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.

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.

A Novel Therapeutic and Prophylactic Vaccine against Tuberculosis Using the Cynomolgus Monkey Model and Mouse Model.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse model compared to the BCG. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality. The BCG prime and HSP65 + IL-12/HVJ vaccine (boost) by the prime-boost method showed a synergistic prophylactic effect in the monkey. Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys.HVJ-Envelope/HSP65 DNA + IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the ESR, and augmented the immuneresponses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.

A Novel Therapeutic and Prophylactic Vaccine (HVJ-Envelope / Hsp65 DNA + IL-12 DNA) against Tuberculosis Using the Cynomolgus Monkey Model.

We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). Furthermore, the BCG priming and HSP65 + IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.

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.

Lung macrophages from bacille Calmette-Guérin-vaccinated guinea pigs suppress T cell proliferation but restrict intracellular growth of M. tuberculosis after recombinant guinea pig interferon-gamma activation.

The guinea pig model of low-dose pulmonary tuberculosis has been used to study the pathogenesis of infection as well as the mechanisms of bacille Calmette-Guérin (BCG) vaccine-induced resistance. We investigated the function of lung cells from naive and BCG-vaccinated guinea pigs after enzymatic digestion of lung tissue with collagenase and DNase I. The total lung digest cells proliferated poorly to purified protein derivative (PPD) but comparatively better to ConA as assessed by [(3)H]-thymidine uptake. However, the non-adherent population obtained after plastic adherence of lung digests showed an enhanced response to concanavalin A (ConA) and PPD. Therefore, proliferation to ConA and PPD of nylon wool-purified T cells co-cultured with peritoneal (PMøs), alveolar (AMøs) or lung macrophages (LMøs) was assessed. Co-cultures of lung T cells and PMøs showed maximum proliferation to PPD, whereas proliferation was suppressed significantly by the addition of AMøs or LMøs. The response of T cells to ConA was unaffected in co-cultures. Incubation of co-cultures with recombinant guinea pig interferon-gamma (rgpIFN-gamma) did not reverse the suppression. In contrast, rgpIFN-gamma-treated plastic adherent LMøs that were non-specific esterase-positive were capable of reducing the intracellular growth of Mycobacterium tuberculosis. Similarly, total, non-adherent and adherent lung digest cells from BCG-vaccinated guinea pigs showed IFN-gamma and tumour necrosis factor (TNF)-alpha mRNA expression in response to ConA, lipopolysaccharide or PPD by reverse transcription-polymerase chain reaction followed by release of TNF protein but not IFN. These studies indicate that rgp-IFN-gamma-treated lung tissue macrophages from BCG-vaccinated guinea pigs are defective for inducing antigen-specific proliferation in T cells, but control the intracellular accumulation of virulent M. tuberculosis.

Clinical and pathologic changes in a guinea pig aerosol challenge model of acute Q fever.

Acute Q fever is a zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii and can manifest as a flu-like illness, pneumonia, or hepatitis. A need exists in Q fever research for animal models mimicking both the typical route of infection (inhalation) and the clinical illness seen in human cases of Q fever. A guinea pig aerosol challenge model was developed using C. burnetii Nine Mile phase I (RSA 493), administered using a specialized chamber designed to deliver droplet nuclei directly to the alveolar spaces. Guinea pigs were given 10(1) to 10(6) organisms and evaluated for 28 days postinfection. Clinical signs included fever, weight loss, respiratory difficulty, and death, with the degree and duration of response corresponding to the dose of organism delivered. Histopathologic evaluation of the lungs of animals infected with a high dose showed coalescing panleukocytic bronchointerstitial pneumonia at 7 days postinfection that resolved to multifocal lymphohistiocytic interstitial pneumonia by 28 days. Guinea pigs receiving a killed whole-cell vaccine prior to challenge with the highest dose of C. burnetii were protected against lethal infection and did not develop fever. Clinical signs and pathological changes noted for these guinea pigs were comparable to those seen in human acute Q fever, making this an accurate and valuable animal model of human disease.

Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.

Gamma interferon (IFN-gamma) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-gamma (gpIFN-gamma) and reported that BCG vaccination induced a significant increase in the IFN-gamma mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-gamma mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-gamma with a histidine tag at the N terminus (His-tagged rgpIFN-gamma) in Escherichia coli. rgpIFN-gamma was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-gamma. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-gamma was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-gamma did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-gamma induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-gamma has been successfully produced and characterized in our laboratory. The study of rgpIFN-gamma will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

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.

Orthopaedic surgeons in Yorkshire--are we ATLS positive?

INTRODUCTION: In 1993, the Major Trauma Working Group of Yorkshire proposed that hospitals should be accredited as Trauma Reception Hospitals with a policy for the response to the arrival of a trauma patient. These requirements include specific criteria for orthopaedics. METHODS: To evaluate if these criteria are being fulfilled, we carried out an audit comparing the response in the hospitals within the Yorkshire deanery to the arrival of major trauma. All consultant and middle-grade orthopaedic surgeons on call for trauma were contacted and questioned as to their ATLS provider status and involvement in the "trauma call". RESULTS: 16 hospitals were included of which 13 have a "trauma team". 191 surgeons (96% response) were included. 175 have completed an ATLS course. Of these, 72 (41%) had out-of-date qualifications. Only 9 (13%) were waiting to revalidate. Variation was seen in the frequency of accident and emergency department attendance by different grades of surgeon for major trauma. DISCUSSION: All hospitals have a response for major trauma although variations occur. The vast majority of orthopaedic surgeons in Yorkshire have been adequately trained in ATLS management (more so than any study has previously shown), particularly the middle grades, who are usually first to attend. The level of revalidation is low and reasons for this are discussed with recommendations for revalidation in the future.

Dietary n-3 polyunsaturated fatty acids suppress splenic CD4(+) T cell function in interleukin (IL)-10(-/-) mice.

Our laboratory has demonstrated that down-regulation of proliferation and cytokine synthesis by CD4(+) T cells in mice fed diets rich in n-3 polyunsaturated fatty acids (PUFA) is highly dependent on the involvement of the co-stimulatory molecule, CD28. It has been reported that the inhibitory cytokine interleukin (IL)-10 acts directly on T cells which up-regulate IL-10 receptor (IL-10R) expression following stimulation via CD28 by efficiently blocking proliferation and cytokine production. Thus, it was hypothesized that dietary n-3 PUFA would suppress T cell function through the effects of IL-10. The proliferation of purified splenic CD4(+) T cells activated in vitro with anti-CD3 and anti-CD28 (alphaCD3/CD28) from conventional mice (C57BL/6) fed either a control corn oil (CO)-enriched diet devoid of n-3 PUFA, docosahexaenoic acid (DHA; 22 : 6) or eicosapentaenoic acid (EPA; 20 : 5) for 14 days was suppressed by dietary DHA and EPA. Surprisingly, a similar trend was seen in IL-10 gene knock-out (IL-10(-/-)) mice fed dietary n-3 PUFA. IL-10R cell surface expression was also significantly down-regulated on CD4(+) T cells from both the C57BL/6 and IL-10(-/-) mice fed dietary n-3 PUFA after 72 h of in vitro stimulation with alphaCD3/CD28. Enzyme-linked immunosorbent assay (ELISA) measurements revealed that C57BL/6 mice fed DHA had significantly reduced interferon (IFN)-gamma and IL-10 levels 48 h post-activation. However, CD4(+) T cells from IL-10(-/-) mice fed dietary n-3 PUFA produced significantly greater levels of IFN-gamma than the CO-fed group. Our data suggest that in the absence of IL-10, CD4(+) T cells from n-3 PUFA-fed mice may up-regulate IFN-gamma. Suppressed CD4(+) T cells from n-3 PUFA-fed C57BL/6 mice may use mechanisms other than IL-10 to down-regulate T cell function.

The relationship between malnutrition and tuberculosis: evidence from studies in humans and experimental animals.

The oral traditions of medicine and public health have it that malnutrition is an important risk factor for the development of tuberculosis (TB). Malnutrition profoundly affects cell-mediated immunity (CMI), and CMI is the principle host defense against TB. It makes biological sense. Although most health professionals readily accept this principle, much of this belief is based on uncontrolled observations such as disaster situations or on backwards logic from the cachexia common among TB patients. In fact, the evidence in humans is surprisingly thin from the perspective of scientific rigor. And few data, if any, quantify the extent of the relative or attributable risk of TB due to malnutrition. Moreover, until recently, data from experimental animals were based on animal models that were largely not relevant to human TB infection and disease. This article reviews the scientific data supporting the contention that malnutrition is an important risk factor for TB concentrating on observations in humans and on experimental animal studies based on a highly relevant animal model. If it is true, malnutrition may account for a greater population attributable risk of TB than HIV infection, and certainly a much more correctable one.

Dietary n-3 PUFA affect TcR-mediated activation of purified murine T cells and accessory cell function in co-cultures.

Diets enriched in n-3 polyunsaturated fatty acids (PUFA) suppress several functions of murine splenic T cells by acting directly on the T cells and/or indirectly on accessory cells. In this study, the relative contribution of highly purified populations of the two cell types to the dietary suppression of T cell function was examined. Mice were fed diets containing different levels of n-3 PUFA; safflower oil (SAF; control containing no n-3 PUFA), fish oil (FO) at 2% and 4%, or 1% purified docosahexaenoic acid (DHA) for 2 weeks. Purified (>90%) T cells were obtained from the spleen, and accessory cells (>95% adherent, esterase-positive) were obtained by peritoneal lavage. Purified T cells or accessory cells from each diet group were co-cultured with the alternative cell type from every other diet group, yielding a total of 16 different co-culture combinations. The T cells were stimulated with either concanavalin A (ConA) or antibodies to the T cell receptor (TcR)/CD3 complex and the costimulatory molecule CD28 (alphaCD3/alphaCD28), and proliferation was measured after four days. Suppression of T cell proliferation in the co-cultures was dependent upon the dose of dietary n-3 PUFA fed to mice from which the T cells were derived, irrespective of the dietary treatment of accessory cell donors. The greatest dietary effect was seen in mice consuming the DHA diet (P = 0.034 in the anova; P=0.0053 in the Trend Test), and was observed with direct stimulation of the T cell receptor and CD28 costimulatory ligand, but not with ConA. A significant dietary effect was also contributed accessory cells (P = 0.033 in the Trend Test). We conclude that dietary n-3 PUFA affect TcR-mediated by T cell activation by both direct and indirect (accessory cell) mechanisms.

Immunological characterization of antigens encoded by the RD1 region of the Mycobacterium tuberculosis genome.

Development of immunoassays specific for the diagnosis of tuberculosis requires antigens unique to Mycobacterium tuberculosis. In a search for such antigens we tested six proteins encoded by RD1, a region present in M. tuberculosis and virulent M. bovis genomes but missing from the DNA of all substrains of M. bovis Bacillus Calmette-Guerin (BCG). The six proteins (Rv3871, Rv3872, Rv3873, MTSA-10, ESAT-6 and Rv3878) were purified to near-homogeneity from recombinant Escherichia coli. When tested for the ability to elicit antibody responses and delayed type hypersensitivity in tuberculous guinea pigs, only two of six antigens, ESAT-6 and MTSA-10, elicited strong skin reactions, while vigorous antibody responses were observed to all six proteins. When antibody responses to RD1 antigens were evaluated in sera from patients having pulmonary tuberculosis and from control subjects (patients having mycobacterioses other than tuberculosis, and healthy persons), a sizeable proportion (25%) of tuberculosis patients but none of the control subjects, had antibodies against MTSA-10 and/or ESAT-6. We conclude that MTSA-10 and ESAT-6 are promising candidates for immunodiagnostic assays specific for tuberculosis.

Respirable PLGA microspheres containing rifampicin for the treatment of tuberculosis: screening in an infectious disease model.

PURPOSE: Targeted delivery of rifampicin loaded microspheres to the alveolar macrophage, the host cell for Mycobacterium tuberculosis (MTB), may be an effective targeted approach to pulmonary tuberculosis therapy. A guinea pig infection model has been adopted as a post-treatment screening method for antimicrobial effect. Insufflation and nebulization methods of drug delivery were evaluated. METHODS: Rifampicin alone (RIF, 1.03-1.72 mg/kg), within poly(lactide-co-glycolide) microspheres (R-PLGA, equivalent to 1.03-1.72 mg/kg) or polymer microparticles alone (PLGA) were administered by insufflation or nebulization, 24 h before bacterial aerosol exposure. Animals were infected with an aerosol containing a small number (2 x 10(5) cfu/mL) of virulent H37Rv strain of MTB. Lung and spleen tissue samples were collected 28 days after infection for quantitative bacteriology and histopathological analysis. RESULTS: There was a dose-effect relationship between insufflated R-PLGA and burden of bacteria in the lungs. In addition, guinea pigs treated with R-PLGA had a significantly smaller number of viable bacteria (P < 0.05), reduced inflammation and lung damage than lactose or saline control, PLGA or RIF treated animals. CONCLUSIONS: These studies indicate the potential of R-PLGA, delivered by insufflation or nebulization directly to the lungs, to affect the early development of pulmonary TB.

Dietary n-3 polyunsaturated fatty acids modulate purified murine T-cell subset activation.

Studies in humans and murine disease models have clearly shown dietary fish oil to possess anti-inflammatory properties, apparently mediated by the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To determine the mechanisms by which dietary EPA and DHA modulate mouse T-cell activation, female C57BL/6 mice were fed diets containing either 2% safflower oil (SAF), 2% fish oil (FO), or a 2% purified EPA/DHA ethyl ester mixture for 14 days. Splenic CD4 T cells ( approximately 90% purity) or CD8 T cells ( approximately 85% purity) were incubated with agonists which act at the plasma membrane receptor level [anti(alpha)-CD3/anti(alpha)-CD28], the intracellular level (PMA/Ionomycin), or at both the receptor and intracellular levels (alphaCD3/PMA). CD4 T cells stimulated with alphaCD3/alphaCD28 or PMA/Ionomycin proliferated and produced principally IL-2 (i.e. a Th1 phenotype), whereas the proliferation of CD4 T cells stimulated with alphaCD3/PMA was apparently driven principally by IL-4 (i.e. a Th2 phenotype). The IL-4 driven proliferation of putative Th2 CD4 cells was enhanced by dietary n-3 fatty acids (P = 0.02). Conversely, IL-2 production by alphaCD3/alpha CD28-stimulated CD4 T cells was reduced in FO-fed animals (P < 0.0001). The alphaCD3/alphaCD28-stimulated CD8 cells cultured from FO-fed animals exhibited a significant decrease (P < 0.05) in proliferation. There were no dietary effects seen in alphaCD3/PMA-stimulated CD8 cells, which produced both IL-2 and IL-4, or in PMA/Ionomycin-stimulated CD8 cells, which produced principally IL-2. These data suggest that dietary n-3 fatty acids down-regulated IL-2 driven CD4 and CD8 activation, while up-regulating the activation of the Th2 CD4 T-cell subset. Thus, the anti-inflammatory effects of n-3 fatty acids may result in both the direct suppression of IL-2-induced Th1 cell activation and the indirect suppression of Th1 cells by the enhanced cross-regulatory function of Th2 cells.