Necrotic Granulomas Research Articles

Lipid Peroxidation and Type I Interferon Coupling Fuels Pathogenic Macrophage Activation Causing Tuberculosis Susceptibility.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNβ superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue

The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Thyroid tuberculosis mimicking carcinoma: A case report and review of the literature

Thyroid tuberculosis (TT) is a very rare condition, even in endemic countries, this can be explained by the high resistance of the thyroid gland to infections. It can be primary or secondary to disseminated infection. A solitary thyroid nodule is the most common reason for consultation, and can present in the form of a cystic nodule. He can even present a picture of a thyroid abscess with pain, fever, and other nonspecific signs and symptoms. We present the case of a 65-year-old woman who consulted for a lower median cervical swelling that had been present for 4 years associated with upper right laterocervical lymphadenopathy mimicking a thyroid carcinoma. As symptoms and imaging features are nonspecific, diagnosis is challenging. Fine-needle aspiration cytology is a useful technique for preoperative diagnosis. Typical caseous necrotic granulomas are the hallmark in histological examination. Thyroid tuberculosis should be considered in the differential diagnosis of a thyroid nodule and of neck mass, in order to prevent unnecessary surgery. Histopathology and culture of MTB remain a key step for the diagnosis.

A Comparison Study of Lymph Node Tuberculosis and Sarcoidosis Involvement to Facilitate Differential Diagnosis and to Establish a Predictive Score for Tuberculosis.

Background: Tuberculosis (TB) and sarcoidosis are two common granulomatous diseases involving lymph nodes. Differential diagnosis is not always easy because pathogen demonstration in tuberculosis is not always possible and both diseases share clinical, radiological and histological patterns. The aim of our study was to identify factors associated with each diagnosis and set up a predictive score for TB. Methods: All cases of lymph node tuberculosis and sarcoidosis were retrospectively reviewed. Demographics, clinical characteristics, laboratory and imaging data, and microbiological and histological results were collected and compared. Results: Among 441 patients screened, 192 patients were included in the final analysis. The multivariate analysis showed that weight loss, necrotic granuloma, normal serum lysozyme level and hypergammaglobulinemia were significantly associated with TB. A risk score of TB was built based on these variables and was able to discriminate TB versus sarcoidosis with an AUC of 0.85 (95% CI: 0.79-0.91). Using the Youden's J statistic, its most discriminant value (-0.36) was associated with a sensitivity of 80% and a specificity of 75%. Conclusions: We developed a score based on weight loss, necrotic granuloma, normal serum lysozyme level and hypergammaglobulinemia with an excellent capacity to discriminate TB versus sarcoidosis. This score needs still to be validated in a multicentric prospective study.

The role of cholesterol and its oxidation products in tuberculosis pathogenesis.

Mycobacterium tuberculosis causes tuberculosis (TB), one of the world's most deadly infections. Lipids play an important role in M. tuberculosis pathogenesis. M. tuberculosis grows intracellularly within lipid-laden macrophages and extracellularly within the cholesterol-rich caseum of necrotic granulomas and pulmonary cavities. Evolved from soil saprophytes that are able to metabolize cholesterol from organic matter in the environment, M. tuberculosis inherited an extensive and highly conserved machinery to metabolize cholesterol. M. tuberculosis uses this machinery to degrade host cholesterol; the products of cholesterol degradation are incorporated into central carbon metabolism and used to generate cell envelope lipids, which play important roles in virulence. The host also modifies cholesterol by enzymatically oxidizing it to a variety of derivatives, collectively called oxysterols, which modulate cholesterol homeostasis and the immune response. Recently, we found that M. tuberculosis converts host cholesterol to an oxidized metabolite, cholestenone, that accumulates in the lungs of individuals with TB. M. tuberculosis encodes cholesterol-modifying enzymes, including a hydroxysteroid dehydrogenase, a putative cholesterol oxidase, and numerous cytochrome P450 monooxygenases. Here, we review what is known about cholesterol and its oxidation products in the pathogenesis of TB. We consider the possibility that the biological function of cholesterol metabolism by M. tuberculosis extends beyond a nutritional role.

Thoracic manifestations of tularaemia: a case series.

Tularaemia is a zoonotic disease caused by Francisella tularensis, a highly virulent bacterium that affects humans and small wild animals. It is transmitted through direct contact with infected animals or indirectly through contaminated soil, water or arthropod bites (e.g. ticks). Primary thoracic manifestations of tularaemia are infrequent and, therefore, a diagnostic challenge for clinicians. We report six tularaemia cases with exclusively thoracic involvement diagnosed in a clinic for pulmonary diseases in Bavaria between 10/2020 and 02/2022. All patients lived or were active in rural areas, four reported a recent tick bite. All patients presented with thoracic lymphadenopathy and pulmonary tumours or consolidations; all underwent bronchoscopy with EBUS-TBNA of lymph nodes, three lung biopsies as well. Five patients showed inflammatory changes in the endobronchial mucosa. The main histological findings were necrotic epithelioid granulomas with remarkable granulocyte infiltration. All cases were identified by positive serology, five by PCR (here identification of F.t. ssp. Holarctica) from biopsy as well. As first-line therapy, oral ciprofloxacin was given (5/6); in 2/6 cases, a combination of quinolone-rifampicin was given. Pulmonary tularaemia may occur after tick bites and without extrathoracic manifestations. In patients who present with thoracic lymphadenopathy and pulmonary consolidations and who are exposed to increased outdoor activities, tularaemia should be included in the diagnostic pathway. Histologically, the presence of neutrophil-granulocyte infiltrations might help to distinguish tularaemia from other granulomatous infections, e.g. tuberculosis. The combination of quinolone-rifampicin rather than i.v. gentamicin reduced length of hospital stay in two patients.

Immune mapping of human tuberculosis and sarcoidosis lung granulomas.

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas "multifocal" granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40-60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

PULMONARY HISTOPLASMOSIS IN A PATIENT WITH CROHN’S DISEASE ON ANTI-TNF INHIBITOR THERAPY

Abstract INTRODUCTION Infliximab, an anti-tumor necrosis factor (TNF) alpha inhibitor, is a commonly used biologic therapy for patients with inflammatory bowel disease (IBD). However, the increased risk of serious infections, including both new and re-activated infections, is a major concern while patients are on this therapy. Often the presence or development of such serious infections necessitates stopping treatment with anti-TNF inhibitors. We describe a case of a patient who developed Histoplasmosis while on infliximab but was later restarted on the same therapy due to risk of severe Crohn’s disease while maintaining him on ongoing Histoplasmosis treatment. CASE DESCRIPTION We describe the case of a 34-year-old male with a past medical history of Crohn’s disease on infliximab, hypertension, depression, and nephrolithiasis who presented to the hospital with dyspnea on exertion, cough, malaise, and night sweats, and was found to have multifocal pulmonary infiltrates on imaging, concerning for atypical pneumonia. He underwent a bronchoscopy and BAL, with pathology reports showing small oval shaped yeast within necrotic granulomas suspicious for Histoplasmosis. He was taken off infliximab and started on liposomal amphotericin for Histoplasma treatment initially, subsequently transitioned to itraconazole for total of 12 months. He was discharged home but was subsequently re-admitted to the hospital with a Crohn’s flare after being taken off infliximab. He was treated with steroids and started on vedolizumab for more gut-selectivity after a multidisciplinary risk/benefit discussion. However, patient was subsequently re-admitted with diarrhea and hematochezia, due to a Crohn’s disease flare while still on steroid taper and having received 2 doses of vedolizumab. He was transitioned to IV steroids and had a colonoscopy which showed large ulcers and mucosal inflammatory changes with a Simple endoscopic score for Crohn’s disease of 18. After a multidisciplinary discussion, he was restarted on infliximab. He was slowly tapered off steroids as his symptoms improved. Repeat colonoscopy after restarting infliximab showed improved but ongoing mucosal inflammatory changes with a Simple endoscopic score for Crohn’s disease of 7. DISCUSSION While anti-TNF inhibitor biologics are commonly used for patients with IBD, opportunistic and fungal infections remain a significant concern while on immunosuppressing biologic therapies, which necessitates stopping the anti-TNF inhibitor. Previously, a study has shown that it is safe to restart anti-TNF therapy after treatment for Histoplasmosis is completed for a 12-month duration. Our case shows that in patients such as ours with severe Crohn’s disease flares while off infliximab, treatment with anti-TNF inhibitors can be resumed after having a multidisciplinary risk/benefit discussion. Figure 1 Colonoscopy findings showing large ulcers and significant mucosal inflammatory changes after patient started on vedolizumab and admitted for Crohn’s Disease flare. Figure 2 Colonoscopy findings showing ongoing mucosal inflammation but improved from prior after restarting treatment with infliximab.

BRONCHOPULMONARY ASPERGILLOSIS WITH COMORBID GRANULOMATOUS POLYANGIITIS IN A PATIENT WHO PRESENTED WITH EXOPHTHALMOS: A CASE REPORT

There have been no reports of the coexistence of allergic bronchopulmonary aspergillosis (ABPA) and granulomatosis with polyangiitis (GPA). The first case of ABPA with comorbid GPA that developed exophthalmos is reported. A 69-year-old man was referred to our hospital for exophthalmos, fever, anorexia and weight loss. The patient had been diagnosed with ABPA six years earlier, which had been repeatedly treated but recurred with oral corticosteroids with or without antifungal therapy. The laboratory data on referral showed elevations of the white blood cell count, C-reactive protein and specific immunoglobulin E against Aspergillus fumigatus, but antineutrophil cytoplasmic antibody was not positive. Urinalysis showed proteinuria. Paranasal sinus and chest computed tomography showed sinusitis with osteochondral destruction, bronchiectasis, mucus plugging, and a pulmonary nodule. Orbital magnetic resonance imaging showed swelling of the medial rectus muscle and peripheral mass. The intraorbital tissue biopsy showed a necrotic granuloma and necrotizing vasculitis. The patient was diagnosed with GPA, on the basis of the Ministry of Health, Labour and Welfare's criteria of Japan. The patient was treated with induction therapy consisting of glucocorticoids and rituximab, and his symptoms improved. Though the pathogenesis common to ABPA and GPA remains unknown, neutrophilic inflammation induced by airway Aspergillus persistent infection might be involved. Study of further cases is needed.

Serological, Morphological and Molecular Diagnosis of Mycobacterium bovis in Cattle at Sulaimani Province of Iraq

Background:Mycobacterium bovis (M. bovis) causes bovine tuberculosis (BTB), is an endemic disease in cattle and poses a high risk of spreading to humans.Objective:This study aimed to determine M. bovis in cattle and assess the similarities between cattle and humans through molecular methods and histopathological examinations.Methodology:Randomly, blood samples from 411 healthy appearance cows (1% of the target population) in five districts in Sulaimani province, Iraq, were collected from January to March 2022. Sera were obtained immediately and used for ELISA test to determine M. bovis. Additionally, the disease prevalence was confirmed by gross lesions at the slaughterhouse and histopathological examination of collected lymph nodes. Moreover, a PCR assay was used to detect M. bovis in suspected cow samples and previously diagnosed human samples. Gene sequencing and phylogenetic tree analysis were also done to determine the molecular differences between animal and human M. bovis.Results:Using an ELISA test, 46 (11.11%) of 414 samples were positive, while 368 (88.89%) were negative without significant differences between the districts (p>0.05). According to postmortem lesions at the slaughterhouse, only three cows were infected with TB, and typical gross lesions were calcified necrotic and multiple well-demarcated granulomas. The molecular test using two primers (CSB2 and oxyR gene) revealed that M. bovis was found in animal and human extra-pulmonary lymph nodes with no molecular change.Conclusion:Healthy cows harbored M. bovis, the causative agent of a contagious disease that spreads and causes a persistent health problem in humans.

Temporal and cellular analysis of granuloma development in mycobacterial infected adult zebrafish.

Because granulomas are a hallmark of tuberculosis pathogenesis, the study of the dynamic changes in their cellular composition and morphological character can facilitate our understanding of tuberculosis pathogenicity. Adult zebrafish infected with Mycobacterium marinum form granulomas that are similar to the granulomas in human patients with tuberculosis and therefore have been used to study host-mycobacterium interactions. Most studies of zebrafish granulomas, however, have focused on necrotic granulomas, while a systematic description of the different stages of granuloma formation in the zebrafish model is lacking. Here, we characterized the stages of granulomas in M. marinum-infected zebrafish, including early immune cell infiltration, nonnecrotizing granulomas, and necrotizing granulomas, using corresponding samples from patients with pulmonary tuberculosis as references. We combined hematoxylin and eosin staining and in situ hybridization to identify the different immune cell types and follow their spatial distribution in the different stages of granuloma development. The macrophages in zebrafish granulomas were shown to belong to distinct subtypes: epithelioid macrophages, foamy macrophages, and multinucleated giant cells. By defining the developmental stages of zebrafish granulomas and the spatial distribution of the different immune cells they contain, this work provides a reference for future studies of mycobacterial granulomas and their immune microenvironments.

Phosphatidylinositolmannoside vaccination induces lipid-specific Th1-responses and partially protects guinea pigs from Mycobacterium tuberculosis challenge

The concept of donor-unrestricted T cells (DURTs) comprises a heterogeneity of lymphoid cells that respond to an abundance of unconventional epitopes in a non-MHC-restricted manner. Vaccinologists strive to harness this so far underexplored branch of the immune system for new vaccines against tuberculosis. A particular division of DURTs are T cells that recognize their cognate lipid antigen in the context of CD1-molecules. Mycobacteria are characterized by a particular lipid-rich cell wall. Several of these lipids have been shown to be presented to T cells via CD1b-molecules. Guinea pigs functionally express CD1b and are hence an appropriate small animal model to study the role of CD1b-restricted, lipid-specific immune responses. In the current study, guinea pigs were vaccinated with BCG or highly-purified, liposome-formulated phosphatidylinositol-hexa-mannoside (PIM6) to assess the effect of CD1-restricted DURTs on the course of infection after virulent Mycobacterium tuberculosis (Mtb) challenge. Robust PIM6-specific T cell-responses were observed both after BCG- and PIM6-vaccination. The cellular response was significantly reduced in the presence of monoclonal, CD1b-blocking antibodies, indicating that a predominant part of this reactivity was CD1b-restricted. When animals were challenged with Mtb, BCG- and PIM6-vaccinated animals showed significantly reduced pathology, smaller necrotic granulomas in lymph node and spleen and reduced bacterial loads. While BCG conferred an almost sterile protection in this setting, compared to control animals’ lesions were reduced roughly by two thirds in PIM6-vaccinated. Comprehensive histological and transcriptional analyses in the draining lymph node revealed that protected animals showed reduced transcription-levels of inflammatory cyto- and chemokines and higher levels of CD1b-expression on professional antigen cells compared to controls. Although BCG as a comparator induced by far stronger effects, our observations in the guinea pig model suggest that CD1b-restricted, PIM6-reactive DURTs contribute to immune-mediated containment of virulent Mtb.

Increased incidence of thyroid disease in patients with sarcoidosis: a systematic review and meta-analysis.

Sarcoidosis is a multiple systemic granulomatous disease, and its main pathological feature is non-caseous necrotic epithelial granuloma. The pathogenesis is not fully understood. The prevalence of thyroid disease is likely higher among individuals with sarcoidosis. However, this association still lacks clinical evidence. To estimate the incidence of thyroid disease in patients with sarcoidosis. A literature search was conducted using PubMed, Web of Science, Embase, and China National Knowledge Infrastructure literature databases. Fixed- or random-effects models were used for analysis according to heterogeneity. The results were subjected to meta-analysis with odds ratios (ORs) and corresponding 95% confidence intervals (CIs). In total, six articles were included in this meta-analysis, which involved 2044 sarcoidosis cases and 5652 controls. The studies found that the incidence of thyroid disease in patients with sarcoidosis was significantly increased compared to the controls (OR 3.28, 95% CI 1.83-5.88). This systematic review is the first to evaluate the incidence of thyroid disease in sarcoidosis patients, which was increased compared with the controls, suggesting that sarcoidosis patients should be screened for thyroid disease.

L-Tyrosine Limits Mycobacterial Survival in Tuberculous Granuloma.

Caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), tuberculosis (TB) remains a massive global public health issue. A well-known and key TB trait is caseous necrotic granuloma, which allows mycobacteria to reactivate and disseminate, thus confounding TB eradication programs. Amino acid (AA) metabolism is key to regulating immune responses in Mtb infections; however, it is currently unclear if AAs can be used to treat tuberculous granulomas. Here, we screened 20 proteinogenic AAs using a Mycobacterium marinum-infected zebrafish granuloma model. Only L-tyrosine simultaneously reduced Mycobacterium marinum (M. marinum) levels in zebrafish larvae and adults and inhibited intracellular pathogen survival levels. Mechanistically, L-tyrosine significantly upregulated interferon-γ (IFN-γ) expression in M. marinum -infected zebrafish adults but not in larvae. Using N-acetylcysteine (NAC) to inhibit reactive oxygen species (ROS), L-tyrosine appeared to inhibit Mtb intracellular survival by promoting ROS production. Thus, L-tyrosine as a non-essential AA may reduce mycobacterial survival in both macrophages and tuberculous granulomas. Our research provides a platform for the clinical development of AAs for active or latent TB patients infected with drug-sensitive or drug-resistant Mtb.

A Novel Tool to Identify Bactericidal Compounds against Vulnerable Targets in Drug-Tolerant M. tuberculosis found in Caseum.

Caseous necrosis is a hallmark of tuberculosis (TB) pathology and creates a niche for drug-tolerant persisters within the host. Cavitary TB and high bacterial burden in caseum require longer treatment duration. An in vitro model that recapitulates the major features of Mycobacterium tuberculosis (Mtb) in caseum would accelerate the identification of compounds with treatment-shortening potential. We have developed a caseum surrogate model consisting of lysed and denatured foamy macrophages. Upon inoculation of Mtb from replicating cultures, the pathogen adapts to the lipid-rich matrix and gradually adopts a nonreplicating state. We determined that the lipid composition of ex vivo caseum and the surrogate matrix are similar. We also observed that Mtb in caseum surrogate accumulates intracellular lipophilic inclusions (ILI), a distinctive characteristic of quiescent and drug-tolerant Mtb. Expression profiling of a representative gene subset revealed common signatures between the models. Comparison of Mtb drug susceptibility in caseum and caseum surrogate revealed that both populations are similarly tolerant to a panel of TB drugs. By screening drug candidates in the surrogate model, we determined that the bedaquiline analogs TBAJ876 and TBAJ587, currently in clinical development, exhibit superior bactericidal against caseum-resident Mtb, both alone and as substitutions for bedaquiline in the bedaquiline-pretomanid-linezolid regimen approved for the treatment of multidrug-resistant TB. In summary, we have developed a physiologically relevant nonreplicating persistence model that reflects the distinct metabolic and drug-tolerant state of Mtb in caseum. IMPORTANCE M. tuberculosis (Mtb) within the caseous core of necrotic granulomas and cavities is extremely drug tolerant and presents a significant hurdle to treatment success and relapse prevention. Many in vitro models of nonreplicating persistence have been developed to characterize the physiologic and metabolic adaptations of Mtb and identify compounds active against this treatment-recalcitrant population. However, there is little consensus on their relevance to in vivo infection. Using lipid-laden macrophage lysates, we have designed and validated a surrogate matrix that closely mimics caseum and in which Mtb develops a phenotype similar to that of nonreplicating bacilli in vivo. The assay is well suited to screen for bactericidal compounds against caseum-resident Mtb in a medium-throughput format, allowing for reduced reliance on resource intensive animal models that present large necrotic lesions and cavities. Importantly, this approach will aid the identification of vulnerable targets in caseum Mtb and can accelerate the development of novel TB drugs with treatment-shortening potential.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-β-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-β-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Adjunctive Integrated Stress Response Inhibition Accelerates Tuberculosis Clearance in Mice.

Despite numerous advances in tuberculosis (TB) drug development, long treatment durations have led to the emergence of multidrug resistance, which poses a major hurdle to global TB control. Shortening treatment time therefore remains a top priority. Host-directed therapies that promote bacterial clearance and/or lung health may improve the efficacy and treatment duration of tuberculosis antibiotics. We recently discovered that inhibition of the integrated stress response, which is abnormally activated in tuberculosis and associated with necrotic granuloma formation, reduced bacterial numbers and lung inflammation in mice. Here, we evaluated the impact of the integrated stress response (ISR) inhibitor ISRIB, administered as an adjunct to standard tuberculosis antibiotics, on bacterial clearance, relapse, and lung pathology in a mouse model of tuberculosis. Throughout the course of treatment, ISRIB robustly lowered bacterial burdens compared to the burdens with standard TB therapy alone and accelerated the time to sterility in mice, as demonstrated by significantly reduced relapse rates after 4 months of treatment. In addition, mice receiving adjunctive ISRIB tended to have reduced lung necrosis and inflammation. Together, our findings identify the ISR pathway as a promising therapeutic target with the potential to shorten TB treatment durations and improve lung health. IMPORTANCE Necrosis of lung lesions is a hallmark of tuberculosis (TB) that promotes bacterial growth, dissemination, and transmission. This process is driven by the persistent hyperactivation of the integrated stress response (ISR) pathway. Here, we show that adjunctive ISR inhibition during standard antibiotic therapy accelerates bacterial clearance and reduces immunopathology in a clinically relevant mouse model of TB, suggesting that host-directed therapies that de-escalate these pathological stress responses may shorten TB treatment durations. Our findings present an important conceptual advance toward overcoming the challenge of improving TB therapy and lowering the global burden of disease.

Π-Π interactions stabilize PeptoMicelle-based formulations of Pretomanid derivatives leading to promising therapy against tuberculosis in zebrafish and mouse models

Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic and had limited bioavailability, we expected that their micellar formulations would overcome these limitations, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model almost completely eradicated the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug-loaded PeptoMicelles significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

A high‐resolution 3D atlas of the spectrum of tuberculous and COVID‐19 lung lesions

Our current understanding of the spectrum of TB and COVID‐19 lesions in the human lung is limited by a reliance on low‐resolution imaging platforms that cannot provide accurate 3D representations of lesion types within the context of the whole lung. To characterize TB and COVID‐19 lesions in 3D, we applied micro/nanocomputed tomography to surgically resected, postmortem, and paraffin‐embedded human lung tissue. We define a spectrum of TB pathologies, including cavitary lesions, calcium deposits outside and inside necrotic granulomas and mycetomas, and vascular rearrangement. We identified an unusual spatial arrangement of vasculature within an entire COVID‐19 lobe, and 3D segmentation of blood vessels revealed microangiopathy associated with hemorrhage. Notably, segmentation of pathological anomalies reveals hidden pathological structures that might otherwise be disregarded, demonstrating a powerful method to visualize pathologies in 3D in TB lung tissue and whole COVID‐19 lobes. These findings provide unexpected new insight into the spatial organization of the spectrum of TB and COVID‐19 lesions within the framework of the entire lung.

S2706 Orofacial Granulomatosis in Crohn's Disease

Introduction: Extra-intestinal manifestations (EIMs) of inflammatory bowel disease (IBD) are rare complications that affect roughly 6-25% of IBD patients. Orofacial granulomatosis (OFG) is a rare manifestation that does not have reliable epidemiologic data and is characterized by orofacial swelling with non-caseating granulomas seen on pathology. Case Description/Methods: A 23-year-old male with a history of Crohn’s colitis presented with loose stools and hematochezia for 7 months and 1 year of left sided lip swelling. He was diagnosed with Crohn’s disease at age 15 and was maintained on oral mesalamine and 6-mercaptopurine (6-MP). At age 16, the patient was evaluated for supraglottic edema. Urgent tonsillectomy was performed, with pathology revealing necrotizing and non-caseating granulomas. He recovered without complication. He achieved endoscopic remission at age 18 and his medications were discontinued. Unfortunately, he was lost to follow-up. During this subsequent presentation, his vitals were within normal limits. Exam was notable for left-sided lip and cheek swelling without mass or lesion. Bloodwork was notable for an ESR of 19 and CRP of 7.5. Lip biopsy showed normal squamous epithelium and non-caseating granulomas, consistent with OFG. Patient was treated for a Crohn’s flare with oral prednisone as a bridge to 6-MP. His OFG was treated with intralesional corticosteroids without relief and has on-going outpatient follow up. His current treatment plan includes further cycles of steroid injections before considering alternative therapies. His most recent colonoscopy showed endoscopic and histologic remission of his luminal Crohn’s while on infliximab. (Figure) Discussion: OFG is frequently seen as a manifestation of a systemic condition such as IBD. OFG is diagnosed by biopsy and clinical suspicion. OFG specific treatments include topical steroids and calcineurin inhibitors. Recent data suggests that a cinnamon/benzoate free diet may provide modest benefit to patients. Adequate treatment of underlying IBD is equally important. This patient has the classic symptoms and histopathologic findings of OFG and likely had undiagnosed OFG for many years given his tonsillar pathology years prior. Despite treatment, this patient has persistent and refractory symptoms. This case highlights a classic presentation of a rare IBD extra-intestinal manifestation and demonstrates how EIMs can occur even with well controlled IBD.Figure 1.: Orofacial Granulomatosis: Lip and Cheek Swelling.