Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals.

Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever.

Molecular and biochemical characterization of recombinant guinea pig tumor necrosis factor-alpha.

Tumor necrosis factor alpha (TNF-α) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-α is essential for the development of a protective immune response to some pathogens, for example, Mycobacterium tuberculosis, by synergizing with other cytokines. However, exorbitant or uncontrolled TNF-α activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-α in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-α has been produced using prokaryotic expression systems. However, it is unknown whether posttranslational modifications which cannot be made in the prokaryotic expression systems may be important for rgpTNF-α structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-α in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-α for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-α lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-α. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-α without concern for the biological integrity.

Studies of a ring-cleaving dioxygenase illuminate the role of cholesterol metabolism in the pathogenesis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a DeltahsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; k(cat)/K(m) = 14.4+/-0.5 microM(-1) s(-1)), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the DeltahsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 A revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design.

Prokaryotic Expression, In Vitro Biological Analysis, and In Silico Structural Evaluation of Guinea Pig IL-4.

Interleukin-4 is a signature cytokine of T-helper type 2 (Th2) cells that play a major role in shaping immune responses. Its role in highly relevant animal model of tuberculosis (TB) like guinea pig has not been studied till date. In the current study, the guinea pig IL-4 gene was cloned and expressed using a prokaryotic expression vector (pET30 a(+)). This approach yielded a recombinant protein of 19 kDa as confirmed by mass spectrometry analysis and named as recombinant guinea pig (rgp)IL-4 protein. The authenticity of the expression of rgpIL-4 protein was further verified through polyclonal anti-IL4 antiserum raised in rabbits that showed specific and strong binding with the recombinant protein. The biological activity of the rgpIL-4 was ascertained in RAW264.7 cells where LPS-treated nitric oxide (NO) production was found to be suppressed in the presence of this protein. The three-dimensional structure of guinea pig IL-4 was predicted by utilizing the template structure of human interleukin-4, which shared a sequence homology of 58%. The homology modeling result showed clear resemblance of guinea pig IL-4 structure with the human IL-4. Taken together, our study indicates that the newly expressed, biologically active rgpIL-4 protein could provide deeper understanding of the immune responses in guinea pig to different infectious diseases like TB and non-infectious ones.

Role of the dosR-dosS two-component regulatory system in Mycobacterium tuberculosis virulence in three animal models.

The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (DeltadosR-dosS [DeltadosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the DeltadosR-S mutant exhibited a growth defect. In the rabbit, the DeltadosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.

The Yin-Yang of TNFalpha in the guinea pig model of tuberculosis.

Tumour necrosis factor alpha (TNF-alpha) is a prototypic pro-inflammatory cytokine that has a central role in the initial host response to Mycobacterium tuberculosis infection. It is a key player in granuloma formation, macrophage activation, bacterial killing, and pathology in vivo. However, the exact mechanism has not been completely understood. This review summarizes the TNFalpha data acquired from the 'gold standard' guinea pig animal model of tuberculosis. While production of TNFalpha is widely accepted as beneficial to the host response, we have found that this hypothesis is just one side of the story. TNFalpha can up-regulate and down-regulate some key pro-inflammatory cytokines (IFNgamma, IL-12p40) and differentially modulate macrophage activation and intracellular bacterial growth. Neutralization of TNFalpha in vivo allows an antiinflammatory TGFbeta-mediated response to develop. Furthermore, BCG vaccination modulates TNFalpha responses directly in the pulmonary granulomas to reduce tissue damage. The bipolar nature of TNFalpha should be considered as knowledge of this critical molecule continues to grow.

Cytokine profiles in primary and secondary pulmonary granulomas of Guinea pigs with tuberculosis.

The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-beta, with some IFN-gamma and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF-alpha mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-beta mRNA with some IL-12p40 and IFN-gamma mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally "vaccinated" by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF-alpha mRNA in unimmunized animals, and mostly TGF-beta mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.

Altered cellular infiltration and cytokine levels during early Mycobacterium tuberculosis sigC mutant infection are associated with late-stage disease attenuation and milder immunopathology in mice.

BACKGROUND: Mouse virulence assessments of certain Mycobacterium tuberculosis mutants have revealed an immunopathology defect in which high tissue CFU counts are observed but the tissue pathology and lethality are reduced. M. tuberculosis mutants which grow and persist in the mouse lungs, but have attenuated disease progression, have the immunopathology (imp) phenotype. The antigenic properties of these strains may alter the progression of disease due to a reduction in host immune cell recruitment to the lungs resulting in disease attenuation and prolonged host survival. RESULTS: In this study we focused on the mouse immune response to one such mutant; the M. tuberculosis Delta sigC mutant. Aerosol infection of DBA/2 and SCID mice with the M. tuberculosis Delta sigC mutant, complemented mutant and wild type strain showed proliferation of mutant bacilli in mouse lungs, but with decreased inflammation and mortality in DBA/2 mice. SCID mice shared the same phenotype as the DBA/2 mice in response to the Delta sigC mutant, however, they succumbed to the infection faster. Bronchoalveolar lavage (BAL) fluid analysis revealed elevated numbers of infiltrating neutrophils in the lungs of mice infected with wild type and complemented Delta sigC mutant strains but not in mice infected with the Delta sigC mutant. In addition, DBA/2 mice infected with the Delta sigC mutant had reduced levels of TNF-alpha, IL-1beta, IL-6 and IFN-gamma in the lungs. Similarly, there was a reduction in proinflammatory cytokines in the lungs of SCID mice. In contrast to the mouse model, the Delta sigC mutant had reduced initial growth in guinea pig lungs. A possible mechanism of attenuation in the Delta sigC mutant may be a reduction in neutrophilic-influx in the alveolar spaces of the lungs, and decreased proinflammatory cytokine secretion. In contrast to mouse data, the M. tuberculosis Delta sigC mutant proliferates slowly in guinea pig lungs, a setting characterized by caseating necrosis. CONCLUSION: Our observations suggest that the immunopathology phenotype is associated with the inability to trigger a strong early immune response, resulting in disease attenuation. While macrophages and T cells have been shown to be important in containing M. tuberculosis disease our study has shown that neutrophils may also play an important role in the containment of this organism.

Dietary eicosapentaenoic acid modulates CTLA-4 expression in murine CD4+ T-cells.

We have demonstrated that downregulation of proliferation by CD4(+) T-cells in mice fed n-3 PUFA diets is dependent on the involvement of CD28. Therefore, we hypothesized that the balance of co-stimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. Mice were fed a control corn oil (CO)-enriched diet devoid of n-3 PUFA, or diets enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 14d. The proliferation of splenic CD4(+) T-cells was suppressed by DHA and EPA following stimulation with anti-CD3 and anti-CD28. Surprisingly, the number of surface CD28 molecules was not reduced in activated CD4(+) T-cells from either group of n-3 PUFA-fed mice. However, in mice fed EPA, CTLA-4 protein levels were enhanced significantly 72 h post-activation (P<0.01). Therefore, we conclude that dietary EPA may suppress CD4(+) T-cell activation by enhancing the downregulatory co-receptor CTLA-4, while not altering the levels of CD28.

Prokaryotic expression and in vitro functional analysis of IL-1ß and MCP-1 from guinea pig.

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1ß) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1ß was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.

Cloning of guinea pig IL-4: reduced IL-4 mRNA after vaccination or Mycobacterium tuberculosis infection.

Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of naïve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freund's Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to naïve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model.

Incorporation of a dietary omega 3 fatty acid impairs murine macrophage responses to Mycobacterium tuberculosis.

BACKGROUND: Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNgamma-mediated signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNgamma, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFalpha, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNgamma receptor with lipid rafts, without affecting surface expression of IFNgamma receptor. CONCLUSIONS/SIGNIFICANCE: We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNgamma, by modulation of IFNgamma receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.

Guinea pig neutrophil-macrophage interactions during infection with Mycobacterium tuberculosis.

We examined the ability of recombinant guinea pig IL-8 (CXCL8) to activate neutrophils upon infection with virulent Mycobacterium tuberculosis. Using a Transwell insert culture system, contact-independent cell cultures were studied in which rgpIL-8-treated neutrophils were infected with virulent M. tuberculosis in the upper well, and AM were cultured in the lower well. IL-1ß and TNF-α mRNA expression was significantly upregulated by AM. Neutralizing anti-rgpTNF-α polyclonal antibody abrogated the response of AM to supernatants from the rgpIL-8-treated, infected neutrophils, while an anti-rgpIL-8 polyclonal antibody had no effect. This suggests that TNF-α produced by rgpIL-8 treated, infected neutrophils may play an important role in the activation of AM in the early response of the host against M. tuberculosis infection. Significant induction of apoptosis in M. tuberculosis-infected neutrophils was observed as compared to the uninfected neutrophils. Feeding of infected, apoptotic neutrophils to AM induced a significant up-regulation of TNF-α and IL-1ß mRNA compared to AM exposed to staurosporine-treated apoptotic neutrophils. Suppressed intracellular mycobacterial growth was also seen in AM fed with infected, apoptotic neutrophils as compared to the AM infected with M. tuberculosis H37Rv alone. Taken together, these data suggest that neutrophil-macrophage interactions may contribute to host defense against M. tuberculosis infection.

The impact of mouse passaging of Mycobacterium tuberculosis strains prior to virulence testing in the mouse and guinea pig aerosol models.

BACKGROUND: It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models. METHODOLOGY/PRINCIPAL FINDINGS: By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates. CONCLUSIONS/SIGNIFICANCE: These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.

Neutralization of TNFalpha alters inflammation in guinea pig tuberculous pleuritis.

Previously, treatment with anti-gpTNFalpha antibody enhanced TNFalpha mRNA expression in pulmonary granulomas microdissected from non-vaccinated guinea pigs, and modified splenic granuloma architecture. In this study, pleural fluid, cells, and granulomatous tissues were collected 3, 5, and 8 days post-pleurisy induction in guinea pigs treated with anti-gpTNFalpha or normal serum control. Neutralizing TNFalpha reduced the percentage of macrophages in the pleural exudate while increasing the proportions of neutrophils and lymphocytes. Cell-associated mycobacterial loads were increased in guinea pigs treated with anti-gpTNFalpha antibody. Cells from the pleural exudate in both treatment groups at day 3 expressed predominantly TNFalpha and IFNgamma mRNA. By day 5, treatment with anti-gpTNFalpha antibody significantly reduced TNFalpha mRNA and increased TGFbeta and iNOS mRNA expression, a transition which did not occur in the control group until day 8. TNFalpha mRNA overwhelmed the cytokine milieu of microdissected pleural granulomas in the control group at day 3 whereas TNFalpha, IFNgamma, and TGFbeta mRNA dominated the anti-gpTNFalpha-treated group. At day 8, granulomas from the control group began shifting towards an anti-inflammatory profile with increased levels of TGFbeta mRNA. Neutralization of TNFalpha hastened the transition to an anti-inflammatory cytokine response in guinea pig pleural granulomas and exudate cells.

Tuberculosis: vaccines in the pipeline.

TB is presenting new challenges as a global health problem, especially with new threats of HIV coinfection and multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis. The current TB vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), is the most widely used vaccine worldwide but its efficacy against pulmonary TB in adults in many high-burden countries is limited. Different vaccine strategies will probably be required for the various needs that exist within a population in which some individuals have been previously immunized with BCG, coinfected with HIV and/or latently infected with M. tuberculosis. In the last 15 years, new strategies to improve or replace BCG in the laboratory have led to several promising vaccine candidates that are actively being evaluated in human clinical trials. Some of these new vaccines may eventually be recommended for travelers to TB high-burden countries. This paper summarizes the progress of vaccine candidates in animal models to improve, replace or augment BCG vaccination.

Vaccination with Bacille-Calmette Guérin promotes mycobacterial control in guinea pig macrophages infected in vivo.

Tuberculosis pleurisy was induced by inoculation of virulent (H37Rv strain or Erdman strain) or attenuated (H37Ra strain) green-fluorescent protein-expressing Mycobacterium tuberculosis into guinea pigs that had or had not been vaccinated with Bacille-Calmette Guérin (BCG). Pleural fluid and cells were analyzed for phagosome-lysosome (P-L) fusion, on the basis of confocal microscopy, intracellular and extracellular bacterial survival, and production of cytokine mRNA. BCG vaccination increased fluid volume and cellular accumulation, significantly enhanced P-L fusion, and significantly decreased intracellular bacterial survival in pleural-effusion macrophages of the guinea pigs infected with the 2 virulent strains. Furthermore, significant increases in interferon-gamma, transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-12p40 cytokine mRNA were seen in the pleural cells of the BCG-vaccinated guinea pigs.

Microdissection of the cytokine milieu of pulmonary granulomas from tuberculous guinea pigs.

Levels of IL-12p40, TNFalpha, TGFbeta, IFNgamma and IL-10 mRNA were assessed by laser capture microdissection followed by quantitative real-time PCR in the pulmonary granulomas of unimmunized and BCG-vaccinated guinea pigs infected by aerosol with virulent Mycobacterium tuberculosis. Lesions microdissected from unimmunized guinea pigs were overwhelmed by the pro-inflammatory TNFalpha mRNA at both 3 and 6 weeks post infection, indicating the struggle to control the mounting infection. The cytokine profile of granulomas from vaccinated guinea pigs shifted from type 1 cytokine mRNA (IFNgamma and IL-12p40) at 3 weeks to a predominantly anti-inflammatory environment (TGFbeta mRNA) at 6 weeks. The relative proportions of cytokine mRNA transcripts in the periphery of the granuloma were different from the centre, reflecting differences in cell composition and architecture. Moreover, analysis of the individual lung lobes at 6 weeks post infection suggests that heterogeneity exists in the cytokine profile between the lobes of the lung.

Accelerated detection of Mycobacterium tuberculosis genes essential for bacterial survival in guinea pigs, compared with mice.

BACKGROUND: Mouse and guinea pig models have been used to identify Mycobacterium tuberculosis mutants attenuated for survival. However, unlike mice, M. tuberculosis-infected guinea pigs form caseating granulomas, which may simulate human disease more closely. METHODS: We used designer arrays for defined mutant analysis, a high-throughput subtractive competition assay, for genotypically defined M. tuberculosis mutants and compared the survival of the same mutant pools in guinea pig and mouse aerosol models. Selected mutants found to be attenuated in either aerosol model were also analyzed in the mouse hollow-fiber model. RESULTS: M. tuberculosis mutants representing 74 genes were tested. Eighteen M. tuberculosis mutants were attenuated for survival in either aerosol model, with 70% of selected mutants also attenuated in the mouse hollow-fiber model. The majority of attenuated mutants in the mouse aerosol model were detected only after 90 days of infection. There was a high degree of concordance between the genes identified by the 2 aerosol models, with detection being significantly earlier in the guinea pig (P<.0003). CONCLUSIONS: We identified M. tuberculosis genes required for survival in mammalian lungs. The majority of mouse late-stage survival mutants were detected significantly earlier in the guinea pig, which suggests that differences in tuberculosis-induced lung pathologic changes may account for this accelerated detection.

Dietary fish oil inhibits antigen-specific murine Th1 cell development by suppression of clonal expansion.

To determine the mechanisms by which dietary fish oil (FO) affects antigen-stimulated Th1 cell development, DO11.10 Rag 2(-/-) T cell receptor transgenic mice were fed a control diet (5% corn oil (CO) or a FO diet (1% CO + 4% FO, (n-3) PUFA) for 2 wk. CD4(+) T cells were cultured under neutral or Th1 polarizing conditions. FO feeding suppressed (P < 0.05) ovalbumin peptide-induced proliferation of nonpolarized CD4(+) T cells. Differentiation in vitro to Th1 cells was not affected by dietary FO, as evidenced by similar percentages of KJ1-26(+), IFN-gamma(+), IL-4(-) Th1 cells in cultures from CO-fed (99%) and FO-fed (97%) mice. However, the absolute number of viable Th1 cells in polarized cultures from FO-fed mice was less than half that observed in CO-fed mice (P < 0.05), indicating that FO inhibits in vitro Th1 clonal expansion. The reduced number of Th1 cells in FO cultures was not a result of increased apoptosis, because similar percentages of apoptotic Th1 cells were observed in cultures from FO- and CO-fed mice. IL-2-induced cell proliferation was significantly decreased in polarized Th1 cells from the FO group; however, the suppressed proliferation was not linked to reduced CD25 surface expression on antigen-stimulated CD4(+) T cells. Adoptively transferred CFSE-labeled DO11.10 CD4(+) cells into immunized mice (Th1 polarizing agents) showed that dietary FO reduced (P < 0.05) the number of cell divisions in vivo. These studies suggest that the attenuated inflammatory response which accompanies FO feeding may be explained, at least in part, by suppression of Th1 clonal expansion.