Lymph nodes-The neglected battlefield in tuberculosis.

Lymph nodes, particularly thoracic lymph nodes, are among the most common sites of extrapulmonary tuberculosis (TB). However, Mycobacterium tuberculosis (Mtb) infection in these organs is understudied. Aside from being sites of initiation of the adaptive immune system, lymph nodes also serve as niches of Mtb growth and persistence. Mtb infection results in granuloma formation that disrupts and-if it becomes large enough-replaces the normal architecture of the lymph node that is vital to its function. In preclinical models, successful TB vaccines appear to prevent spread of Mtb from the lungs to the lymph nodes. Reactivation of latent TB can start in the lymph nodes resulting in dissemination of the bacteria to the lungs and other organs. Involvement of the lymph nodes may improve Bacille Calmette-Guerin (BCG) vaccine efficacy. Lastly, drug penetration to the lymph nodes is poor compared to blood, lung tissue, and lung granulomas. Future studies on evaluating the efficacy of vaccines and anti-TB drug treatments should include consideration of the effects on thoracic lymph nodes and not just the lungs.

Lymph nodes are sites of prolonged bacterial persistence during Mycobacterium tuberculosis infection in macaques.

Tuberculosis is commonly considered a chronic lung disease, however, extrapulmonary infection can occur in any organ. Even though lymph nodes (LN) are among the most common sites of extrapulmonary Mycobacterium tuberculosis (Mtb) infection, and thoracic LNs are frequently infected in humans, bacterial dynamics and the effect of Mtb infection in LN structure and function is relatively unstudied. We surveyed thoracic LNs from Mtb-infected cynomolgus and rhesus macaques analyzing PET CT scans, bacterial burden, LN structure and immune function. FDG avidity correlated with the presence of live bacteria in LNs at necropsy. Lymph nodes have different trajectories (increasing, maintaining, decreasing in PET activity over time) even within the same animal. Rhesus macaques are more susceptible to Mtb infection than cynomolgus macaques and this is in part due to more extensive LN pathology. Here, we show that Mtb grows to the same level in cynomolgus and rhesus macaque LNs, however, cynomolgus macaques control Mtb at later time points post-infection while rhesus macaques do not. Notably, compared to lung granulomas, LNs are generally poor at killing Mtb, even with drug treatment. Granulomas that form in LNs lack B cell-rich tertiary lymphoid structures, disrupt LN structure by pushing out T cells and B cells, introduce large numbers of macrophages that can serve as niches for Mtb, and destroy normal vasculature. Our data support that LNs are not only sites of antigen presentation and immune activation during infection, but also serve as important sites for persistence of significant numbers of Mtb bacilli.

Clostridium difficile Enterocolitis in a Captive Geoffroy's Spider Monkey (Ateles geoffroyi) and Common Marmosets (Callithrix jacchus).

Clostridium difficile is a well-documented cause of enterocolitis in several species, including humans, with limited documentation in New World nonhuman primates. We report several cases of C. difficile-associated pseudomembranous enterocolitis, including a case in a Geoffroy's spider monkey (Ateles geoffroyi) and several cases in common marmosets (Callithrix jacchus). The histologic lesions included a spectrum of severity, with most cases characterized by the classic "volcano" lesions described in humans and several other animal species. C. difficile was isolated from the colon of the spider monkey, while the presence of toxin A or toxin B or of the genes of toxin A or B by polymerase chain reaction served as corroborative evidence in several affected marmosets. C. difficile should be considered a cause of enterocolitis in these species.

Safe use of anti-CD154 monoclonal antibody in pig islet xenotransplantation in monkeys.

Anti-CD154mAb is a powerful co-stimulation blockade agent that is efficacious in preventing rejection, even in xenogeneic settings. It has been used in the majority of successful long-term pig-to-non-human primate islet transplantation models. However, its clinical use was halted as a result of thromboembolic complications that were also observed in preclinical and clinical organ transplantation models. An anti-CD154mAb was administered to 14 streptozotocin-induced diabetic cynomolgus monkey recipients of porcine islets, some of which received the agent for many months. Monkeys were monitored for complications, and blood monitoring was carried out frequently. After euthanasia, multiple biopsies of all organs were examined for histological features of thromboembolism. Anti-CD154mAb prevented rejection of genetically engineered pig islets in all monkeys. No significant complications were attributable specifically to anti-CD154mAb. There was no evidence of thromboembolism in multiple histological sections from all major organs, including the brain. Our data suggest that in diabetic monkeys with pig islet grafts, anti-CD154mAb would appear to be an effective and safe therapy, and is not associated with thromboembolic complications.

CD4+ T cells are homeostatic regulators during Mtb reinfection.

Immunological priming - either in the context of prior infection or vaccination - elicits protective responses against subsequent Mycobacterium tuberculosis (Mtb) infection. However, the changes that occur in the lung cellular milieu post-primary Mtb infection and their contributions to protection upon reinfection remain poorly understood. Here, using clinical and microbiological endpoints in a non-human primate reinfection model, we demonstrate that prior Mtb infection elicits a long-lasting protective response against subsequent Mtb exposure and that the depletion of CD4+ T cells prior to Mtb rechallenge significantly abrogates this protection. Leveraging microbiologic, PET-CT, flow cytometric, and single-cell RNA-seq data from primary infection, reinfection, and reinfection-CD4+ T cell depleted granulomas, we identify differential cellular and microbial features of control. The data collectively demonstrate that the presence of CD4+ T cells in the setting of reinfection results in a reduced inflammatory lung milieu characterized by reprogrammed CD8+ T cell activity, reduced neutrophilia, and blunted type-1 immune signaling among myeloid cells, mitigating Mtb disease severity. These results open avenues for developing vaccines and therapeutics that not only target CD4+ and CD8+ T cells, but also modulate innate immune cells to limit Mtb disease.

Evaluation of IL-1 Blockade as an Adjunct to Linezolid Therapy for Tuberculosis in Mice and Macaques.

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during M. tuberculosis infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area.

Incidence of Neoplasia in Pigs and Its Relevance to Clinical Organ Xenotransplantation.

As clinical pig organ xenotransplantation draws closer, more attention is being paid to diseases that affect pigs and those that provide a potential risk to human recipients of pig organs. Neoplasia arising from the pig organ graft is one such concern. Various tumors and other neoplastic diseases are well known to show increased incidence in organ allotransplant recipients receiving immunosuppressive therapy. Whether this effect will prove to be the case after xenotransplantation has not yet been established. Malignant tumors in young pigs are rare, with lymphosarcoma, nephroblastoma, and melanoma being the most common. The combination of noninvasive techniques and intraoperative examination of the pig organ likely will readily confirm that a pig organ graft is tumor-free before xenotransplantation. Posttransplantion lymphoproliferative disorder (PTLD) is a concern after allotransplantation, but the incidence after solid organ allotransplantation is low when compared with hematopoietic cell allotransplantation (for example, bone marrow transplantation), unless immunosuppressive therapy is particularly intensive. Organ-source pigs used for clinical xenotransplantation will be bred and housed under designated pathogen-free conditions and will be free of the γ-herpesvirus that is a key factor in the development of PTLD in pigs. Therefore if a recipient of a pig xenograft develops PTLD, it will almost certainly be of recipient origin. The increasing availability of organs from pigs genetically-engineered to protect them from the human immune response likely will diminish the need for intensive immunosuppressive therapy. Considering the low incidence of malignant disease in young pigs, donor-derived malignancy is likely to be rare in patients who receive pig organ grafts. However, if the graft remains viable for many years, the incidence of graft malignancy may increase.

Life-supporting Kidney Xenotransplantation From Genetically Engineered Pigs in Baboons: A Comparison of Two Immunosuppressive Regimens.

BACKGROUND: The aims of this study were to evaluate the efficacy of US Food and Drug Administration-approved drugs in genetically engineered pig-to-baboon kidney xenotransplantation and compare the results with those using an anti-CD40 monoclonal antibody (mAb)-based regimen. METHODS: Ten life-supporting kidney transplants were carried out in baboons using α1,3-galactosyltransferase gene-knockout/CD46 pigs with various other genetic manipulations aimed at controlling coagulation dysregulation. Eight transplants resulted in informative data. Immunosuppressive therapy consisted of induction with antithymocyte globulin and anti-CD20mAb, and maintenance based on either (1) CTLA4-Ig and/or tacrolimus (+rapamycin or mycophenolate mofetil) (GroupA [US Food and Drug Administration-approved regimens], n = 4) or (2) anti-CD40mAb + rapamycin (GroupB, n = 4). All baboons received corticosteroids, interleukin-6R blockade, and tumor necrosis factor-α blockade. Baboons were followed by clinical and laboratory monitoring of kidney function, coagulation, and immune parameters. At euthanasia, morphological and immunohistochemical studies were performed on the kidney grafts. RESULTS: The median survival in GroupB was 186 days (range 90-260), which was significantly longer than in GroupA; median 14 days (range 12-32) (P < 0.01). Only GroupA baboons developed consumptive coagulopathy and the histopathological features of thrombotic microangiopathic glomerulopathy and interstitial arterial vasculitis. CONCLUSIONS: Recognizing that the pig donors in each group differed in some genetic modifications, these data indicate that maintenance immunosuppression including anti-CD40mAb may be important to prevent pig kidney graft failure.

Physiologic Aspects of Pig Kidney Transplantation in Nonhuman Primates.

Xenotransplantation can provide a solution to the current shortage of human organs for patients with terminal renal failure. The increasing availability of genetically engineered pigs, effective immunosuppressive therapy, and antiinflammatory therapy help to protect pig tissues from the primate immune response and can correct molecular incompatibilities. Life-supporting pig kidney xenografts have survived in NHP for more than 6 mo in the absence of markers of consumptive coagulopathy. However, few reports have focused on the physiologic aspects of life-supporting pig kidney xenografts. We have reviewed the literature regarding pig kidney xenotransplantation in NHP. The available data indicate (1) normal serum creatinine, (2) normal serum electrolytes, except for a trend toward increased calcium levels and a transient rise in phosphate followed by a fall to slightly subnormal values, (3) minimal or modest proteinuria without hypoalbuminemia (suggesting that previous reports of proteinuria likely were due to a low-grade immune response rather than physiologic incompatibilities), (4) possible discrepancies between pig erythropoietin and the primate erythropoietin receptor, and (5) significant early increase in kidney graft size, which might result from persistent effects of pig growth hormone. Further study is required regarding identification and investigation of physiologic incompatibilities. However, current evidence suggests that, in the absence of an immune response, a transplanted pig kidney likely would satisfactorily support a human patient.

Transient and permanent arterial occlusions modeling poststroke epilepsy in aging rats.

The pathophysiological mechanisms of epileptogenesis following ischemic stroke in the aged brain are not well understood, largely due to limited developments in animal modeling of poststroke epilepsy (PSE). A recent study in our laboratory (Kelly et al., 2018) using transient (3 h) unilateral middle cerebral artery (MCA) and common carotid artery (CCA) occlusion (MCA/CCAo) in 4- and 20-month-old Fischer (F344) rats resulted in epileptic seizures in both age groups; age and infarction factors independently had effects on seizure frequency. We hypothesized that permanent unilateral MCA/CCAo, a simpler model, was capable of producing results comparable to those of transient MCA/CCAo. In this study, we performed permanent MCA/CCAo and compared it to transient MCA/CCAo in 76 4-, 12-, and 20-month-old F344 rats; 41 (54%) animals experienced early, unexpected mortality. The remaining 35 (46%) animals had depth electrodes implanted. Prior to implantation of depth electrodes, 9 (26%) of these 35 animals (26%) were monitored periodically by video alone before video-EEG monitoring (17,837 h total) to assess the potential development of PSE. No EEG recordings were obtained from 12- or 20-month-old transient occlusion or 20-month-old permanent occlusion animals due to premature deaths. Five animals (14%) demonstrated epileptic seizure activity after MCA/CCAo: one 4-month-old transient occlusion animal, one 4-month-old permanent occlusion animal, and three 12-month-old permanent occlusion animals. Of these 5 animals, all but the 4-month-old permanent animal demonstrated 1-4 Hz spike-wave discharges variably associated with inactivity or frank motor arrest, and 2 animals (4- and 12-month-old permanent) demonstrated generalized ictal EEG discharges associated with grade 5 convulsive activity. All animals monitored with video-EEG demonstrated generalized 7-9 Hz spike-wave discharges, innate in F344 animals and distinct from lesion-induced epileptic seizures. Gross inspection of brains revealed variability in lesion presence and size among age groups and occlusion types. Comparison of infarct volumes of permanent MCA/CCAo animals (2.9 ± 1.29 mm3, n = 6) with those of transient MCA/CCAo animals (1.7 ± 0.31 mm3, n = 3) was not significant (p = 0.44) due to the small sample size. Timm staining revealed no evidence of mossy fiber sprouting in 7 animals tested, only one of which was known to be epileptic (4-month-old transient). These results provide evidence of focal nonconvulsive electrographic ictal discharges and behavioral seizures in both permanent and transient MCA/CCAo animals lesioned at 4- or 12-months-of-age and support the use of arterial ligation as a viable method for modeling PSE.

B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy.

BACKGROUND: In the pig-to-baboon artery patch model with no immunosuppressive therapy, a graft from an α1,3-galactosyltransferase gene-knockout (GTKO) pig elicits a significant anti-nonGal IgG response, indicating sensitization to the graft. A costimulation blockade-based regimen, e.g., anti-CD154mAb or anti-CD40mAb, prevents sensitization. However, neither of these agents is currently FDA-approved. The aim of the present study was to determine the efficacy of FDA-approved agents on the T and B cell responses. METHODS: Artery patch xenotransplantation in baboons was carried out using GTKO/CD46 pigs with (n = 2) or without (n = 1) the mutant transgene for CIITA-knockdown. Immunosuppressive therapy consisted of induction with ATG and anti-CD20mAb, and maintenance with different combinations of CTLA4-Ig, tacrolimus, and rapamycin. In addition, all 3 baboons received daily corticosteroids, the IL-6R blocker, tocilizumab, at regular intervals, and the TNF-α blocker, etanercept, for the first 2 weeks. Recipient blood was monitored for anti-nonGal antibody levels by flow cytometry (using GTKO/CD46 pig aortic endothelial cells), and mixed lymphocyte reaction (MLR). CD22+B cell profiles (naïve [IgD+/CD27-], non-switched memory [IgD+/CD27+], and switched memory [IgD-/CD27+] B cell subsets) were measured by flow cytometry. At 6 months, the baboons were euthanized and the grafts were examined histologically. RESULTS: No elicited anti-pig antibodies developed in any baboon. The frequency of naïve memory B cells increased significantly (from 34% to 90%, p = 0.0015), but there was a significant decrease in switched memory B cells (from 17% to 0.5%, p = 0.015). MLR showed no increase in the proliferative T cell response in those baboons that had received CTLA4-Ig (n = 2). Histological examination showed few or no features of rejection in any graft. CONCLUSIONS: The data suggest that immunosuppressive therapy with only FDA-approved agents may be adequate to prevent an adaptive immune response to a genetically-engineered pig graft, particularly if CTLA4-Ig is included in the regimen, in part because the development of donor-specific memory B cells is inhibited.

Data on B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy.

This report is related to the research article entitled "B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy" (Yamamoto et al., in press). Herein we provide the data regarding pig artery patch xenotransplantation into the baboon׳s aorta, trough levels of tacrolimus and rapamycin in the blood after transplantation, analysis of B cell phenotype on the basis of IgD and CD27 expression in the blood, and analysis of T cell phenotype on the basis of CD28 and CD95 expression in the blood.

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Active transforming growth factor-ß is associated with phenotypic changes in granulomas after drug treatment in pulmonary tuberculosis.

BACKGROUND: Tuberculosis (TB) chemotherapy clears bacterial burden in the lungs of patients and allows the tuberculous lesions to heal through a fibrotic process. The healing process leaves pulmonary scar tissue that can impair lung function. The goal of this study was to identify fibrotic mediators as a stepping-stone to begin exploring mechanisms of tissue repair in TB. METHODS: Hematoxylin and eosin staining and Masson's trichrome stain were utilized to determine levels of collagenization in tuberculous granulomas from non-human primates. Immunohistochemistry was then employed to further interrogate these granulomas for markers associated with fibrogenesis, including transforming growth factor-ß (TGFß), α-smooth muscle actin (αSMA), phosphorylated SMAD-2/3, and CD163. These markers were compared across states of drug treatment using one-way ANOVA, and Pearson's test was used to determine the association of these markers with one another. RESULTS: TGFß and αSMA were present in granulomas from primates with active TB disease. These molecules were reduced in abundance after TB chemotherapy. Phosphorylated SMAD-2/3, a signaling intermediate of TGFß, was observed in greater amounts after 1 month of drug treatment than in active disease, suggesting that this particular pathway is blocked in active disease. Collagen production during tissue repair is strongly associated with TGFß in this model, but not with CD163+ macrophages. CONCLUSIONS: Tissue repair and fibrosis in TB that occurs during drug treatment is associated with active TGFß that is produced during active disease. Further work will identify mechanisms of fibrosis and work towards mitigating lung impairment with treatments that target those mechanisms.

Very Low Doses of Mycobacterium tuberculosis Yield Diverse Host Outcomes in Common Marmosets (Callithrix jacchus).

Identifying and refining small-animal models of tuberculosis that recapitulate aspects of human Mycobacterium tuberculosis infection can contribute to advancing our understanding of critical facets of the disease. To study the effects of very low-dose infections with 2 strains of M. tuberculosis on disease progression and survival in common marmosets, animals were challenged with strains Erdman and CDC1551 at doses ranging from 1 to 12 cfu. These data revealed that the susceptibility of marmosets to M. tuberculosis infection is influenced by strain virulence and initial dose. Marmoset infection with the Erdman strain, even at very low doses, resulted in rapid disease progression associated with severe weight loss, extensive pathology, and poor survival. By contrast, challenge with the less virulent CDC1551 strain resulted in slower disease progression, delayed weight loss, and prolonged survival. One marmoset infected with CDC1551 at a very low dose (approximately 1 cfu) was able to contain and control M. tuberculosis infection in a subclinical state that persisted as long as 300 d. These findings underscore the critical importance of understanding the heterogeneity in host outcome that can arise in association with different infectious doses and strains in the marmoset model of tuberculosis.

Mortality associated with melarsomine dihydrochloride administration in two North American river otters (Lontra canadensis) and a red panda (Ailurus fulgens fulgens).

Two adult North American river otters (Lontra canadensis) and an adult red panda (Ailurus fulgens fulgens) at three separate institutions died within 22 hr after receiving single 2.5- to 2.7-mg/kg doses of melarsomine dihydrochloride administered in the epaxial musculature as a treatment for filarid nematodes. One otter had a suspected Dirofilaria immitis infection, the other had a confirmed D. lutrae infection, and the red panda had a confirmed Dirofilaria sp. infection, presumably with D. immitis. Postmortem examinations revealed similar gross lesions, although they were less severe in the red panda. The trachea and primary bronchi contained abundant foamy fluid, the lungs were mottled with areas of consolidation, and the pulmonary parenchyma exuded abundant fluid at the cut section. Histologic evaluation revealed acute pulmonary edema, which resulted in respiratory failure and death. There may have been direct pulmonary cellular toxicity of melarsomine dihydrochloride or a severe systemic anaphylactic reaction to antigens released after parasite death. An idiosyncratic drug reaction or a low therapeutic index of melarsomine probably caused the death of the three individuals. Melarsomine dihydrochloride use should be avoided in North American river otters and red pandas.

Subglottic stenosis examined as a fibrotic response to airway injury characterized by altered mucosal fibroblast activity.

OBJECTIVE: To investigate the association between mucosal fibroblast activity and subglottic stenosis (SGS) development. DESIGN: Prospective study of an animal model of SGS. SETTING: Academic research laboratory. SUBJECTS: New Zealand white rabbits were assigned to either the cricothyroidotomy and carbon dioxide laser injury group or the cricothyroidotomy and silver nitrate injury group. Airways were excised for histologic analysis and the establishment of primary fibroblast cultures. Lesions from surgical excision of established SGS and subglottic tissue were used to analyze SGS recurrence. INTERVENTIONS: The subglottis was approached via cricothyroidotomy and was subjected to either carbon dioxide laser or silver nitrate injury before closure. The SGS lesions were excised at 8 to 10 weeks and were used to establish explants for fibroblast culture. The animals underwent recovery for an additional 14 days to follow recurrence of SGS. After 14 days, all the animals were killed humanely, and subglottic tissue was harvested for histologic evaluation. Rates of migration and contraction of SGS and normal airway fibroblasts were assayed using established in vitro methods under basal conditions and with prostaglandin E(2) treatment. MAIN OUTCOME MEASURES: For in vivo studies, injury, healing, and scarring of the mucosa and cartilage were the primary measures. For cultured fibroblast experiments, cellular responses of fibroblasts from normal and stenosed mucosa were compared and contrasted. RESULTS: Mucosal injury resulted in acute fibroplasia and chronic SGS, surgical excision of mature SGS at 8 weeks resulted in rapid recurrence of stenosis, and SGS-derived fibroblasts were relatively refractory to the effects of prostaglandin E(2) on migration and contraction. CONCLUSIONS: Subglottic stenosis represents a fibrotic airway repair process that involves fibroblasts that produce recurrent, excessive scar formation. We suggest that SGS development and recurrence may be partially dictated by altered fibroblast responsiveness to antifibroplastic signals during mucosal repair.

Absence of Mycobacterium avium subsp. paratuberculosis in the microdissected granulomas of Crohn's disease.

The etiology of Crohn's disease remains unknown with inflammatory, infectious, and/or genetic causes suspected. Granulomatous inflammation is a characteristic feature of the disorder, resembling the tissue response to mycobacterium. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent in Johne's disease, a chronic ulcerative intestinal condition in cattle, and has been implicated as a likely candidate. We carefully microdissected the granulomas from the paraffin-embedded resection specimens of 18 patients with well-established Crohn's disease. The DNA obtained was PCR amplified for the IS900 and IS1311 repeat elements of MAP, PCR product size maintained at 101 and 124 base pairs, respectively. Archival tissue from bovine Johne's disease was used as a positive control. MAP-specific DNA, confirmed by sequencing and comparison with prototype strain sequence, was appropriately amplified from the positive control. None of the Crohn's disease cases yielded a positive amplification product, failing to support a role for the organism in the pathogenesis of this illness.