Validation of Muscle Fiber Architecture of the Human Tongue Revealed by Diffusion Magnetic Resonance Imaging With Histology Verification.

PURPOSE: The goal of this study is to validate the muscle architecture derived from both ex vivo and in vivo diffusion-weighted magnetic resonance imaging (dMRI) of the human tongue with histology of an ex vivo tongue. METHOD: dMRI was acquired with a 200-direction high angular resolution diffusion imaging (HARDI) diffusion scheme for both a postmortem head (imaged within 48 hr after death) and a healthy volunteer. After MRI, the postmortem head was fixed and the tongue excised for hematoxylin and eosin (H&E) staining and histology imaging. Structure tensor images were generated from the stained images to better demonstrate muscle fiber orientations. The tongue muscle fiber orientations, estimated from dMRI, were visualized using the tractogram, a novel representation of crossing fiber orientations, and compared against the histology images of the ex vivo tongue. RESULTS: Muscle fibers identified in the tractograms showed good correspondence with those appearing in the histology images. We further demonstrated tongue muscle architecture in in vivo tractograms for the entire tongue. CONCLUSION: The study demonstrates that dMRI can accurately reveal the complex muscle architecture of the human tongue and may potentially benefit planning and evaluation of oral surgery and research on speech and swallowing.

The effect of anti-alcoholic gastric ulcer before and after vinegar-processed Yuanhu Zhitong prescription based on spectral-effect relationship.

The aim of this study was to screen out the active ingredients of Yuanhu Zhitong prescription (YZP) before and after vinegar processing to play an anti-alcoholic gastric ulcer role through spectrum-effect relationship. First, the fingerprint of 16 batches of YZP was studied using the ultra-high-performance liquid chromatography-quadrupole mass spectrometry detector analysis (UPLC-QDA) method. Second, gastric lesion was induced by anhydrous ethanol. The degree of gastric mucosa injury was evaluated by hematoxylin and eosin staining, and the contents of malondialdehyde and tumor necrosis factor α and superoxide dismutase were detected using the enzyme-linked immunosorbent assay kit. Sixteen batches of YZP were analyzed using the spectrum-effect relationship method. Finally, absorption, distribution, metabolism, and excretion (ADME) was used to evaluate the bioavailability of potential compounds. The results showed that the UPLC-QDA method could successfully establish the fingerprint of YZP. Hematoxylin and eosin staining and biochemical indicators showed that YZP had obvious anti-alcoholic gastric ulcer action. Coptisine chloride, corydaline, berberine chloride, palmatine, imperatorin, and phellopterin were screened out using the spectrum-effect method, and all of them possessed good bioavailability. The results of this study suggest that YZP plays an anti-ulcer role by exerting antioxidant and anti-inflammatory effects through six main active components.

Segmentation of HE-stained meningioma pathological images based on pseudo-labels.

Biomedical research is inseparable from the analysis of various histopathological images, and hematoxylin-eosin (HE)-stained images are one of the most basic and widely used types. However, at present, machine learning based approaches of the analysis of this kind of images are highly relied on manual labeling of images for training. Fully automated processing of HE-stained images remains a challenging task due to the high degree of color intensity, size and shape uncertainty of the stained cells. For this problem, we propose a fully automatic pixel-wise semantic segmentation method based on pseudo-labels, which concerns to significantly reduce the manual cell sketching and labeling work before machine learning, and guarantees the accuracy of segmentation. First, we collect reliable training samples in a unsupervised manner based on K-means clustering results; second, we use full mixup strategy to enhance the training images and to obtain the U-Net model for the nuclei segmentation from the background. The experimental results based on the meningioma pathology image dataset show that the proposed method has good performance and the pathological features obtained statistically based on the segmentation results can be used to assist in the clinical grading of meningiomas. Compared with other machine learning strategies, it can provide a reliable reference for clinical research more effectively.

Change in rat meibomian gland structure with external pressure on the eyelid.

PURPOSE: This study investigated structural changes in rat meibomian glands following repeated and sustained application of external pressure on the eyelids using a magnet and then subsequent removal of the external pressure. METHODS: Twenty-eight Sprague-Dawley rats were used. The upper eyelid was externally compressed using a pair of magnets. One magnet was placed inside the upper eyelid, another was placed outside the eyelid, and varying periods of pressure were investigated. Untreated eyes were used as controls. Meibography was performed, and the transverse eyelid tissue sections were stained with hematoxylin and eosin and anti-cytokeratin 5 antibody at one hour, two and four weeks after removing the magnets. RESULTS: Meibography showed increased meibomian gland loss (30.0 ± 5.0%), and tissue sections showed decreased area of secretory acini (0.04 ± 0.08 mm2) at one hour after applying external pressure using magnets versus in the control eyes (5.0 ± 5.0% and 0.08 ± 0.08 mm2, respectively). On the other hand, there was no meibomian gland loss or reduction of the area of secretory acini at two and four weeks after removing the magnets in comparison with the control eyes. CONCLUSIONS: Repeated and sustained application of external pressure on the eyelid could induce meibomian gland loss; however, this meibomian gland loss can be restored when the external pressure is removed. Therefore, the repeated application of external pressure on the eyelid is a safe treatment method for obstructive MGD.

Use of Immunostaining for the diagnosis of Lymphovascular invasion in superficial Barrett's esophageal adenocarcinoma.

BACKGROUND: The prevalence of Barrett's esophageal adenocarcinoma (BEA) is increasing in Japan. Accurate assessment of lymphovascular invasion (LVI) after endoscopic resection or surgery is essential in evaluating treatment response. This study aimed to assess the usefulness of immunostaining in determining the extent of LVI in superficial BEA. METHODS: We retrospectively included 41 patients who underwent endoscopic resection or surgery between January 2007 and July 2018. In all cases, 3-µm serial sections from paraffin-embedded resected specimens were used for hematoxylin and eosin (H-E) staining and immunostaining for D2-40 and CD31. Two specialized gastrointestinal pathologists (T.Y. and T.T.), blinded to clinical information, independently evaluated the extent of LVI from these specimens. The LVI-positivity rate was evaluated with respect to the depth of invasion, changes in the positivity rate on immunostaining, pathological characteristics of patients with LVI, lymph node metastasis or relapse, and course after treatment. RESULTS: H-E staining alone identified LVI in 7 patients (positivity rate: 17.1%). Depths of invasion were categorized based on extension to the submucosa (SM) or deeper. On immunostaining for D2-40 and CD31, additional positivity was detected in 2 patients with SM1 and 1 SM3, respectively; LVI was detected in 10 patients (positivity rate: 24.4%). LVI-positivity rates with invasion of the superficial muscularis mucosa (SMM)/lamina propria mucosa (LPM)/deep muscularis mucosa (DMM), SM 1, 2, and 3 were 0, 75, 28.6, and 55.6%, respectively. CONCLUSIONS: Combined H-E staining and immunostaining is useful in diagnosing LVI in superficial BEA, particularly in endoscopically resected specimens.

Applicability of eucalyptus wood waste extracts for the histological staining of connective tissue.

Biological staining of tissue is a crucial procedure in histotechnology. Rudimentary methods for section preparation have often used stains from natural products, although use of synthetic dyes has become the contemporary standard. Artificial dyes increase the operating costs of a laboratory as well as increase the environmental and personnel risks during manufacturing and usage. These considerations have stimulated research to find alternative natural stains from the wide diversity of plant species. The present study investigated the effect of Eucalyptus sp. (Myrtaceae) wood waste extract on histological staining of animal tissues, using a series of pigment concentrations, pH conditions, and temperatures. Eucalyptus wooden slivers were dried, milled, and 1 g, 2 g, and 4 g of the fine powder was subjected to 50% ethanol extraction for 2 days. Staining tests were then performed on formalin fixed paraffin embedded (FFPE) sections. Increasing acetic acid concentrations (1%, 2% and 4%) were added to the extracts and compared to an acid-free extract. Staining was performed at both ambient room temperature (RT) and 60°C. Connective tissue acidophilic components were well-contrasted and a hematoxylin counterstain demonstrated distinct structural differences between matrix and cell nuclei. Therefore, the present findings demonstrate the potential utility of the eucalyptus wood extracts application as a natural stain alternative for routine histology.

AIM2 gene silencing attenuates diabetic cardiomyopathy in type 2 diabetic rat model.

AIMS: Absent in melanoma 2 (AIM2) is a cytosolic DNA sensor which plays an important role in inflammasome formation and is involved in various cellular functions including pyroptosis, fibrosis, and tissue injury. Our study aimed to investigate whether AIM2 plays a role in diabetic cardiomyopathy (DCM) and to explore its potential molecular mechanism. MAIN METHODS: Sprague-Dawley rats were randomly divided into 4 groups: Control, Diabetes Mellitus (DM), DM + shAIM2, and DM + shNC. The cardiac function of rats was measured. Hematoxylin and eosin staining, Masson's staining, sinus red staining, and immunohistochemistry were performed. H9c2 cardiomyocytes were cultured in DMEM and stimulated with high-glucose treatment (25 mmol/l). The level of reactive oxygen species (ROS) was measured. AIM2-siRNA were used to inhibit the expression of AIM2. TUNEL assay and EthD-III staining were used to measure cell death. The expression levels of AIM2, ASC, caspase-1, IL-1ß, and GSDMD-N were measured by western blotting. KEY FINDINGS: In the streptozotocin-induced diabetic rat model, AIM2 expression was significantly increased in heart tissue compared with the control. Also, diabetic rats exhibited severe left ventricular dysfunction including metabolic disorder, cardiac fibrosis, and cardiomyocyte death. Gene silencing of AIM2 alleviated cardiac dysfunction which resulted from metabolic disorder and ventricular remodelling. In vitro, treatment of H9C2 cardiomyoblasts with HG significantly increased AIM2, while ROS inhibition reduced the level of AIM2. AIM2-siRNA alleviated GSDMD-N-related pyroptosis in H9c2 cardiomyoblasts. SIGNIFICANCE: Our results indicate that AIM2 plays an important role in cell death and fibrosis in HG-induced, ROS-mediated diabetic cardiomyopathy via the GSDMD pathway.

Is it time for one-step nucleic acid amplification (OSNA) in colorectal cancer? A systematic review and meta-analysis.

BACKGROUND: Lymph node metastasis (LNM) is prognostic in colorectal cancer (CRC). However, evaluation by routine haematoxylin and eosin histology (HE) limits nodal examination and is subjective. Missed LNMs from tissue allocation bias (TAB) might under-stage disease, leading to under-treatment. One-step nucleic acid amplification (OSNA) for CK19 messenger ribonucleic acid (mRNA), a marker of LNM, analyses the whole node. The aim of the present systematic review and meta-analysis was to assess recent studies on OSNA versus HE and its implications for CRC staging and treatment. METHODS: Databases including OVID, Medline and Google Scholar were searched for OSNA, LNM and CRC. Study results were pooled using a random-effects model. Summary receiver operator curves (SROC) assessed OSNA's performance in detecting LNM when compared to routine HE histology. RESULTS: Five case-control studies analysing 4080 nodes from 622 patients were included. The summary estimates of pooled results for OSNA were sensitivity 0.90 [95% confidence interval (CI) 0.86-0.93], specificity 0.94 (95% CI 0.93-0.95) and diagnostic odds ratio 179.5 (CI 58.35-552.2, p < 0.0001). The SROC curve indicated a maximum joint sensitivity and specificity of 0.88 and area under the curve of 0.94, p < 0.0001. On average, 5.4% HE-negative nodes were upstaged by OSNA. CONCLUSIONS: OSNA is as good as routine HE. It may avoid TAB and offer a more objective and standardised assay of LNM. However, for upstaging, its usefulness as an adjunct to HE or superiority to HE requires further assessment of the benefits, if any, of adjuvant therapy in patients upstaged by OSNA.

Sweet’s syndrome-like eruption in association with the exacerbation of Behçet’s disease after the Great East Japan Earthquake / Erupción tipo síndrome de Sweet asociada al empeoramiento de la enfermedad de Behçet después del Gran terremoto de Japón oriental

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Nevo spilus asociado a nevus azul agminado: una combinación infrecuente / Nevus Spilus Associated with Agminated Blue Nevus: A Rare Combination

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Comparación entre las biopsias gástricas con fijación inmediata y diferida para la detección de Helicobacter pylori / A comparison of immediate and delayed fixation of gastric biopsies for the detection of Helicobacter pylori

Objetivo. Comparar las técnicas de fijación inmediata y diferida en la detección de Helicobacter pylori (Hp) en biopsias de mucosa gástrica. Material y métodos. Se seleccionaron aleatoriamente las biopsias de mucosa gástrica de 86 pacientes que acudieron al servicio de Gastroenterología del Hospital Belén de Trujillo entre noviembre del 2013 y junio del 2014. A cada paciente se le extrajeron 4 fragmentos de mucosa gástrica: 2 fueron fijados con formol al 10% inmediatamente y los otros 2 fueron fijados después de 24 h (fijación diferida). Ambas muestras, fueron procesadas con la técnica convencional de inclusión en parafina, coloración con hematoxilina-eosina (H&E), y como patrón de oro Warthin-Starry. Resultados. Se encontró que la presencia de Hp en las biopsias coloreadas con H&E fue mayor en las biopsias con fijación diferida (96,5%; kappa: 1,0) que en las biopsias con fijación inmediata (80,2%; kappa: 0,839); sin embargo, no hubo diferencias estadísticamente significativas (p > 0,05). Con respecto al valor diagnóstico, la técnica de fijación diferida presentó una sensibilidad del 100%, una especificidad del 100%, un valor predictivo positivo del 100% y un valor predictivo negativo del 100%, mientras que la técnica de fijación inmediata para la detección del Hp en biopsias gástricas presentó una sensibilidad del 100%, una especificidad del 76,5%, un valor predictivo positivo del 94,5% y un valor predictivo negativo del 100%. Conclusiones. La técnica de fijación diferida mostró mayor valor que la técnica de fijación inmediata en la detección del Hp en biopsias de mucosa gástrica, aunque la diferencia no fue estadísticamente significativa (AU)

Objective. To compare the techniques of immediate fixation and delayed fixation in detecting Helicobacter pylori (Hp) in gastric mucosal biopsies. Material and methods. We randomly selected gastric mucosal biopsies from 86 patients who attended the gastroenterology division at Belen Hospital (Trujillo) between November 2013 and June 2014. Four fragments of gastric mucosa were extracted from each patient, two were fixed immediately with 10% formaldehyde, and the other two were fixed after 24 hours (delayed fixation). Both samples were processed with the conventional technique of paraffin embedding and staining with hematoxylin-eosin (H&E) using Warthin-Starry as the gold standard. The presence of Hp was investigated in both fixation techniques. Results. It was found that the presence of Hp in H&E stained biopsies was greater in biopsies with delayed fixation (96.5%; kappa: 1,0) than in biopsies with immediate fixation (80.2%; kappa: 0,839); nevertheless, there was no statistically significant difference (p > 0.05). With respect to diagnostic value, the delayed fixation technique had a sensitivity of 100%, a specificity of 100%, a positive predictive value of 100%, and a negative of 100%; while the immediate fixation technique for detection of Hp in gastric biopsies had a sensitivity of 100%, a specificity of 76.5%, a positive predictive value of 94.5% and a negative of 100%. Conclusions. Delayed fixation technique showed higher values in the detection of Hp than the immediate fixation technique in gastric mucosal biopsies, although the difference was not statistically significant (AU)

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats.

Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners.

Nefropatía mesangial y síndrome antisintetasa: una forma curiosa de asociación / Mesangial nephropathy and anti-syntethase syndrome: An odd association

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Analysis of tumor mitotic rate in thin metastatic melanomas compared with thin melanomas without metastasis using both the hematoxylin and eosin and anti-phosphohistone 3 IHC stain.

Studies have suggested that elevated tumor mitotic rate (MR) is linked to overall survival in thin melanoma. Recently, promising data regarding anti-phosphohistone 3 (pHH3) immunohistochemistry and its ability to aid in calculation of MR have emerged. The authors retrospectively analyzed original biopsies from 13 thin melanomas with positive sentinel node (SN) status and 16 thin melanomas with negative SN status. Both anti-pHH3 immunohistochemistry and the hematoxylin and eosin (H&E) stain were used to evaluate MR by 2 dermatopathologists blinded to SN status using the "hot spot" method. Intraclass coefficient values were attained to measure interobserver concordance and reliability of the pHH3 stain. By generating a receiver operating characteristic curve and analyzing the overall area under the curve, pHH3 was found to have good interobserver reliability. The relationship between MR and SN involvement was also evaluated, but this correlation was not statistically significant.

Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections.

The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer.

[Morphometric characteristics of tularemia infectious process in the organism of immunized guinea pigs].

AIM: Morphometric characteristic of organ and system state of guinea pigs immunized with live tularemia vaccine during infection with virulent culture of tularemia. MATERIALS AND METHODS: Morphometric study of histological material from immunized guinea pigs infected subcutaneously at day 30 with a culture of virulent tularemia strain was performed. A standard scheme of sampling and preparation of morphological material and staining of final semifine section with hematoxylin and eosin, impregnation with silver by Masson in Gamperl and Grimelius modificationwas used. Morphometric study was performed by using "Densitomorphometry" program. RESULTS: Morphometric parameters that characterize functional state of organs and systems in immunized, immunized with consequent infection and infected guinea pigs were established. Reactive processes that take place in the infected animal organism against the background of prior immunization fit into the range of adaptation-compensation reactions. CONCLUSION: The morphometric study carried out allowed to adequately evaluate the state of functionally important systems of the organism of experimental animals, this allows to consider perspective the wider use of morphometric analysis in experimental morphology.

Histopathological investigation in porcine infected with torque teno sus virus type 2 by inoculation.

BACKGROUND: Porcine torque teno sus virus (TTSuV) is a small icosahedral and non-enveloped virus which contains a single-stranded (ssDNA), circular and negative DNA genome and infects mainly vertebrates and is currently classified into the 'floating' genus Anellovirus of Circoviridae with two species. Viral DNA of both porcine TTSuV species has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States, Europe and Asia. However, there exists no information about histopathological lesions caused by infection with porcine TTSuV2. METHODS: Porcine liver tissue homogenate with 1 ml of 6.91 × 107 genomic copies viral loads of porcine TTSuV2 that had positive result for torque teno sus virus type 2 and negative result for torque teno sus virus type 1 and porcine pseudorabies virus type 2 were used to inoculate specific pathogen-free piglets by intramuscular route and humanely killed at 3,7,10,14,17,21 and 24 days post inoculation (dpi), the control pigs were injected intramuscularly with 1 ml of sterile DMEM and humanely killed the end of the study for histopathological examination routinely processed, respectively. RESULTS: All porcine TTSuV2 inoculated piglets were clinic asymptomatic but developed myocardial fibroklasts and endocardium, interstitial pneumonia, membranous glomerular nephropathy, and modest inflammatory cells infiltration in portal areas in the liver, foci of hemorrhage in some pancreas islet, a tiny amount red blood cells in venule of muscularis mucosae and outer longitudinal muscle, rarely red blood cells in the microvasculation and infiltration of inflammatory cells (lymphocytes and eosinophils) of tonsil and hilar lymph nodes, infiltration of inflammatory lymphocytes and necrosis or degeneration and focal gliosis of lymphocytes in the paracortical zone after inoculation with porcine TTSuV2-containing tissue homogenate. CONCLUSIONS: Analysis of these presentations revealed that porcine TTSuV2 was readily transmitted to TTSuV-negative swine and that infection was associated with characteristic pathologic changes in specific pathogen-free piglets inoculated with porcine TTSuV2. Those results indicated no markedly histopathological changes happened in those parenchymatous organs, especially the digestive system and immune system when the specific pathogen-free pigs were infected with porcine TTSuV2, hence, to some extent, it was not remarkable pathological agent for domestic pigs at least. So, porcine TTSuV2 could be an unrecognized pathogenic viral infectious etiology of swine. This study indicated a directly related description of lesions responsible for TTSuV2 infection in swine.

Tectonic lamellar keratoplasty with acellular corneal stroma in high-risk corneal transplantation.

PURPOSE: Tectonic lamellar keratoplasty (TLKP) is a primary surgical procedure to improve the condition of the recipient bed in high-risk corneal transplantation. It is usually performed for a secondary optical penetrating keratoplasty (PKP). The present study was undertaken to explore a new strategy for TLKP using acellular corneal stroma (ACS) prepared by decellularization. METHODS: ACS for TLKP was prepared from cat cornea by decellularization. The efficiency of the decellularization was examined by hematoxylin and eosin (H&E) staining and through DNA content analysis. Twenty-eight New Zealand white rabbits, as recipients, were assigned to one of two groups that had different material for their TLKP. The TLKP was combined with a central optical PKP as a single-stage procedure. Either ACS or fresh cat corneal lamella, 11.25 mm in diameter, was used for the TLKP in these two groups. After TLKP, a 6.5-mm full-thickness cat cornea was placed in the central cornea of each recipient rabbit for PKP. Clinical outcomes and the histology of the transplants were compared post-operatively. RESULTS: ACS for TLKP prolonged the survival of the transplants. The mean survival time of the transplants in the ACS group (36.4±4.3 days) was longer than for those in the control group (14.0±2.2 days, p<0.05). The ACS group showed a significantly smaller neovascularization area compared to the control group. The areas of corneal neovascularization were 5.3±1.1 mm² and 45.2±4.9 mm² (p<0.05), respectively, after two weeks, and 25.1±4.7 mm² and 105.3±12.4 mm² (p<0.05), respectively, after four weeks. Histology revealed that fewer inflammatory cells were infiltrating the transplants in the ACS group than those in the control group. CONCLUSIONS: The use of ACS for TLKP prolonged the survival of corneal transplants, reduced corneal neovascularization, and prevented from infiltration of inflammatory cells. It is a feasible and effective strategy to prolong the survival of transplants in high-risk corneal transplantation.

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

Phenotyping the claudin 11 deficiency in testis: from histology to immunohistochemistry.

The testis is a heterogeneous organ that comprises a number of cell types, including germ cells at -different stages in their maturation, differentiated neighbor nursing cells, and endocrine somatic cells. Despite such cellular heterogeneity the testis is highly organized, with germ cell development and differentiation being compartmentalized into the interconnected tubular network of the seminiferous epithelium. Intratesticular scaffolds rely heavily on the basement membrane of the seminiferous tubules while germ cell development inside the seminiferous epithelium is critically dependent on the Blood Testis Barrier (BTB). The BTB is a macromolecular tight junction complex generated by somatic Sertoli cells within the seminiferous epithelium. The BTB divides the seminiferous epithelium into two compartments: the basal compartment, which delineates a niche for the proliferation and renewal of spermatogonia; and the adluminal compartment, where differentiating germ cells undergo meiosis and spermiogenesis. The BTB is unique in mammalian tissues because it is cyclically reconstructed during the spermatogenic cycle as preleptotene spermatocytes migrate from the basal compartment to the adluminal compartment and enter meiosis. In mouse, the loss of the BTB in the absence of the claudin 11 protein causes azoospermia and leads to infertility. Specifically, cldn11 deficiency results in sloughing of the cells of the seminiferous epithelium into the lumen. Understanding this pathophysiology has involved histological examination of the tissue defects as well as immunohistological characterization. Here, we present a comparative study of several modifications to the classical Hematoxylin-Eosin stain that may improve the diagnostic usefulness of this technique, as well as the use of several selective markers to identify testicular cell types.