Cod Liver Oil, but Not Retinoic Acid, Treatment Restores Bone Thickness in a Vitamin A-Deficient Rat.

Vitamin A plays a prominent role for maintaining optimal bone status, but its impact upon the bone in response to vitamin A deficiency is not well defined. The purpose of this study was to evaluate how replenishing vitamin A by either whole food cod liver oil (COD) or the active metabolite of vitamin A, retinoic acid (RA), altered bone thickness of vitamin A-deficient (VAD) rats. Weanling rats were administered a control diet (CTRL) or VAD diet for 9 weeks. This was followed by four weeks of treatment in which the VAD group was divided into the following 4 subgroups: (1) VAD (9 weeks)-VAD (4 weeks); (2) VAD-CTRL; (3) VAD-COD; and (4) VAD-RA. Compared to controls, VAD rats had thicker bones which showed marked dysplasia. VAD-rats treated with COD produced a thinner bone that was not significantly different from that of untreated rats. In contrast, RA did not significantly change the thicker bone, and also had significantly greater periosteal and endosteal osteoblast numbers compared to VAD-COD. Active osteoclasts were not detected in VAD rats, nor during the treatment period. These findings suggest that the abnormal bone thickness in VAD rats appears to be more effectively restored to bone thickness of untreated control rats when treated with COD.

MCPIP1 deficiency in mice results in severe anemia related to autoimmune mechanisms.

Autoimmune gastritis is an organ-specific autoimmune disease of the stomach associated with pernicious anemia. The previous work from us and other groups identified MCPIP1 as an essential factor controlling inflammation and immune homeostasis. MCPIP1(-/-) developed severe anemia. However, the mechanisms underlying this phenotype remain unclear. In the present study, we found that MCPIP1 deficiency in mice resulted in severe anemia related to autoimmune mechanisms. Although MCPIP1 deficiency did not affect erythropoiesis per se, the erythropoiesis in MCPIP1(-/-) bone marrow erythroblasts was significantly attenuated due to iron and vitamin B12 (VB12) deficiency, which was mainly resulted from autoimmunity-associated gastritis and parietal cell loss. Consistently, exogenous supplement of iron and VB12 greatly improved the anemia phenotype of MCPIP1(-/-) mice. Finally, we have evidence suggesting that autoimmune hemolysis may also contribute to anemia phenotype of MCPIP1(-/-) mice. Taken together, our study suggests that MCPIP1 deficiency in mice leads to the development of autoimmune gastritis and pernicious anemia. Thus, MCPIP1(-/-) mice may be a good mouse model for investigating the pathogenesis of pernicious anemia and testing the efficacy of some potential drugs for treatment of this disease.

Targeted disruption of MCPIP1/Zc3h12a results in fatal inflammatory disease.

Previous studies using MCP-induced protein 1 (MCPIP1)/Zc3h12a-deficient mice suggest that MCPIP1 is an important regulator of inflammation and immune homeostasis. However, the characterization of the immunological phenotype of MCPIP1-deficient mice has not been detailed. In this study, we performed evaluation through histological, flow cytometric, enzyme-linked immunosorbent assay and real-time PCR analysis and found that targeted disruption of MCPIP1 gene leads to fatal, highly aggressive and widespread immune-related lesions. In addition to previously observed growth retardation, splenomegaly, lymphoadenopathy, severe anemia and premature death, MCPIP1-deficient mice showed disorganization of lymphoid organs, including spleen, lymph nodes and thymus, and massive infiltration of lymphocytes, macrophages and neutrophils into many other non-lymphoid organs, primarily in lungs and liver. Flow cytometric analysis found significant increase in activated and differentiated T cells in peripheral blood and spleen of MCPIP1-deficient mice. Moreover, heightened production of inflammatory cytokines from activated macrophages and T cells were observed in MCPIP1-deficient mice. Interestingly, treatment of MCPIP1-deficient mice with antibiotics resulted in significant improvement of life span and a decrease in inflammatory syndrome. Taken together, these results suggest a prominent role for MCPIP1 in the control of inflammation and immune homeostasis.

Effect of exendin (exenatide)--GLP 1 receptor agonist on the thyroid and parathyroid gland in a rat model.

Exenatide or Exendin-4 is a 39-amino acid agonist of the glucagon like peptide (GLP-1) receptor approved for the adjunctive treatment for type 2 diabetes. Recent reports suggest that GLP-1 agonists may also have distant effects including C-cell thyroid hyperplasia. The aim of this study was to evaluate the effect of exendin-4 on the thyroid and parathyroid cells in a rat model. Rat thyroids were stained for calcitonin, H&E and for carcinoembryonic antigen (CEA). Thyroid C-cell hyperplasia was graded on H&E stained slides using cell size and secretory granule numbers, morphological features of the parathyroid glands and the serum calcium concentrations of the rats were also evaluated. Counts of stained cells/high power field and intensity of staining were recorded by two pathologists. Data were analyzed by ANOVA/post-tests. C cell hypertrophy was elevated in exenatide-treated vs. untreated animals (22.5 ± 8.7 vs. 10.5 ± 2.7 cells/HPF). CEA staining failed to show effects by exendin. Calcitonin staining was significantly elevated in exenatide treated controls (P<0.001). Parathyroid glands were histologically normal in both groups, and serum calcium levels were within normal range in all animals. In summary, exenatide was associated with C cell hyperplasia and increased calcitonin staining of thyroids, but was unrelated to CEA levels. These data raise important concerns about the effects of exenatide which, given its wide clinical use, should be clarified with urgency.

The putative tumor suppressor Zc3h12d modulates toll-like receptor signaling in macrophages.

Toll-like receptors (TLR) are pivotal in macrophage activation. The molecular mechanisms controlling TLR signaling and macrophage activation are not completely understood. Zc3h12d is originally identified as a possible tumor suppressor gene. However, its function remains unknown. We here report that Zc3h12d negatively regulates TLR signaling and macrophage activation. Zc3h12d was enriched in spleen, lung and lymph node. In macrophages, the expression of Zc3h12d was remarkably induced by TLR ligands through JNK and NF-κB signal pathways. On the other hand, overexpression of Zc3h12d significantly inhibited TLR2 and TLR4 activation-induced JNK, ERK and NF-κB signaling as well as macrophage inflammation. Similar to Zc3h12a/MCPIP1, Zc3h12d also decreased the global cellular protein ubiquitination. These findings suggest that Zc3h12d is a novel negative feedback regulator of TLR signaling and macrophage activation and thus may play a role in host immunity and inflammatory diseases.

Monocyte chemotactic protein-induced protein 1 (MCPIP1) suppresses stress granule formation and determines apoptosis under stress.

It is unclear how stress granule (SG) formation and cellular apoptosis are coordinately regulated. MCPIP1 (monocyte chemotactic protein-induced protein 1), also known as Zc3h12a, is a critical regulator of the inflammatory response and immune homeostasis. However, the role of MCPIP1 in stress response remains unknown. Here, we report that overexpression of MCPIP1 inhibited the assembly of SGs in response to various stresses. Conversely, MCPIP1-deficient splenocytes developed more SGs even without stress. On the other hand, overexpression of MCPIP1 sensitized RAW 264.7 cells to apoptosis under stress, whereas MCPIP1-deficient cells were resistant to stress-induced apoptosis. Mutagenesis study showed that the ability of MCPIP1 to repress SG formation is dependent on its deubiquitinating activity. Consistently, MCPIP1 negatively regulated stress-induced phosphorylation of eIF2α and thus released stress-induced inhibition of protein translation. However, MCPIP1 also inhibited 15-deoxy-Δ(12,14)-prostaglandin J(2)-induced SG formation, which was reported to be independent of eIF2α phosphorylation. Taken together, these results suggest that MCPIP1 coordinates SG formation and apoptosis during cellular stress and may play a critical role in immune homeostasis and resolution of macrophage inflammation.

Treatment with omega-3 fatty acids but not exendin-4 improves hepatic steatosis.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in the Western world. The aim of this study was to evaluate the biochemical and histological effects of omega-3 fatty acid and exendin-4 treatment on NAFLD in an animal model. METHODS: Sixty-three 8-week-old outbred Sprague-Dawley male rats were used for this study. Three animals were used as procedure controls, and 30 rats were fed a methionine and choline deficient (MCD) diet and 30 were fed a regular chow diet. In each group of 30 animals, 10 served as controls, 10 received exendin-4, and 10 received omega-3 fatty acids. After 75 days of treatment, the animals were euthanized, the tissues and serum were harvested, and the livers were formalin-fixed for histology. RESULTS: The MCD diet was exceptionally efficient at producing fatty livers. The MCD control animals had a liver steatosis score of 38+/-6.7 (of 50 possible); treatment with exendin-4 was not associated with a significant reduction of steatosis (44+/-5.16, P=0.07) and the omega-3 fatty acid treatment was associated with a significant decrease in the liver steatosis score (15.6+/-13.46, P<0.001) compared with both the controls and the exendin-4 groups. The omega-3 fatty acid treatment increased serum aspartate aminotransferase significantly, whereas exendin-4 had no effect. CONCLUSION: In an animal model of NAFLD, the omega-3 fatty acid therapy was associated with significant improvement in hepatic steatosis compared with exendin-4. These data suggest that omega-3 fatty acid supplements may have a potential therapeutic role in patients with NAFLD.

Fat embolism: evolution of histopathological changes in the rat lung.

The pathophysiology of Fat Embolism Syndrome (FES) is poorly understood and subject to some controversy. Evaluation of the evolution of histological changes in the lungs of patients with FES is impractical. The current theories of FES were established through acute clinical observations and acute animal experiments, but sequential changes in the histology of lungs over a prolonged period have not been made. The progressive effects of fat embolization of the lungs were examined in a rat model over a period of 11 days. Triolein, a major bone marrow fat, was administered to conscious Sprague-Dawley rats via the caudal vein. Rats were euthanized at 24, 48, 96 h, and 11 days, but some died within a few hours. Histomorphometric evaluations of lung tissue were made, including stains for fat, collagen, and smooth muscle actin. Arterial and arteriolar patency decreased progressively up to 96 h, but returned toward normal after 11 days. A striking finding was the very early presence of inflammation and fibrosis after only several hours, persisting up to 11 days. The results of this study provide evidence of both very early and prolonged changes due to fat embolization.

Development of colour-producing beta-keratin nanostructures in avian feather barbs.

The non-iridescent structural colours of avian feather barbs are produced by coherent light scattering from amorphous (i.e. quasi-ordered) nanostructures of beta-keratin and air in the medullary cells of feather barb rami. Known barb nanostructures belong to two distinct morphological classes. 'Channel' nanostructures consist of beta-keratin bars and air channels of elongate, tortuous and twisting forms. 'Spherical' nanostructures consist of highly spherical air cavities that are surrounded by thin beta-keratin bars and sometimes interconnected by tiny passages. Using transmission electron microscopy, we observe that the colour-producing channel-type nanostructures of medullary beta-keratin in feathers of the blue-and-yellow macaw (Ara ararauna, Psittacidae) develop by intracellular self-assembly; the process proceeds in the absence of any biological prepattern created by the cell membrane, endoplasmic reticulum or cellular intermediate filaments. We examine the hypothesis that the shape and size of these self-assembled, intracellular nanostructures are determined by phase separation of beta-keratin protein from the cytoplasm of the cell. The shapes of a broad sample of colour-producing channel-type nanostructures from nine avian species are very similar to those self-assembled during the phase separation of an unstable mixture, a process called spinodal decomposition (SD). In contrast, the shapes of a sample of spherical-type nanostructures from feather barbs of six species show a poor match to SD. However, spherical nanostructures show a strong morphological similarity to morphologies produced by phase separation of a metastable mixture, called nucleation and growth. We propose that colour-producing, intracellular, spongy medullary beta-keratin nanostructures develop their characteristic sizes and shapes by phase separation during protein polymerization. We discuss the possible role of capillary flow through drying of medullary cells in the development of the hollow morphology of typical and spongy feather medullary cells.

Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux.

PURPOSE: The purpose of this manuscript is to investigate the presence of nucleoside/nucleotide efflux transporter in cornea and to evaluate the role in ocular drug efflux. METHODS: RT-PCR, immunoprecipitation followed by Western blot analysis and immunostaining were employed to establish molecular presence of multidrug resistance associated protein 5 (MRP5) on cornea. Corneal efflux by MRP5 was studied with bis(POM)-PMEA and acyclovir using rabbit and human corneal epithelial cells along with MRP5 over expressing cells (MDCKII-MRP5). Ex vivo studies using excised rabbit cornea and in vivo ocular microdialysis in male New Zealand white rabbits were used to further evaluate the role of MRP5 in conferring ocular drug resistance. RESULTS: RT-PCR confirms the expression of MRP5 in both rabbit and human corneal epithelial cells along with MDCKII-MRP5 cells. Immunoprecipitation followed by Western blot analysis using a rat (M511-54) monoclonal antibody that reacts with human epitope confirms the expression of MRP5 protein in human corneal epithelial cells and MDCKII-MRP5 cells. Immunostaining performed on human cornea indicates the localization of this efflux pump on both epithelium and endothelium. Efflux studies reveal that depletion of ATP decreased PMEA efflux significantly. MRP5 inhibitors also diminished PMEA and acyclovir efflux. However, depletion of glutathione did not alter efflux. MDR1 and MRP2 did not contribute to PMEA efflux. However, MRP2 is involved in acyclovir efflux while MDR1 do not participate in this process. TLC/autoradiography suggested the conversion of bis(POM)-PMEA to PMEA in rabbit and human corneal epithelial cells. Two well known antiglaucoma drugs, bimatoprost and latanoprost were rapidly effluxed by MRP5. Ex vivo study on intact rabbit corneas demonstrated accumulation of PMEA in cornea in the presence of ATP-depleting medium. In vivo ocular pharmacokinetics also revealed a significant increase in maximum aqueous humor concentration (C(max)) and area under the aqueous humor time curve (AUC) of acyclovir in the presence of MK-571, a specific MRP inhibitor. CONCLUSIONS: Taken together immunolocalization on human cornea, in vitro efflux in human, rabbit corneal and MRP5 over expressing cells, ex vivo and in vivo studies in intact rabbit cornea suggest that MRP5 on cornea can significantly lower the permeability of antiviral and glaucoma drugs. These findings may be valuable in developing formulation strategies to optimize ocular bioavailability of topically administered ocular agents.

Pulsed high intensity focused ultrasound mediated nanoparticle delivery: mechanisms and efficacy in murine muscle.

High intensity focused ultrasound (HIFU) is generally thought to interact with biological tissues in two ways: hyperthermia (heat) and acoustic cavitation. Pulsed mode HIFU has recently been demonstrated to increase the efficacy of a variety of drug therapies. Generally, it is presumed that the treatment acts to temporarily increase the permeability of the tissue to the therapeutic agent, however, the precise mechanism remains in dispute. In this article, we present evidence precluding hyperthermia as a principal mechanism for enhancing delivery, using a quantitative analysis of systemically administered fluorescent nanoparticles delivered to muscle in the calves of mice. Comparisons were carried out on the degree of enhancement between an equivalent heat treatment, delivered without ultrasound, and that of the pulsed-HIFU itself. In the murine calf muscle, Pulsed-HIFU treatment resulted in a significant increase in distribution of 200 nm particles (p < 0.016, n = 6), while the equivalent thermal dose showed no significant increase. Additional studies using this tissue/agent model also demonstrated that the pulsed HIFU enhancing effects persist for more than 24 h, which is longer than that of hyperthermia and acoustic cavitation, and offers the possibility of a novel third mechanism for mediating delivery.

Voltage-gated sodium channel Nav1.1, Nav1.3 and beta1 subunit were up-regulated in the hippocampus of spontaneously epileptic rat.

The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsions and absence-like seizures from the age of 8 weeks. Since the first point mutation in the voltage-gated sodium channel (VGSC) beta(1) subunit in human generalized epilepsy with febrile seizures plus (GEFS+) was identified, more and more types of genetic epilepsy have been causally suggested to be related to gene changes in VGSC. However, there are no reports that can elucidate the effects of VGSC in SER. The present study was undertaken to detect sodium channel I alpha-isoform (Na(v)1.1), sodium channel III alpha-isoform (Na(v)1.3) and beta(1) subunit from both the level of mRNA and protein in SERs hippocampus compared with control Wistar rats. In this study, the mRNA expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit in SERs hippocampus were significantly higher than those in control rats hippocampus by real-time RT-PCR; The protein distributions and expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit in SERs hippocampus were detected by immunofluorescence, immunohistochemistry and western blot, and the protein expressions of Na(v)1.1, Na(v)1.3 and beta(1) subunit were significantly increased. In conclusion, our study suggested for the first time that sodium channel Na(v)1.1, Na(v)1.3 and beta(1) subunit up-regulation at the mRNA and protein levels of SER hippocampus might contribute to the generation of epileptiform activity and underlie the observed seizure phenotype in SER. The results of this study may be of value in revealing components of the molecular mechanisms of hippocampal excitation that are related to genetic epilepsy.

Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux.

Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium, thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the P-glycoprotein (P-gp) in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multidrug resistance associated proteins (MRPs) contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40-HCEC and rPCEC were selected for in vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [(3)H]-cyclosporine-A and [(14)C]-erythromycin, which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at approximately 272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band approximately 181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at approximately 190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [(3)H]-cyclosporine-A and [(14)C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40-HCEC and rPCEC. Similarly a significant elevation in (A-->B) permeability of [(3)H]-cyclosporine-A and [(14)C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A-->B transport of [(3)H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding.

Phthalate-Induced Liver Protection against Deleterious Effects of the Th1 Response: A Potentially Serious Health Hazard.

Infection with Mycobacterium tuberculosis (TB) induces pulmonary immunopathology mediated by classical Th1 type of acquired immunity with hepatic involvement in up to 80% of disseminated cases. Since PPAR agonists cause immune responses characterized by a decrease in the secretion of Th1 cytokines, we investigated the impact of activating these receptors on hepatic pathology associated with a well-characterized model of Th1-type pulmonary response. Male Fischer 344 rats were either maintained on a drug-free diet (groups I and II), or a diet containing diethylhexylphthalate (DEHP), a compound transformed in vivo to metabolites known to activate PPARs, for 21 days (groups III and IV). Subsequently, animals were primed with Mycobacterium bovis purified protein derivative (PPD) in a Complete Freund's Adjuvant. Fifteen days later, animals in groups II and IV were challenged with Sepharose 4B beads covalently coupled with PPD, while animals in groups I and III received blank Sepharose beads. Animals with Th1 response (group II) showed a marked structural disruption in the hepatic lobule. Remarkably, these alterations were conspicuously absent in animals which received DEHP (group IV), despite noticeable accumulation of T cells in the periportal triads. Immunostaining and confocal microscopy revealed hepatic accumulation of IFNgamma+ Th1 and IL-4+ Th2 cells in animals from groups II and IV, respectively. Our data suggest a PPARalpha-mediated suppression of the development of a Th1 immune response in the liver, resulting in hepatoprotective effect. However, potentially negative consequences of PPAR activation, such as decreased ability of the immune system to fight infection and interference with the efficacy of vaccines designed to evoke Th1 immune responses, remain to be investigated.

Anatomically diverse butterfly scales all produce structural colours by coherent scattering.

The structural colours of butterflies and moths (Lepidoptera) have been attributed to a diversity of physical mechanisms, including multilayer interference, diffraction, Bragg scattering, Tyndall scattering and Rayleigh scattering. We used fibre optic spectrophotometry, transmission electron microscopy (TEM) and 2D Fourier analysis to investigate the physical mechanisms of structural colour production in twelve lepidopteran species from four families, representing all of the previously proposed anatomical and optical classes of butterfly nanostructure. The 2D Fourier analyses of TEMs of colour producing butterfly scales document that all species are appropriately nanostructured to produce visible colours by coherent scattering, i.e. differential interference and reinforcement of scattered, visible wavelengths. Previously hypothesized to produce a blue colour by incoherent, Tyndall scattering, the scales of Papilio zalmoxis are not appropriately nanostructured for incoherent scattering. Rather, available data indicate that the blue of P. zalmoxis is a fluorescent pigmentary colour. Despite their nanoscale anatomical diversity, all structurally coloured butterfly scales share a single fundamental physical color production mechanism - coherent scattering. Recognition of this commonality provides a new perspective on how the nanostructure and optical properties of structurally coloured butterfly scales evolved and diversified among and within lepidopteran clades.

Neutrophil-anti-microbial interaction in the established infection: effect on Staphylococcus aureus.

OBJECTIVES: To determine if differences in drug-related Staphylococcus aureus killing, associated in vivo with neutropenia, is neutrophil-related in vitro, and the mechanisms of this interaction. METHODS: To evaluate the influence of living neutrophils on drug-S. aureus interactions, cell wall enzymes, the PBPs, were isolated and their binding to five (beta lactam and other) antibiotics was evaluated following incubation (or not) with neutrophils. S. aureus killing by the test drugs was assayed in growth media of sterile filtered abscess fluid, either neutropenic infected or normal infected. At MBCs for the test isolate, each drug or saline control was incubated with S. aureus 10(6)and dilution-plated. RESULTS: Neutrophil incubation with S. aureus eliminated the S. aureus PBP-2 band in all Western blots irrespective of the drug used to tag the PBPs. Time-kill of S. aureus grown in neutropenic or normal abscess fluid showed greater kill by all drugs in neutropenic abscess fluid (p=0.029 6h incubation). Killing difference between the media correlates with drug PBP-2 activity. CONCLUSIONS: Drug activity against S. aureus in vitro is changed by neutrophil incubation. The neutrophil-induced loss of S. aureus PBP-2 drug binding suggests novel host-bacterial interaction that may impinge on drug treatment of S. aureus infections.

Transplant-related bronchiolitis obliterans (BOS) demonstrates unique cytokine profiles compared to toxicant-induced BOS.

Bronchiolitis obliterans (BOS - bronchiolitis obliterans syndrome - clinical diagnosis; CBO-histopathologic diagnosis), is a chronic disease process of fibrosis and cellular deposition in airways, complicating long term survival following lung transplantation. BOS is also the result of sporadic toxicant exposure, with airway signs, symptoms, and histology indistinguishable from allograft rejection. This study establishes a transplant BOS model in MHC-mismatched rats and compares their cytokine profiles and histopathology to that of our established toxicant-induced BOS model. Both models result in lung histopathology similar to human disease. Cytokines and inflammation markers that are elevated in human transplant BOS (TGFbeta, iNOS, IFNgamma) were also elevated significantly in both models. Anti-nuclear antibody was absent from all sera in transplant or toxicant models exhibiting advanced airway pathology. The cytokine osteopontin was highly elevated in BAL early in toxicant-induced BOS, but increased late in the transplant-induced BOS model. The data show that BOS is a disease of a pathologic endpoint that is induced by different triggers and processes. The highly elevated BAL osteopontin early in the toxicant-induced BOS model suggests a need for evaluation in the diagnostic setting.

The effect of treatment with a protease inhibitor on mycobacterial infection.

Mycobacterial infection occurs frequently in patients that receive protease inhibitors, which are drugs used to treat AIDS, but are known for metabolic effects. Proteases of microbial antigens have been recognized as important regulators of host inflammation and cellular response. To evaluate protease inhibitor effect on a mycobacterial infection, a pilot animal model was established. Mycobacterium bovis (bacillus Calmette-Guerin, or BCG) infection was compared in rats that received ritonavir and those that did not. Tissues and serum from one drug-treated and one control were analyzed weekly. Fewer acid-fast bacilli (AFBs) were consistently found in the drug-treated group by 3 separate measures: culture of tissue homogenates on solid media, tissue granuloma counts on organ sections, and staining of tissues for AFBs. Possible mechanisms of the observed relative resistance to BCG infection in ritonavir-treated rats were explored, by evaluating M. bovis cell wall lipids and proteins and by measuring infection-related cytokines in treated and control animals.