Early Granulomas Research Articles

Kupffer cell and recruited macrophage heterogeneity orchestrate granuloma maturation and hepatic immunity in visceral leishmaniasis.

In murine models of visceral leishmaniasis (VL), parasitization of resident Kupffer cells (resKCs) is responsible for early growth of Leishmania infantum in the liver, which leads to granuloma formation and eventual parasite control. We employed the chronic VL model, and revealed an open niche established by KCs death and their migration outside of the sinusoids, resulting in their gradual replacement by monocyte-derived KCs (moKCs). While early granulomas were composed of resKCs, late granulomas were found outside of the sinusoids and contained resKC-derived macrophages, and monocyte-derived macrophages (momacs). ResKCs and moKCs within the sinusoids were identified as homeostatic/regulatory cells, while resKC-derived macrophages and momacs within late granulomas were pro-inflammatory. Despite the infection being largely confined to the resKC-derived macrophages, in the absence of monocyte recruitment, parasite control was strongly compromised. Macrophage heterogeneity, involving migration and reprogramming of resKCs, along with recruitment of monocyte-derived cells, is a hallmark of granuloma maturation and hepatic immunity in VL.

Histopathologic patterns of female genital tuberculosis with clinical correlation: a 10-year (2013–2022) retrospective cross-sectional study

ObjectiveUnderdiagnosis of female genital tuberculosis (FGTB) often leads to infertility. In this study, we aimed to determine the site and histopathologic patterns of FGTB and its correlation with clinical presentation and acid-fast bacilli (AFB) status.MethodsA retrospective cross-sectional study was conducted on 122 patients with a histopathological diagnosis of FGTB at the Department of Pathology, College of Health Sciences (CHS), Tikur Anbessa Specialized Hospital (TASH), Addis Ababa University (AAU), from January 1, 2013, to August 30, 2022.ResultsFemale genital tuberculosis was found in 0.94% of the gynecology specimens examined. The most common presentations were menstrual disturbance, abdominopelvic pain, and infertility. Among patients with FGTB, 4.6% exhibited misleading clinical and radiologic findings, leading to suspicion of malignancy and subsequent aggressive surgical management. The endometrium was the most frequently affected organ, followed by the fallopian tube, ovary, cervix, and vulva. In the majority of tuberculous endometritis cases (53.3%), histopathology revealed early-stage granulomas. Acid-fast bacilli were found in a significant proportion (42.6%) of FGTB tissues with TB histopathology. The ovary had the highest rate of AFB detection, followed by the fallopian tube, endometrium, and cervix.ConclusionFemale genital tuberculosis should be considered in reproductive-age women presenting with menstrual irregularities, abdominopelvic pain, infertility, or an abdominopelvic mass. The endometrium is commonly affected, displaying early granulomas with low AFB positivity.

Histopathological Cellular and Diagnostic Features of Tuberculous Lymphadenitis

Background: Tuberculosis (TB) remains a global health problem with 25% of the world’s population infected from the causative agent Mycobacterium tuberculosis(MTB). Indian ranks first amongst the eight countries reported to have highest burden of MTB across the globe. TB is categorized into pulmonary and extrapulmonary(EPTB); the most common clinical manifestation of EPTB being tuberculous lymphadenitis(LNTB). LNTB generally occurs due to reactivation of latent infection and cervical lymph nodes are the most common sites.
 Objective: The present research work was conducted to explore disease spectrum of LNTB highlighting specific histopathologic features.
 Materials and Methods: Twenty-one patients with clinically diagnosed LNTB were included and one lymph node biopsy was obtained from each patient. The formalin-fixed paraffin tissue sections were subjected to standard hematoxylin & eosin staining (H & E stain) to understand different histologic features of LNTB.
 Results: LNTB displayed a disease spectrum which can be categorized into four broad categories as: 1. Early granulomas(n=2); 2. Non-caseating granulomas(n=12); 3. Caseating granulomas(n=6); 4. Massive extensive caseation without Langhan’s giant cells(n=1). The well-formed, non-necrotic, epithelioid cell granulomas were prominent feature of LNTB in our study.
 Conclusion: The above study supports the existence of a disease spectrum of LNTB with histologic features ranging from early granulomas to massive caseation necrosis. This can aid clinicians for better diagnosis of LNTB so to aid for early detection and an appropriate treatment of the disease.

A fresh look at mycobacterial pathogenicity with the zebrafish host model.

The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real‐time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models.

IMMUNOPATHOLOGICAL EFFECT OF SENSITIZED TRANSFER FACTOR ON THE ORGANS OF WHITE MICE AGAINST SALMONELLA TYPHI CHALLENGE INFECTION.

In an experimental study to evaluate the immunopathological effect of transfer factor on the reticuloendothelial organs of white mice and their protection against their challenge infection with Salmonella typhi. The results of this study were showed the followings: 1. Transfer factor recipient group: it was showed an early granulomatous lesions in the liver. Reactive hyperplasia in the T cell regions of the spleen and mediastinal lymph node. The early granulomas were persisted during 7th day and slightly regressed on 14th day postinoculation. 2. Infected group with S. typhi: It was showed a multifocal microabscesses consisted of aggregates of neutrophils in the focal area of necrosis, which was evident during 7th day and gradually transform into granulomas on 14th day postinoculation. 3. Transfer factor recipient and challenge infection group: It was showed well developed granulomatous reactions, which indicate an emergence of cellular immunity (delayed type hypersensitivity reaction). These granulomas were more evident on 7th day and slightly regressed on 14th day postinoculation; providing a transfer factor role in tissue reaction and termination of infection.

Effectiveness and safety of 0.5% timolol solution in the treatment of pyogenic granuloma: A randomized, double-blind and placebo-controlled study.

Introduction Pyogenic granulomas are benign vascular lesions of the skin and mucosa which are often a source of concern because of their recurrent bleeding even with minimal trauma. Current treatment for pyogenic granuloma is ablative; no medical therapy is standardized to date. Timolol, due to its vasoconstrictive effect, vascular growth factor inhibition and apoptosis promotion properties, is a potential therapeutic option. Objectives: To assess the effectiveness and safety of topical timolol in the treatment of pyogenic granulomas. Methods A two-centre, double-blind and placebo-controlled trial (Registration CTRI/2019/04/018581) was conducted. Patients of either sex were recruited with pyogenic granuloma lesions of less than eight weeks duration. Topical treatment with 0.5% timolol or matching glycerin placebo was continued for six weeks. Changes in color, size, bleeding tendency, physicians' and patients' global assessments and adverse events were assessed. Results Forty subjects were randomized between the two groups which were comparable in age, sex, duration of illness and baseline lesion size.Significant improvement was noted with timolol, with color change from first follow-up onwards and lesion size reduction from second follow-up onward. Patients' assessment of bleeding tendency also showed imrovement from the second visit onward. Between-group comparison showed significant difference with respect to percentage reduction in size (timolol 40.9%, placebo 3.4%; P = 0.002). Rescue treatment (electrosurgery) was required in five patients on placebo and in one in the timolol group (P = 0.182). Complete resolution occurred in 2 (10%) patients with timolol and in no patients on placebo (P = 0.231). Limitations: We observed effects of treatment for only six weeks. Conclusion Topical timolol may be a treatment option for early pyogenic granulomas but complete resolution is unlikely in six weeks. Studies of longer duration are required to assess resolution and recurrence rates.

MCherry-expressing Mycobacterium tuberculosis gives insight into host cell tropism and immune pressure at the single bacterium level in macaques with tuberculosis

Abstract Tuberculosis is caused by Mycobacterium tuberculosis (Mtb) and is associated with granuloma formation in the lungs and other tissues. These lesions can limit bacterial replication but also contain host cells for Mtb and support bacterial persistence and dissemination in poorly controlled disease. Macrophages are often viewed as primary host cells for Mtb but work in mice demonstrates that other cell types host Mtb in acute infection. Less is known about host cell tropism in human TB, especially in the early stages of infection but these early interactions may have implications for later stages of disease. To identify which cells are infected in early and established TB, we infected two cynomolgus macaques with an mCherry-expressing Mtb strain and used flow cytometry and microscopy to identify and phenotype the infected cells at four- and ten-weeks post infection. This approach also allowed us to compare bacterial mCherry expression and size to determine if granulomas exert different levels of pressure on Mtb that vary by timepoint or lesion type. We found that granulomas contained numerous Mtb-infected neutrophils and macrophages at four weeks post infection but fewer infected neutrophils at ten weeks post infection. When the fluorescence and size of individual bacteria were compared across granulomas and timepoints, we found that bacteria in high Mtb burden granulomas were more fluorescent than bacteria in granulomas that contained fewer bacteria, and the bacteria in the four-week granulomas were longer, suggesting that early granulomas are better at supporting Mtb growth than granulomas in established disease. These results provide new insights into Mtb-host cell dynamics in a nonhuman primate model that replicates human TB.

Histopathological and microbiological study of porcine lymphadenitis: contributions to diagnosis and control of the disease

Tuberculosis like lesions (TBL) in free-range pigs are characterised by presenting a marked heterogeneity in pathology and microbiology features, with a notorious role of Mycobacterium tuberculosis complex (MTC), Trueperella pyogenes and different Streptococcus species. However, the capacity of these microorganism to spread to different organic cavities leading to a generalised disease is unknown. Therefore, this study evaluated the organic distribution of these agents in free-range pig carcasses whole condemned due to generalised TBL.A total of 37 totally condemned animals were analysed, and samples of lymph nodes and organs were obtained (n = 262) and subjected to histopathological and microbiological examination. In addition, T. pyogenes and streptococci species were further characterised by PFGE analysis. Two different patterns were evidenced with lack or occasional lesions in superficial inguinal (SILN) and popliteal (PLN) lymph nodes and advanced lesions in submandibular (SLN) (35/36) and gastrohepatic (GHLN) (33/35) lymph nodes (stages III and IV). Early stage granulomas (stage I and II) prevailed in lungs (16/20), liver (14/31) and spleen (7/18). The microbiological analysis revealed that MTC, detected by qPCR, was present in 31 out of 37 animals and 90 (90/262) samples. In 26 out of the 31 pigs, MTC was detected from two or more organs. SLN (24/31) and GHLN (19/31) were the MTC+ organs most frequently detected, with 29 out of 31 MTC+ pigs detected as positive in one or both samples, which points out that both lymph nodes must be included in the sampling of surveillance programs. Other pathogens, such as T. pyogenes and Streptococcus spp., were also involved in generalised lymphadenitis, being frequently isolated from SLN and other organs, such as liver (T. pyogenes), tonsils or lung (Streptococcus spp.). A wide genetic diversity among streptococci was observed, showing the ubiquitous character of these pathogens, however, the isolation of a single clone of T. pyogenes from different organic locations from animals with generalised TBL was a common finding of this study, highlighting that the role of this pathogen in porcine lymphadenitis may be underestimated. These results should be considered in future studies on the pathogenesis and control of porcine lymphadenitis.

The Many Faces of Cardiac Sarcoidosis.

The objective of this study was to review and illustrate the sometimes diagnostically challenging features of cardiac sarcoidosis. We emphasize variable phenotypes presented at explant and biopsy evaluation and review literature regarding ancillary clinical and pathologic studies to enhance diagnostic accuracy. A literature review was performed and two cardiac sarcoidosis cases were illustrated. Our cases and literature review demonstrate the pathologic spectrum of cardiac sarcoidosis. Irregular left ventricular free wall involvement is most common, followed by the interventricular septum and right ventricle. Although granulomas are often composed of tight epithelioid macrophage aggregates, early granulomas comprise loosely associated macrophages with lymphocyte predominance. Chronic disease leads to fibrosis and end-stage heart failure. Sampling errors and variable histology cause low endomyocardial biopsy sensitivity. Current guidelines use clinical, radiologic, and immunohistologic criteria for diagnosing cardiac sarcoidosis. Knowledge of these guidelines will assist pathologists in making accurate diagnosis of this disease.

Spleen and head kidney differential gene expression patterns in trout infected with Lactococcus garvieae correlate with spleen granulomas

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.

Mycobacterium tuberculosis WhiB3 maintains redox homeostasis and survival in response to reactive oxygen and nitrogen species

Mycobacterium tuberculosis (Mtb) survives under oxidatively and nitosatively hostile niches inside host phagocytes. In other bacteria, adaptation to these stresses is dependent upon the redox sensitive two component systems (e.g., ArcAB) and transcription factors (e.g., FNR/SoxR). However, these factors are absent in Mtb. Therefore, it is not completely understood how Mtb maintains survival and redox balance in response to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Here, we present evidences that a 4Fe-4S-cofactor containing redox-sensitive transcription factor (WhiB3) is exploited by Mtb to adapt under ROS and RNS stress. We show that MtbΔwhiB3 is acutely sensitive to oxidants and to nitrosative agents. Using a genetic biosensor of cytoplasmic redox state (Mrx1-roGFP2) of Mtb, we show that WhiB3 facilitates recovery from ROS (cumene hydroperoxide and hydrogen peroxide) and RNS (acidified nitrite and peroxynitrite). Also, MtbΔwhiB3 displayed reduced survival inside RAW 264.7 macrophages. Consistent with the role of WhiB3 in modulating host-pathogen interaction, we discovered that WhiB3 coordinates the formation of early human granulomas during interaction of Mtb with human peripheral blood mononuclear cells (PBMCs). Altogether, our study provides empirical proof that WhiB3 is required to mitigate redox stress induced by ROS and RNS, which may be important to activate host/bacterial pathways required for the granuloma development and maintenance.

A zebrafish model for ocular tuberculosis.

Ocular tuberculosis (TB) commonly causes severe inflammation and vision loss in TB-endemic countries. The mechanism by which tuberculous infection becomes established in the eye is poorly understood. We have developed the zebrafish larva infected with Mycobacterium marinum as a model to study the early pathogenesis of ocular TB. We find that hematogenous bacterial seeding of the eye occurs despite a functional blood retinal barrier. Prototypical early granulomas form in response to bacteria in the eye. These granulomas involve the retinal vasculature and retinal pigment epithelium-choroid complex which are characteristic locations for human ocular TB. We find that peripheral blood monocytes are recruited to the nascent ocular granuloma further suggesting that the immune privileged nature of the eye is breached by this inflammatory focus.

Ultrasonic appearances of epididymal spermatic granuloma

Objective To explore the relationship between ultrasonic appearances of epididymal spermatic granuloma and its pathologic characteristics. Methods The sonographic features of 32 cases of epididymal spermatic granuloma confirmed by pathology were retrospectively analyzed. Results A total of 34 epididymal spermatic granuloma nodules in 32 cases included 4 located in caput epididymidis and 30 located in cauda epididymidis, and maximum diameter of nodules were about 0.5-2.3 cm. Sonographic and pathologic appearances of nodules were: ①mostly low echo type, 21 nodules mostly showed the obscure boundary and quite abundant blood flow signals, early granulomas formed with interstitial fibrosis inapparently under-microscope; ②inhomogeneous echo type, 12 nodules mostly showed the barely clear boundary and blood flow signals from small amount to abundant amount, granulomas of different maturites distributed dispersedly with interstitial fibrosis of varying degrees under-microscope or single giant lesions formed; ③mostly high echo type, 1 nodule showed the obscure boundary and inapparent blood flow signals, center of the nodules distributed by a large amount of dense fibrous tissue under-microscope. Anechoic ethmoid sinus seen in 29 nodules was section of dilated epididymal ductus. Dotted strong echoes seen in 6 nodules were calcification of sperms in dilated epididymal ductus and necrotic tissue. Conclusions Ultrasonic appearances of epididymal spermatic granuloma are closely related to its pathologic characteristics. To recognize both relations contributes to improve the diagnostic value of ultrasound. Key words: Ultrasonography; Spermatic Granuloma; Epididymis

Histological morphology and pathological changes in liver of rats naturally infected with larval stage Cysticercus fasciolaris of Taeniae taeniaeformis

The aim of this study is to describe the morphology of Cysticercus fasciolaris by using light microscopy, and the pathological changes in the liver of rats naturally infected. A total of 50 liver specimens of local rats (Wister rats) were collected for examination. The gross lesions showed the presence of single or multiple cysts. Microscopic findings revealed the presence of larvae within the cysts which represent the larvae Cysticercus fasciolaris of the adult parasite Taeniae taeniaeformis which inhabited the small intestine of the domestic cats surrounded by fibrous connective tissue infiltrated with inflammatory cells (mononuclear cells and plasma cells). These lesions cause pressure atrophy to the adjacent hepatic parenchyma. In advanced hepatic infection there is a tendency to undergo neoplastic changes (fibroma). Other pathological lesions seen in the liver parenchyma were necrosis, apoptosis with infiltration of chronic inflammatory cells in the portal area, in addition; to formation of early granulomas with congestion of blood vessels which contain neutrophiles in their lumina with extensive area of hemorrhages in liver parenchyma. In conclusion the C. fasciolaris infction induce hepatic neoplasia in rat livers (fibroma) in advance cases of heavy infection, which could be developed to fibrosarcoma in future.

Multinucleated giant cell cytokine expression in pulmonary granulomas of cattle experimentally infected with Mycobacterium bovis

Regardless of host, pathogenic mycobacteria of the Mycobacterium tuberculosis complex such as Mycobacterium bovis, induce a characteristic lesion known as a granuloma, tubercle or tuberculoid granuloma. Granulomas represent a distinct host response to chronic antigenic stimuli, such as foreign bodies, certain bacterial components, or persistent pathogens such as M. bovis. Granulomas are composed of specific cell types including epithelioid macrophages, lymphocytes and a morphologically distinctive cell type, the multinucleated giant cell. Multinucleated giant cells are formed by the fusion of multiple macrophages; however, their function remains unclear. In humans, giant cells in tuberculous granulomas have been shown to express various cytokines, chemokines and enzymes important to the formation and maintenance of the granuloma. The objective of this study was to quantitatively assess multinucleated giant cell cytokine expression in bovine tuberculoid granulomas; focusing on cytokines of suspected relevance to bovine tuberculosis. Using calves experimentally infected with M. bovis, in situ cytokine expression was quantitatively assessed using RNAScope® for the following cytokines TNF-α, IFN-γ, TGF-β, IL-17A and IL-10. Multinucleated giant cells in bovine tuberculoid granulomas expressed all examined cytokines to varying degrees, with differential expression of TGF-β, IL-17A and IL-10 in giant cells from early versus late stage granulomas. There was a modest, positive correlation between the level of cytokine expression and cell size or number of nuclei. These results suggest that multinucleated giant cells are active participants within bovine tuberculoid granulomas, contributing to the cytokine milieu necessary to form and maintain granulomas.

Therapeutic Potential of Mesenchymal Stem Cells on Early and Late Experimental Hepatic Schistosomiasis Model.

Cell-based therapy is emerging as a promising therapeutic approach for a wide range of liver diseases. This study aimed to investigate the regenerative and antifibrotic therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) in an early and late experimental hepatic schistosomiasis model. BM-MSCs were isolated from 6-wk-old BALB/c donor male mice, then grown and propagated in culture until cell count was 5-8 × 10(6)/ml. MSCs were then separated and injected into Schistosoma mansoni -infected female BALB/c mice on their 6, 10, 14, and 18 wk post-infection. Mice were sacrificed on the fourth and eighth week after BM-MSCs transplantation in each group. Homing of BM-MSCs was confirmed by PCR detection of male Y-chromosome gene (sry) in the liver tissue of the recipient female mice. The regenerative and antifibrotic potential of BM-MSCs was assessed by histopathological examination, morphometric analysis, electron microscopy, and liver function tests. Schistosoma-infected mice, which were treated with BM-MSCs, showed a decrease in the granuloma size, percentage and density of the fibrotic area, formation of new hepatocytes, and improvement of the liver function tests. Immunohistochemical examination of alpha-smooth muscle actin revealed a significant decrease in the immunoreactive hepatic stellate cells in mice treated with MSCs. Early granulomas (acute infection) showed better response to MSC injection than did later granulomas (chronic infection). Dosing and timing of MSCs transplantation should undergo more investigations in long-term experiments before application to the clinical field. This study is the first to assess and compare the effect of MSCs treatment on early and late granulomas.

Somatostatin negatively regulates parasite burden and granulomatous responses in cysticercosis.

Cysticercosis is an infection of tissues with the larval cysts of the cestode, Taenia solium. While live parasites elicit little or no inflammation, dying parasites initiate a granulomatous reaction presenting as painful muscle nodules or seizures when cysts are located in the brain. We previously showed in the T. crassiceps murine model of cysticercosis that substance P (SP), a neuropeptide, was detected in early granulomas and was responsible for promoting granuloma formation, while somatostatin (SOM), another neuropeptide and immunomodulatory hormone, was detected in late granulomas; SOM's contribution to granuloma formation was not examined. In the current studies, we used somatostatin knockout (SOM−/−) mice to examine the hypothesis that SOM downmodulates granulomatous inflammation in cysticercosis, thereby promoting parasite growth. Our results demonstrated that parasite burden was reduced 5.9-fold in SOM−/− mice compared to WT mice (P < 0.05). This reduction in parasite burden in SOM−/− mice was accompanied by a 95% increase in size of their granulomas (P < 0.05), which contained a 1.5-fold increase in levels of IFN-γ and a 26-fold decrease in levels of IL-1β (P < 0.05 for both) compared to granulomas from WT mice. Thus, SOM regulates both parasite burden and granulomatous inflammation perhaps through modulating granuloma production of IFN-γ and IL-1β.

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum.

Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB), indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.

S-shaped tracheostoma in pediatric tracheostomy using three-dimensional sigma-plasty—A novel technique to possibly reduce procedure related complications

ObjectivesWe have designed a technique of tracheostomy in pediatric patients with S-shaped incision on the tracheal wall which we think, provides a larger cross-sectional area of stoma and facilitates easier insertion of tracheostomy tube and thus helped in reducing early and late complications associated with it in our series. TechniqueThe trachea was exposed in midline by a vertical skin incision. In order to make S-shaped tracheostoma, second tracheal ring was identified. The conventional vertical incision was made in second tracheal ring and then extended at both its ends laterally in the inter-cartilaginous space parallel to the tracheal cartilage in the opposite direction to make the incision S-shaped. The trachea was dilated with tracheal dilator and appropriate size of tracheostomy tube was then placed into the trachea. ResultsS-shaped tracheostomy was performed in 40 children with mean age of 6.36 years (age range is 2–12) required for airway maintenance or prolonged ventilatory support. The incidence of early complications was quite less in our patients (ranged from 0 to 5%). There was no incidence of excessive intra-operative bleeding or injury to surrounding structures causing subcutaneous emphysema or vocal cord palsy. One patient developing pneumothorax after the procedure was managed conservatively. There was no incidence of tracheo-esophageal fistula, suprastomal collapse or difficulty in decannulation on 9 months of follow up related to our technique. However, one of the patients developed early trachietis and cutaneous peristomal granulomas and 2 patients developed late trachietis which was treated conservatively. ConclusionS-shaped tracheoplasty, a new pediatric tracheotomy technique has resulted in a quantifiable reduction in the risk of the early and late complications in our series. Hence, we feel that this new technique is a better alternative to existing methods but larger randomized controlled studies are required before universal adoption of this technique.

Immunohistochemical evaluation of surfactant proteins and lymphocyte phenotypes in the lungs of cattle with natural tuberculosis

The aim of the present study was to evaluate the immunohistochemical expression of pulmonary surfactant proteins (SP-A, SP-B, SP-C) and lymphocytic phenotypes in the lungs of 12 cattle with natural tuberculosis. Grossly, the disease-affected cattle revealed numerous granulomas in the lung lobes. Histopathological examination found multiple lung granulomas with typical cellular elements. Type II pneumocytes with adenomatous proliferation around the granulomas were strongly immunopositive for SP-A and SP-B compared to normal type II cells. Clara cells showed also cytoplasmic immunopositivity for these surfactant proteins. Positive immunolabelling for proSP-C was detected exclusively in the normal and proliferative type II pneumocytes, and the reaction was marked in the perinuclear area of the cells. CD3+ T and CD79αcy+ B lymphocytes were predominantly localized in the fibrotic capsule margin of advanced granulomas, in greater numbers than in the early granulomas. In conclusion, the study found that type II pneumocytes proliferated highly and surrounded the tuberculous granulomas in the lungs, that hyperplastic type II pneumocytes synthesized and secreted larger amounts of surfactant proteins than the normal type II cells, and that SP-A might have played an important role in host defence against the mycobacterial agents. Additionally, the presence of high numbers of CD3+ T cells throughout the granulomas confirmed the dominance of a cellular immune response in cattle tuberculosis.