramide kinetics in primary cultures of activated T-lymphocytes.

T cell activation results in the generation of diacylglycerol (DAG), the physiological activator of protein kinase C. Recently, ceramide, a bioactive lipid intracellular second messenger, has been shown to play a positive role in T cell proliferation. Most studies examining mitogen induction of DAG and ceramide in T cells have been conducted in cell lines over short periods of time (0-30 min) relative to the 2-3-h time frame required for commitment to proliferation. Therefore, we examined T cell mitogen-induced DAG and ceramide kinetics under physiologically relevant conditions during the initial 2 h of culture. Freshly isolated murine splenic lymphocytes were stimulated with the T cell-specific mitogen, concanavalin A (Con A). Our results show that Con A induced a multiphasic DAG response with significant peaks in DAG mass occurring at 2, 20 and 120 min. Concomitantly, ceramide mass was significantly increased 2 min following Con A addition and remained elevated until 120 min. Addition of C8-ceramide (10 microM) to lymphocyte cultures significantly enhanced mitogen-induced proliferation. These results demonstrate that DAG is continuously produced by activated T lymphocytes in a multiphasic fashion, and that ceramide is a positive effector molecule with respect to murine T cell proliferation. These results establish a foundation for further examination of the relationship between DAG, ceramide and T cell activation.

Role of immunoglobulin classes in experimental histoplasmosis in bats.

Pooled normal bat serum was separated by gel filtration to give fractions rich in IgG-, Iga- and IgM-like proteins. These fractions were analogous to the corresponding human immunoglobulin classes by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Rabbits were immunized with the fractions and the antisera absorbed. Neotropical bats (Artibeus lituratus) were infected with Histoplasma capsulatum and serum samples were collected weekly and tested for specific serologic response to the fungus. A radial immunodiffusion test was devised to monitor changes in concentrations of IgG, IgA and IgM in the same sera. Bats infected with a low dose of fungus had significantly increased levels of IgM and IgA between 2-6 weeks post-infection. Bats receiving a high dose maintained elevated levels of IgM and IgA through the end of the study. Significantly elevated levels of IgG were not detected until late in the disease (8-9 weeks). In bats with histoplasmosis, IgM and IgA appeared to contribute primarily to the early positive serologies, while precipitating antibodies of the IgG class were detectable later in the disease. These results are similar to the serologic profile seen in human histoplasmosis, and extend our understanding of comparative immune responses in an important wildlife reservoir of human mycotic pathogens.