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The pathophysiology of dengue may be influenced by antibodies released during infection. Several autoimmune diseases are accompanied by antinuclear antibodies (ANAs) but 8-10% of the general population have positive ANA tests. To test the hypothesis that an ANA-positive test indicates an immune dysregulated state that modifies the risk for certain clinical disorders in people with or without an autoimmune disease, we examined the various ANA profiles and their relationships to various autoimmune disorders, as well as the severity of these relationships, in patients infected with dengue fever. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods were used. Indirect immunofluorescence assay (IIFA) and line immunoassay (LIA) were performed to detect and differentiate the ANAs among dengue infected patients. Out of 135 dengue virus-positive patients, 94.07% were positive by ELISA and 5.93% positive by RT-PCR method. ANAs by IIFA and LIA were detected in 54.8% and 18.5% of the dengue positive patients, respectively, and 10.3% and 7.1% of the 126 dengue negative patients, respectively. This study showed that dengue was associated with an increased risk of autoimmune myositis and mixed connective tissue disease (MCTD), a rare complication of dengue. The risk of other autoimmune diseases did not seem to increase after DENV infection.

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The formation of precise numbers of neuronal connections, known as synapses, is crucial for brain function. Therefore, synaptogenesis mechanisms have been one of the main focuses of neuroscience. Immunohistochemistry is a common tool for visualizing synapses. Thus, quantifying the numbers of synapses from light microscopy images enables screening the impacts of experimental manipulations on synapse development. Despite its utility, this approach is paired with low-throughput analysis methods that are challenging to learn, and the results are variable between experimenters, especially when analyzing noisy images of brain tissue. We developed an open-source ImageJ-based software, SynBot, to address these technical bottlenecks by automating the analysis. SynBot incorporates the advanced algorithms ilastik and SynQuant for accurate thresholding for synaptic puncta identification, and the code can easily be modified by users. The use of this software will allow for rapid and reproducible screening of synaptic phenotypes in healthy and diseased nervous systems.

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Giardia duodenalis is a common parasite of the gastrointestinal tract of dogs, with an especially high prevalence in dogs <1-y-old. Methods for detecting G. duodenalis are point-of-care (POC) tests such as lateral-flow tests or fecal flotation. The Vetscan Imagyst (Zoetis) is a new POC device for the detection of G. duodenalis in fecal samples using zinc sulfate flotation, automated slide scanning, and image recognition with artificial intelligence. Vetscan results are the number of Giardia cysts per coverslip. We compared the performance of the Vetscan and another POC test (SNAP Giardia test; Idexx) with a direct immunofluorescence assay (IFA) performed in a specialized parasitology laboratory as the reference test. We included 164 dogs <19-mo-old. We used pooled fecal samples from 3 defecations gained within 2-3 d and tested the repeatability of the Vetscan by triplicate measurement. Compared to IFA, Vetscan had a diagnostic sensitivity of 88.4% and specificity of 98.1%; SNAP had a diagnostic sensitivity of 74.4% and specificity of 98.1%. A variation coefficient of 67.0% was determined for the Vetscan results. The performance of the Vetscan is acceptable for the qualitative evaluation of fecal samples (Giardia positive or negative), and the device can be used by untrained personnel. Given its high variation coefficient, we do not recommend the Vetscan for monitoring the number of cysts.

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Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium Ecytonucleospora hepatopenaei (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against Cryptosporidium parvum, a coccidian parasite that infects calves (Bos taurus), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (Penaeus vannamei). EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of C. parvum has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease.

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Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

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The Antisecretory Factor (AF) is a protein that can reduce intestinal hypersecretion and various inflammation disorders in vivo. Discovered in many mammalian tissues and plasma, its mechanism of action remains unknown. Interestingly, its induction has been found to counteract vertigo in patients with Méniere's disease. This suggests an inherent ability to control body balance and posture, an activity that may play a role in cerebellar function. Therefore, it may be worthwhile to investigate whether this activity can inhibit neuronal cells involved in cerebellar circuitries and its potential action on enteric nervous system ganglia, which could explain its antisecretory effect in the intestine. Previously, we studied the role of AF on GABAA receptors in cerebellar granule cells, taking advantage of electrophysiology and evaluating the effects of the administration of AF-16, an AF peptide. Treatment with AF-16 increased GABAA receptor responses, especially those containing the α6 subunit. Here, we performed immunofluorescence experiments by staining α1 and α6 subunits before and after incubation with AF-16, analyzed super-resolved images comparing pre- and post-treatment maps and critically examined these experimental results with our previous electrophysiological data to shed light on the mechanisms of action of AF protein on GABAA receptor subpopulations, specifically the "fast" receptors of αn ß2/3 γ2 composition that contain either the α1 or the α6 subunit. The results indicate that the α6 subunit is redistributed, with a decrease in neurites and an increase in soma. Conversely, the α1 subunit shows opposite results, with an increase in neurites and a decrease in soma.

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Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

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PROBLEM: Endometrial immune cells are essential for maintaining homeostasis and the endometrial receptivity to embryo implantation. Understanding regional variations in endometrial immune cell populations is crucial for comprehending normal endometrial function and the pathophysiology of endometrial disorders. Despite previous studies focusing on the overall immune cell composition and function in the endometrium, regional variations in premenopausal women remain unclear. METHOD OF STUDY: Endometrial biopsies were obtained from four regions (anterior, posterior, left lateral, and right lateral) of premenopausal women undergoing hysteroscopy with no abnormalities. A 15-color human endometrial immune cell-focused flow cytometry panel was used for analysis. High-dimensional flow cytometry combined with a clustering algorithm was employed to unravel the complexity of endometrial immune cells. Additionally, multiplex immunofluorescent was performed for further validation. RESULTS: Our findings revealed no significant variation in the distribution and abundance of immune cells across different regions under normal conditions during the proliferative phase. Each region harbored similar immune cell subtypes, indicating a consistent immune microenvironment. However, when comparing normal regions to areas with focal hemorrhage, significant differences were observed. An increase in CD8+ T cells highlights the impact of localized abnormalities on the immune microenvironment. CONCLUSIONS: Our study demonstrates that the endometrial immune cell landscape is consistent across different anatomical regions during the proliferative phase in premenopausal women. This finding has important implications for understanding normal endometrial function and the pathophysiology of endometrial disorders.

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We present a unique case of an 89-year-old male with Alzheimer's disease who developed hemorrhagic blisters on his palms, which ruptured with time and were followed by pruritic erythematous lesions across his chest, upper back, lower abdomen, and thighs. The patient was diagnosed with dyshidrosiform bullous pemphigoid (DBP), an uncommon variant of the autoimmune condition bullous pemphigoid characterized by cutaneous and mucosal blistering, which commonly appears as vesiculobullous eruptions in the palmoplantar areas and may spread to other parts of the body. Less than 100 cases of DBP have been documented in the medical literature. Since DBP is difficult to identify and treat due to its clinical appearance similar to pompholyx, we reviewed the treatment of DBP and included clinical images and direct immunofluorescence (DIF) staining technique images to better establish the diagnosis.

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Cell imaging assays utilising fluorescence stains are essential for observing sub-cellular organelles and their responses to perturbations. Immunofluorescent staining process is routinely in labs, however the recent innovations in generative AI is challenging the idea of wet lab immunofluorescence (IF) staining. This is especially true when the availability and cost of specific fluorescence dyes is a problem to some labs. Furthermore, staining process takes time and leads to inter-intra-technician and hinders downstream image and data analysis, and the reusability of image data for other projects. Recent studies showed the use of generated synthetic IF images from brightfield (BF) images using generative AI algorithms in the literature. Therefore, in this study, we benchmark and compare five models from three types of IF generation backbones-CNN, GAN, and diffusion models-using a publicly available dataset. This paper not only serves as a comparative study to determine the best-performing model but also proposes a comprehensive analysis pipeline for evaluating the efficacy of generators in IF image synthesis. We highlighted the potential of deep learning-based generators for IF image synthesis, while also discussed potential issues and future research directions. Although generative AI shows promise in simplifying cell phenotyping using only BF images with IF staining, further research and validations are needed to address the key challenges of model generalisability, batch effects, feature relevance and computational costs.

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Background: The utilization of smartphone-assisted evaluation is emerging in the field of histopathology. This technique improves the adequacy of samples at the bedside, avoids procedure-related complications, reduces unnecessary repeat biopsies, and saves the cost of the procedure. This study aims to compare the number of glomeruli in a renal biopsy specimen obtained by an ultrasound-guided percutaneous needle biopsy, counted at the bedside using a smartphone fitted with a 16-megapixel macro lens (Bedside method) with that observed under a light microscope after the processing of the biopsy specimen (LM method). Materials and Methods: In this prospective cohort study, 24 consecutive adult patients (48 kidney biopsy samples) who underwent kidney biopsies were enrolled. All specimens were extracted by an ultrasound-guided percutaneous renal biopsy from the lower pole of the left kidney. Patients' demographics and clinical data were prospectively collected. The number of glomeruli in all the biopsy specimens was counted using a smartphone fitted with a 16-megapixel macro lens at the bedside (Bedside method) and subsequently under a light microscope by a pathologist after processing the biopsy specimen (LM method). Seven or more glomeruli in the specimen were considered adequate in our study. Results: The mean age of patients at biopsy was 46.9 ± 16 years with slightly male predominance (54.2%). A total of 47 specimens were obtained from 24 patients. Of the 24 patients, 22 had native kidney biopsy and 2 had renal allograft biopsy. The average number of cores obtained per patient was 1.96. The length of core specimens ranged from 1.5 to 2 cm. A good agreement was found between bedside adequacy and slide adequacy, κ =0.684, P = 0.000. The positive agreement rate and negative agreement rate were 91.4% and 23.1%, respectively. Conclusion: In the modern era of technology, the smartphone is a good tool to evaluate the adequacy of biopsy specimens at the bedside.

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Artificial intelligence (AI) is increasingly being used in medicine to enhance the speed and accuracy of disease diagnosis and treatment. AI-based image analysis is expected to play a crucial role in future healthcare facilities and laboratories, offering improved precision and cost-effectiveness. As technology advances, the requirement for specialized software knowledge to utilize AI applications is diminishing. Our study will examine the advantages and challenges of employing AI-based image analysis in the field of immunology and will investigate whether physicians without software expertise can use MS Azure Portal for ANA IIF test classification and image analysis. This is the first study to perform Hep-2 image analysis using MS Azure Portal. We will also assess the potential for AI applications to aid physicians in interpreting ANA IIF results in immunology laboratories. The study was designed in four stages by two specialists. Stage 1: creation of an image library, Stage 2: finding an artificial intelligence application, Stage 3: uploading images and training artificial intelligence, Stage 4: performance analysis of the artificial intelligence application. In the first training, the average pattern identification accuracy for 72 testing images was 81.94%. After the second training, this accuracy increased to 87.5%. Patterns Precision improved from 71.42 to 79.96% after the second training. As a result, the number of correctly identified patterns and their accuracy increased with the second training process. Artificial intelligence-based image analysis shows promising potential. This technology is expected to become essential in healthcare facility laboratories, offering higher accuracy rates and lower costs.

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BACKGROUND: Older patients who are predisposed to bullous pemphigoid (BP) may exhibit reluctance to undergo skin biopsy due to potential complications. OBJECTIVES: This study aimed to conduct a comparative evaluation among histology, direct immunofluorescence (DIF), and indirect immunofluorescence (IIF) to determine the optimal diagnostic tool in elderly patients. METHODS: A retrospective study was conducted on 841 patients suspected of having BP. All cases were initially classified as BP and non-BP in accordance with the diagnostic criteria. Student's t-test and chi-squared test examined differences between the 2 groups. We evaluated the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio detected by the 3 tools. We stratified the analysis by age to compare the performance of the diagnostic tools and examined the risk factors associated with BP using logistic regression. RESULTS: Overall, histology exhibited the highest sensitivity (89.4%), while DIF demonstrated the highest specificity (67.1%). In the elderly, the IIF test exhibited the highest specificity (57.5%), the highest positive likelihood ratio (2.047), and the lowest negative likelihood ratio (0.226). Among patients taking Dipeptidyl Peptidase-4 (DPP-4) inhibitors, IIF demonstrated the highest positive likelihood ratio (3.194) and the second-lowest negative likelihood ratio (0.235). CONCLUSIONS: In cases that elderly patients suspected of having BP are reluctant to undergo skin biopsy, IIF demonstrates the optimal diagnostic method due to its highest positive likelihood ratio, the lowest negative likelihood ratio among the 3 diagnostic measures. Moreover, IIF is found to be a more effective tool for detecting BP in patients using DPP-4 inhibitors.

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BACKGROUND: Nephronophthisis (NPH) comprises a heterogeneous group of inherited renal ciliopathies clinically characterized by progressive kidney failure. So far, definite diagnosis is based on molecular testing only. Here, we studied the feasibility of NPHP1 and NPHP4 immunostaining of nasal epithelial cells to secure and accelerate the diagnosis of NPH. METHODS: Samples of 86 individuals with genetically determined renal ciliopathies were analyzed for NPHP1 localization using immunofluorescence microscopy (IF). A sub-cohort of 35 individuals was also analyzed for NPHP4 localization. Western blotting was performed to confirm IF results. RESULTS: NPHP1 and NPHP4 were both absent in all individuals with disease-causing NPHP1 variants including one with a homozygous missense variant (c.1027G > A; p.Gly343Arg) formerly classified as a "variant of unknown significance." In individuals with an NPHP4 genotype, we observed a complete absence of NPHP4 while NPHP1 was severely reduced. IF results were confirmed by immunoblotting. Variants in other genes related to renal ciliopathies did not show any impact on NPHP1/NPHP4 expression. Aberrant immunostaining in two genetically unsolved individuals gave rise for a further genetic workup resulting in a genetic diagnosis for both with disease-causing variants in NPHP1 and NPHP4, respectively. CONCLUSIONS: IF of patient-derived respiratory epithelial cells may help to secure and accelerate the diagnosis of nephronophthisis-both by verifying inconclusive genetic results and by stratifying genetic diagnostic approaches. Furthermore, we provide in vivo evidence for the interaction of NPHP1 and NPHP4 in a functional module.

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The assessment of chemotherapy response in osteosarcoma (OS), based on the average percentage of viable cells, is limited, as it overlooks the spatial heterogeneity of tumor cell response (foci of resistant cells), immune microenvironment, and bone microarchitecture. Despite the resulting positive classification for response to chemotherapy, some patients experience early metastatic recurrence, demonstrating that our conventional tools for evaluating treatment response are insufficient. We studied the interactions between tumor cells, immune cells (lymphocytes, histiocytes, and osteoclasts), and bone extracellular matrix (ECM) in 18 surgical resection samples of OS using multiplex and conventional immunohistochemistry (IHC: CD8, CD163, CD68, and SATB2), combined with multiscale characterization approaches in territories of good and poor response (GRT/PRT) to treatment. GRT and PRT were defined as subregions with <10% and ≥10% of viable tumor cells, respectively. Local correlations between bone ECM porosity and density of immune cells were assessed in these territories. Immune cell density was then correlated to overall patient survival. Two patterns were identified for histiocytes and osteoclasts. In poor responder patients, CD68 osteoclast density exceeded that of CD163 histiocytes but was not related to bone ECM load. Conversely, in good responder patients, CD163 histiocytes were more numerous than CD68 osteoclasts. For both of them, a significant negative local correlation with bone ECM porosity was found (P < ,01). Moreover, in PRT, multinucleated osteoclasts were rounded and intermingled with tumor cells, whereas in GRT, they were elongated and found in close contact with bone trabeculae. CD8 levels were always low in metastatic patients, and those initially considered good responders rapidly died from their disease. The specific recruitment of histiocytes and osteoclasts within the bone ECM, and the level of CD8 represent new features of OS response to treatment. The associated prognostic signatures should be integrated into the therapeutic stratification algorithm of patients after surgery.

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BACKGROUND: Chronic photosensitivity dermatitis (CPD) (also named actinic reticuloid) is an unusual disease classically referred often in elderly men. Affected patients have severely itchy, thickened dry skin in areas exposed to the sun throughout the years. METHOD: A Caucasian female patient who worked most of her life outside who had "chronic dermatitis" in her neck started planting chrysanthemum in her garden on a sunny day. Later, she presented edema, erythema, papules, and a few vesicles in her neck with severe pruritus. STUDIES: A skin biopsy revealed the diagnosis of CPD, along with positive testing for ultraviolet B (UVB), minimal erythema doses (MED) for UVB (MEDB) UVA (MEDA) and PhotoPath. RESULTS: Direct immunofluorescence (DIF) stains using anti-human antibodies against fibrinogen, albumin, IgG, IgM, lambda, kappa, and C3c and C1q were positive at the base membrane area of the dermal epidermal junction, in the papillary dermis, as well as the neurovascular bundles in all the dermis and the extracellular matrix, especially those under the blisters. CONCLUSION: With this case, we suggest not forgetting the importance of using DIF in reactivated CPD cases in addition to the photo patch testing.

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OBJECTIVE: Atopic dermatitis (AD) is characterized by compositional and structural changes to the skin at lesional sites. Alteration to the levels and organization of both protein and lipid components are associated with disease status and lead to impaired barrier and hydration. Corneodesmosin (CDSN) and the arrangement and length of the intercellular lipid lamellae (ICLL) are altered in disrupted skin states. The aim of this research was to profile the distribution of CDSN and the ICLL in the stratum corneum (SC) at lesional and non-lesional sites in AD-prone skin and to investigate the impact of an eczema calming lotion containing petroleum jelly, fatty acids, and colloidal oatmeal. METHODS: An IRB-approved study was conducted with participants with active AD. From a small subset of participants, tape strips were collected from lesional and non-lesional sites on the arm, prior to and after twice daily application, over 4 weeks of an eczema calming lotion containing petroleum jelly, fatty acids, and colloidal oatmeal. Fluorescent antibody staining was used to investigate the distribution of CDSN. Transmission electron microscopy (TEM) was used to characterize the ICLL. RESULTS: The distribution/coverage of CDSN was similar between lesional and non-lesional sites at baseline; application of the lotion resulted in a more defined honeycomb/peripheral distribution. Normalized ICLL (nICLL) was lower in baseline samples from lesional sites relative to non-lesional sites. Application of the lotion increased this parameter by the end of the study at all sites. CONCLUSION: The eczema calming lotion containing petroleum jelly, fatty acids and colloidal oatmeal provided changes in corneodesmosomal proteins distribution and ICLL, consistent with improvements in corneocyte maturation and improved barrier function in the skin of individuals with atopic dermatitis.

OBJECTIF: La dermatite atopique (DA) est caractérisée par des modifications de la composition et de la structure de la peau au niveau des sites lésionnels. L'altération des taux et de l'organisation des composants protéiques et lipidiques est associée au statut de la maladie, et entraîne une altération de la barrière et de l'hydratation. La cornéodesmosine (CDSN), et la disposition et la longueur des lamelles lipidiques intercellulaires (LLIC) sont altérées dans les états cutanés perturbés. L'objectif de cette étude était d'établir le profil de la distribution de la CDSN et des LLIC dans la couche cornée (CC) au niveau des sites lésionnels et non lésionnels dans la peau sujette à la DA, et d'étudier l'impact d'une lotion apaisante contre l'eczéma contenant de la vaseline, des acides gras et de l'avoine colloïdale. MÉTHODES: Une étude approuvée par un CPP a été menée auprès de participants atteints de DA active. Dans un petit sous­ensemble de participants, des bandes adhésives ont été prélevées sur des sites lésionnels et non lésionnels du bras, avant et après l'application deux fois par jour pendant 4 semaines d'une lotion apaisante contre l'eczéma contenant de la vaseline, des acides gras et de l'avoine colloïdale. Une coloration par anticorps fluorescents a été utilisée pour étudier la distribution de la CDSN. La microscopie électronique en transmission (MET) a été utilisée pour caractériser les LLIC. RÉSULTATS: La distribution/couverture de la CDSN était similaire entre les sites lésionnels et non lésionnels à l'entrée dans l'étude; l'application de la lotion a entraîné une distribution en nid d'abeille/périphérique plus définie. Le taux normalisé de LLIC (LLICn) était plus faible dans les échantillons prélevés à l'entrée dans l'étude au niveau des sites lésionnels par rapport aux sites non lésionnels. L'application de la lotion a augmenté ce paramètre à la fin de l'étude pour tous les sites. CONCLUSIONS: La lotion apaisante contre l'eczéma contenant de la vaseline, des acides gras et de l'avoine colloïdale a entraîné des changements dans la distribution des protéines cornéodesmosomales et des LLIC, ce qui correspond à des améliorations de la maturation des cornéocytes et de la fonction de barrière de la peau des personnes atteintes de dermatite atopique.

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