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Aβ42 is one of the most extensively studied blood and Cerebrospinal fluid (CSF) biomarkers for the diagnosis of symptomatic and prodromal Alzheimer’s disease (AD). Because of the heterogeneity and transient nature of Aβ42 oligomers (Aβ42Os), the development of technologies for dynamically detecting changes in the blood or CSF levels of Aβ42 monomers (Aβ42Ms) and Aβ42Os is essential for the accurate diagnosis of AD. The currently commonly used Aβ42 ELISA test kits usually mis-detected the elevated Aβ42Os, leading to incomplete analysis and underestimation of soluble Aβ42, resulting in a comprised performance in AD diagnosis. Herein, we developed a dual-target lateral flow immunoassay (dLFI) using anti-Aβ42 monoclonal antibodies 1F12 and 2C6 for the rapid and point-of-care detection of Aβ42Ms and Aβ42Os in blood samples within 30 min for AD diagnosis. By naked eye observation, the visual detection limit of Aβ42Ms or/and Aβ42Os in dLFI was 154 pg/mL. The test results for dLFI were similar to those observed in the enzyme-linked immunosorbent assay (ELISA). Therefore, this paper-based dLFI provides a practical and rapid method for the on-site detection of two biomarkers in blood or CSF samples without the need for additional expertise or equipment. Graphical Abstract

Background Exogenous materials may be encountered in skin biopsies as contamination. Contamination may occur during the biopsy procedure in the clinic or during tissue processing in the laboratory. Although the experienced pathologist may often instinctively ignore clear examples of contamination, such tissue may be a source of confusion for young pathologists and those in training. Foreign materials can typically be recognized as exogenous by morphology, polarizability, and the presence or absence of a tissue reaction, but we have rarely encountered situations in which exogenous materials could be misinterpreted as organisms, either by their morphologic appearance or staining properties. Methods Exogenous materials used during skin biopsy and grossing were embedded in a nonhuman tissue scaffold and prepared into histologic slides. Hematoxylin and eosin (H&E), periodic acid-Schiff with diastase, and Grocott methenamine silver stains were performed, and each material was evaluated under polarized microscopy. Results Exogenous materials were divided into the following 3 categories with shared morphologic appearances and staining properties: suture materials, plant-based materials, and synthetic materials. Conclusion We present a comprehensive characterization of the morphologic and cytochemical staining properties of multiple exogenous materials that may contaminate a skin biopsy. This characterization will aid the pathologist by providing a mechanism to identify potential contaminants in skin biopsies.

Objectives To report clinical features, gross post mortem and histopathological findings from an investigation into sudden or unexpected death in rabbits that was undertaken during an outbreak of rabbit haemorrhagic disease. Materials and methods Using a standard protocol, veterinarians were invited to submit case histories and results of their post mortem examination of pet rabbits that died unexpectedly. Histopathological examination of heart, lungs, liver, spleen and kidney samples was collated with macroscopic appearance and clinical details. Results Hepatocellular necrosis, characteristic of rabbit haemorrhagic disease, was observed in 185 of 300 (62%) submissions, often accompanied by glomerular thrombosis and changes in other organs. Evidence of rabbit haemorrhagic disease was not apparent on histopathology in 113 of 300 (38%) rabbits. Gross post mortem examination by veterinary practitioners did not always reflect reported histopathological changes. No macroscopic abnormalities were seen in 78/185 (42%) of rabbit haemorrhagic disease cases. Rapid death and death of other rabbits in the household were common features of rabbit haemorrhagic disease. Ante mortem clinical signs included anorexia, collapse, lethargy, seizures, icterus, bleeding from the mouth, dyspnoea, hypothermia, pyrexia, bradycardia or poor blood clotting. Clinical importance Rabbit haemorrhagic disease can be suspected from a history of sudden death, especially if multiple rabbits are affected. There is not always macroscopic evidence of the disease but histopathology is useful to support or refute a diagnosis of rabbit haemorrhagic disease and provide information about other causes of death.

Significance: Seborrheic keratoses (SKs) are harmless pigmented skin lesions (PSLs) that may be confused clinically not only with other benign conditions but also with cutaneous melanoma (CM). As SKs are one of the most common neoplasms in adults, the importance of their correct diagnosis is high. Misclassifying SK as malignant is not rare and leads to a high number of unnecessary biopsies. On the other hand, misdiagnosing CM as SK may have a large impact on prognosis or therapy. Aim: In the non-invasive technique of dermatofluoroscopy, the fluorophores in melanocytes and keratinocytes are excited in vivo with nanosecond laser pulses and the resulting spectrally resolved, melanin-dominated fluorescence signals are used to differentiate between pigmented benign lesions and CM. Approach: In this single-center, non-interventional study, 33 PSLs of 20 patients were scanned with dermatofluoroscopy in vivo. For all included cases, dermatofluoroscopic signals were compared to pathology classification. Results: The characteristic spectral features of SK were identified, where the signals are dominated by keratin, NAD(P)H, and melanin. The fluorescence spectra of SKs differed substantially from those of CM: a characteristic spectrum of SK has been identified in 27 of 28 SKs. Conclusions: The high-accuracy differential diagnosis between CM and SK is possible with dermatofluoroscopy.

Significance: As one part of the central nervous system, the retina manifests neurovascular defects in Alzheimer’s disease (AD). Quantitative imaging of retinal neurovascular abnormalities may promise a new method for early diagnosis and treatment assessment of AD. Previous imaging studies of transgenic AD mouse models have been limited to the central part of the retina. Given that the pathological hallmarks of AD frequently appear in different peripheral quadrants, a comprehensive regional investigation is needed for a better understanding of the retinal degeneration associated with AD-like pathology. Aim: We aim to demonstrate concurrent optical coherence tomography (OCT) and OCT angiography (OCTA) of retinal neuronal and vascular abnormalities in the 5XFAD mouse model and to investigate region-specific retinal degeneration. Approach: A custom-built OCT system was used for retinal imaging. Retinal thickness, vessel width, and vessel density were quantitatively measured. The artery and vein (AV) were classified for differential AV analysis, and trilaminar vascular plexuses were segmented for depth-resolved density measurement. Results: It was observed that inner and outer retinal thicknesses were explicitly reduced in the dorsal and temporal quadrants, respectively, in 5XFAD mice. A significant arterial narrowing in 5XFAD mice was also observed. Moreover, overall capillary density consistently showed a decreasing trend in 5XFAD mice, but regional specificity was not identified. Conclusions: Quadrant- and layer-specific neurovascular degeneration was observed in 5XFAD mice. Concurrent OCT and OCTA promise a noninvasive method for quantitative monitoring of AD progression and treatment assessment.

Significance: Three-dimensional (3D) vascular and metabolic imaging (VMI) of whole organs in rodents provides critical and important (patho)physiological information in studying animal models of vascular network. Aim: Autofluorescence metabolic imaging has been used to evaluate mitochondrial metabolites such as nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FAD). Leveraging these autofluorescence images of whole organs of rodents, we have developed a 3D vascular segmentation technique to delineate the anatomy of the vasculature as well as mitochondrial metabolic distribution. Approach: By measuring fluorescence from naturally occurring mitochondrial metabolites combined with light-absorbing properties of hemoglobin, we detected the 3D structure of the vascular tree of rodent lungs, kidneys, hearts, and livers using VMI. For lung VMI, an exogenous fluorescent dye was injected into the trachea for inflation and to separate the airways, confirming no overlap between the segmented vessels and airways. Results: The kidney vasculature from genetically engineered rats expressing endothelial-specific red fluorescent protein TdTomato confirmed a significant overlap with VMI. This approach abided by the “minimum work” hypothesis of the vascular network fitting to Murray’s law. Finally, the vascular segmentation approach confirmed the vascular regression in rats, induced by ionizing radiation. Conclusions: Simultaneous vascular and metabolic information extracted from the VMI provides quantitative diagnostic markers without the confounding effects of vascular stains, fillers, or contrast agents.

For the validation of optical diagnostic technologies, experimental results need to be benchmarked against the gold standard. Currently, the gold standard for tissue characterization is assessment of hematoxylin and eosin (H&E)-stained sections by a pathologist. When processing tissue into H&E sections, the shape of the tissue deforms with respect to the initial shape when it was optically measured. We demonstrate the importance of accounting for these tissue deformations when correlating optical measurement with routinely acquired histopathology. We propose a method to register the tissue in the H&E sections to the optical measurements, which corrects for these tissue deformations. We compare the registered H&E sections to H&E sections that were registered with an algorithm that does not account for tissue deformations by evaluating both the shape and the composition of the tissue and using microcomputer tomography data as an independent measure. The proposed method, which did account for tissue deformations, was more accurate than the method that did not account for tissue deformations. These results emphasize the need for a registration method that accounts for tissue deformations, such as the method presented in this study, which can aid in validating optical techniques for clinical use. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.

Significance: The study has confirmed the feasibility of using ultraviolet (UV) excitation to visualize and quantify desmoplasia in fresh tumor tissue of pancreatic adenocarcinoma (PDAC) in an orthotopic xenograft mouse model, which provides a useful imaging platform to evaluate acute therapeutic responses. Aim: Stromal network of collagen prominent in PDAC tumors is examined by imaging fresh tissue samples stained with histological dyes. Fluorescence signals are color-transferred to mimic Masson’s trichrome staining. Approach: Murine tumor samples were stained with Hoechst, eosin, and rhodamine B and excited at 275-nm. Fluorescence signals in the visible spectrum were captured by a CMOS color camera with high contrast and resolution at whole-tumor slice field of view. Results: Fluorescence imaging using UV excitation is capable of visualizing collagen deposition in PDAC tumors. Both fluorescence and histology data showed collagen content of up to 30%. The collagen modulation effect due to photodynamic priming treatment was observed showing 13% of collagen reduction. Necrosis area is visible and perfusion imaging using Texas Red dextran is feasible. Conclusions: The study demonstrates collagen visualization in fresh PDAC tumor samples using UV excitation. This imaging platform also provides quantitative stromal information from fiber analysis and visibility of necrosis and perfusion, suitable for therapeutic response assessment of photodynamic therapy.

Significance: Lupus nephritis (LuN) is a chronic inflammatory kidney disease. The cellular mechanisms by which LuN progresses to kidney failure are poorly characterized. Automated instance segmentation of immune cells in immunofluorescence images of LuN can probe these cellular interactions. Aim: Our specific goal is to quantify how sample fixation and staining panel design impact automated instance segmentation and characterization of immune cells. Approach: Convolutional neural networks (CNNs) were trained to segment immune cells in fluorescence confocal images of LuN biopsies. Three datasets were used to probe the effects of fixation methods on cell features and the effects of one-marker versus two-marker per cell staining panels on CNN performance. Results: Networks trained for multi-class instance segmentation on fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) samples stained with a two-marker panel had sensitivities of 0.87 and 0.91 and specificities of 0.82 and 0.88, respectively. Training on samples with a one-marker panel reduced sensitivity (0.72). Cell size and intercellular distances were significantly smaller in FFPE samples compared to fresh frozen (Kolmogorov–Smirnov, p≪0.0001). Conclusions: Fixation method significantly reduces cell size and intercellular distances in LuN biopsies. The use of two markers to identify cell subsets showed improved CNN sensitivity relative to using a single marker.

Significance: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. With a worldwide incidence rate of 2 to 3 per 100,000 people, it accounts for more than 60% of all brain cancers; currently, its 5-year survival rate is

Significance: Early detection of epithelial cancers and precancers/neoplasia in the presence of benign lesions is challenging due to the lack of robust in vivo imaging and biopsy guidance techniques. Label-free nonlinear optical microscopy (NLOM) has shown promise for optical biopsy through the detection of cellular and extracellular signatures of neoplasia. Although in vivo microscopy techniques continue to be developed, the surface area imaged in microscopy is limited by the field of view. FDA-approved widefield fluorescence (WF) imaging systems that capture autofluorescence signatures of neoplasia provide molecular information at large fields of view, which may complement the cytologic and architectural information provided by NLOM. Aim: A multimodal imaging approach with high-sensitivity WF and high-resolution NLOM was investigated to identify and distinguish image-based features of neoplasia from normal and benign lesions. Approach: In vivo label-free WF imaging and NLOM was performed in preclinical hamster models of oral neoplasia and inflammation. Analyses of WF imaging, NLOM imaging, and dual modality (WF combined with NLOM) were performed. Results: WF imaging showed increased red-to-green autofluorescence ratio in neoplasia compared to inflammation and normal oral mucosa (p

Significance: The blood–brain barrier (BBB) is a major obstacle to detecting and treating brain tumors. Overcoming this challenge will facilitate the early and accurate detection of brain lesions and guide surgical resection of tumors. Aim: We generated an orthotopic brain tumor model that simulates the pathophysiology of gliomas at early stages; determine the BBB integrity and breakdown over the time course of tumor progression using generic and cancer-targeted near-infrared (NIR) fluorescent molecular probes. Approach: We developed an intracranial tumor xenograft model that rapidly reestablished BBB integrity and monitored tumor progression by bioluminescence imaging. Sham control mice were injected with phosphate-buffered saline only. Fluorescence molecular tomography (FMT) was used to quantify the uptake of tumor-targeted and passive NIR fluorescent imaging agents in orthotopic glioma (U87-GL-GFP PDE7B H217Q cells) tumor model. Cancer-induced and transient (with focused ultrasound, FUS) disruption of BBB integrity was monitored with NIR fluorescent dyes. Results: Stereotactic injection of 50,000 cells into mouse brain allowed rapid reestablishment of BBB integrity within a week, as determined by the inability of both tumor-targeted and generic NIR imaging agents to extravasate into the brain. Tumor-induced BBB disruption was observed 7 weeks after tumor implantation. FUS achieved a similar effect at any time point after reestablishing BBB integrity. While tumor uptake and retention of the passive NIR dye, indocyanine green, was negligible, both actively tumor-targeting agents exhibited selective accumulation in the tumor region. The tumor-targeting molecular probe that clears rapidly from nontumor brain tissue exhibits higher contrast than the analogous vascular-targeting agent and helps delineate tumors from sham control. Conclusions: We highlight the utility of FMT imaging for longitudinal assessment of brain tumors and the interplay between the stages of BBB disruption and molecular probe retention in tumors, with potential application to other neurological diseases.

Significance: A key risk faced by oncological surgeons continues to be complete removal of tumor. Currently, there is no intraoperative imaging device to detect kidney tumors during excision. Aim: We are evaluating molecular chemical imaging (MCI) as a technology for real-time tumor detection and margin assessment during tumor removal surgeries. Approach: In exploratory studies, we evaluate visible near infrared (Vis-NIR) MCI for differentiating tumor from adjacent tissue in ex vivo human kidney specimens, and in anaesthetized mice with breast or lung tumor xenografts. Differentiation of tumor from nontumor tissues is made possible with diffuse reflectance spectroscopic signatures and hyperspectral imaging technology. Tumor detection is achieved by score image generation to localize the tumor, followed by application of computer vision algorithms to define tumor border. Results: Performance of a partial least squares discriminant analysis (PLS-DA) model for kidney tumor in a 22-patient study is 0.96 for area under the receiver operating characteristic curve. A PLS-DA model for in vivo breast and lung tumor xenografts performs with 100% sensitivity, 83% specificity, and 89% accuracy. Conclusion: Detection of cancer in surgically resected human kidney tissues is demonstrated ex vivo with Vis-NIR MCI, and in vivo on mice with breast or lung xenografts.

Background Contrast-enhanced ultrasonography (CEUS) features of primary hepatobiliary neoplasms have been reported in dogs but no information is available in cats. Methods Qualitative and quantitative features of bile duct adenomas (BDAs, n=20), bile duct carcinomas (BDCs, n=16), and hepatocellular carcinomas (HCCs, n=8) are described in 44 cats. Results There was an overlap in CEUS qualitative features between different histotypes, both in wash-in and wash-out phases. Distinction between different neoplasms based only on the CEUS qualitative features was not possible. At peak of enhancement, the BDAs, BDCs and HCCs showed a large range of echogenicities, from hypoenhancement to hyperenhancement, in comparison to the liver parenchyma. Eight of 20 BDAs showed inhomogeneous hyperenhancement during wash-in, which is a feature reported as typical of malignant lesions in dogs. BDC had a significantly faster wash-in compared with both BDA and HCC but the diagnostic accuracy of all the included quantitative variables was only moderate. No significant differences in the wash-out quantitative features of BDA and BDC were evident. Conclusion There is poor evidence that CEUS may be used to distinguish between different primary hepatobiliary neoplasms in cats.

Despite decades of epidemiological research, it remains uncertain whether ionizing radiation can cause lymphomas. Most epidemiological studies of lymphoma risk following non-uniform exposure used dose to red bone marrow (RBM), constituting a small fraction of the lymphocytes, as a surrogate of dose to the lymphocytes. We developed a method to estimate dose to the lymphocytes using the reference distribution of lymphocytes throughout the body and Monte Carlo simulations of computational human phantoms. We applied our method to estimating lymphocyte doses for a pediatric CT patient cohort in the United Kingdom. Estimated dose to the RBM was greater than lymphocyte dose for most scan types (up to 2.6-fold higher, a 5-year-old brain scan) except abdomen scan (RBM dose was about half the lymphocyte dose, a 5-year-old abdomen scan). The lymphocyte dose in the UK cohort showed that T-spine and whole body scans delivered the highest lymphocyte doses (up to 22.4 mGy).

Significance: Detection and characterization of circulating tumor cells (CTCs), a key determinant of metastasis, are critical for determining risk of disease progression, understanding metastatic pathways, and facilitating early clinical intervention. Aim: We aim to demonstrate label-free imaging of suspected melanoma CTCs. Approach: We use a linear-array-based photoacoustic tomography system (LA-PAT) to detect melanoma CTCs, quantify their contrast-to-noise ratios (CNRs), and measure their flow velocities in most of the superficial veins in humans. Results: With LA-PAT, we successfully imaged suspected melanoma CTCs in patients in vivo, with a CNR >9. CTCs were detected in 3 of 16 patients with stage III or IV melanoma. Among the three CTC-positive patients, two had disease progression; among the 13 CTC-negative patients, 4 showed disease progression. Conclusions: We suggest that LA-PAT can detect suspected melanoma CTCs in patients in vivo and has potential clinical applications for disease monitoring in melanoma.

Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. With a high concentration in basal cell cytoplasm, THG contrast provided by melanin overshadows other THG sources in human skin studies. For better understanding of the THG signals in keratinocytes without the influence of melanin, an in vivo THG microscopy (THGM) study was first conducted on vitiliginous skin. As a result, the THG-brightness ratio between the melanin-lacking cytoplasm of basal cells and collagen fibers is about 1.106 at the dermal–epidermal junctions of vitiliginous skin, indicating high sensitivity of THGM for the presence of melanin. We further applied the in vivo THGM to assist evaluating the therapeutic outcome from the histopathological point of view for those showed no improvement under narrowband ultraviolet B therapy based on the seven-point Physician Global Assessment score. Our clinical study indicates the high potential of THGM to assist the histopathological assessment of the therapeutic efficacy of vitiligo treatments.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

Inexpensive near-infrared microscopy (NIRM) was developed as a convenient technique to detect the medulla loss of scalp hair while reducing analytical time with easy sample preparation, leading to a field screening tool for breast cancer. NIRM has been evaluated as an alternative to synchrotron-based nanoscopy and to the relatively expensive method of conventional infrared microscopy to determine the degree and pattern of medulla loss of scalp hairs of patients with breast cancer and benign diseases, as well as normal healthy individuals. NIR imaging showed a strong, scattering-based hyperintense contrast of the medulla compared to the fully attenuated cortex in medullated healthy hair. Complete medulla loss (CML) per hair strand was more extensively (60.9 ± 10.2 %) (p

Conventional techniques are insufficient precisely to describe the internal structure, the heterogeneous cell populations, and the dynamics of biological processes occurring in diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to help avoid postoperative liver failure. We analyze the progression of both acute (cholestasis) and chronic (fibrosis) liver pathology using multiphoton microscopy with fluorescence lifetime imaging and second-harmonic generation modes combined with time-of-flight secondary ion mass spectrometry chemical analysis to obtain new data about pathological changes to hepatocytes at the cellular and molecular levels. All of these techniques allow the study of cellular metabolism, lipid composition, and collagen structure without staining the biological materials or the incorporation of fluorescent or other markers, enabling the use of these methods in a clinical situation. The combination of multiphoton microscopy and mass spectrometry provides more complete information about the liver structure and function than could be assessed using either method individually. The data can be used both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique for liver quality analysis in order to plan surgery and to help avoid postoperative liver failure in clinic.

Detection of Leishmania in cutaneous lesions is possible by visualization of amastigotes. Detection of Leishmania DNA by PCR presents greater sensitivity, and PCR has been used to diagnose cutaneous leishmaniosis in humans using noninvasive clinical specimens.Study I: to determine if Leishmania DNA could be efficiently extracted and amplified from archived Diff-QuikSamples from cutaneous lesions of 54 dogs.Study I: Leishmania-qPCR was performed on 19 glass slides (from nine dogs) with cytologically visible amastigotes. Fifteen slides with no visible amastigotes, obtained from 12 dogs seronegative for Leishmania by ELISA, served as controls. Study II: Leishmania-qPCR was performed on glass slides and FPI from cutaneous lesions compatible with clinical leishmaniosis in 33 dogs.Study I: all slides with visible amastigotes had positive qPCR, whereas all control slides yielded negative results. Study II: of 13 dogs definitively diagnosed with clinical leishmaniosis, eight had visible amastigotes on cytology, whereas Leishmania-qPCR was positive on 11 glass slides and 13 FPI. Leishmaniosis was ruled out by standard methods in 20 dogs, four of which yielded positive qPCR on FPI and/or glass slides.Leishmania-DNA can be detected efficiently by qPCR from cutaneous cytological specimens and FPI to diagnose Leishmania infection in dogs with cutaneous lesions suggestive of CanL.Détection de Leishmania dans les lésions cutanées est possible par visualisation des amastigotes. La détection de l’ADN de Leishmania par PCR présente une meilleure sensibilité et la PCR a été utilisée pour diagnostiquer la leishmaniose cutanée de l'homme à l'aide de techniques cliniques non invasives.Etude I : déterminer si l’ADN de Leishmania peut être efficacement extraite et amplifiée de lames colorées au Diff-QuikEchantillons de lésions cutanées de 54 chiens. MATÉRIEL ET MÉTHODE: Etude I : la PCRq a été réalisée sur 19 lames (de 9 chiens) avec des amastigotes visibles à la cytologie. Quinze lames sans amastigote visible, obtenu de 12 chiens séronégatifs pour Leishmania par ELISA, ont servi de contrôle. Etude II : Une PCRq Leishmania a été réalisée sur des lames et FPI de lésions cutanées compatibles avec une leishmaniose clinique chez 33 chiens. RÉSULTATS: Etude I : toutes les lames avec amastigotes visibles avaient une PCRq positive tandis que les lames contrôles étaient négatives. Etude II : sur les 13 chiens diagnostiqués définitivement comme leishmaniose clinique, huit avaient des amastigotes visibles à la cytologie tandis que la PCRq Leishmania était positive sur 11 des lames et 13 FPI. LA leishmaniose a été exclue par méthodes standard chez 20 chiens, dont quatre présentaient une PCRq positive sur FPI et/ou lames.L’ADN de Leishmania peut être détectée efficacement par PCRq de cytologies cutanées et FPI pour le diagnostic de leishmaniose chez les chiens présentant des lésions cutanées évocatrices de CanL.INTRODUCCIÓN: la visualización de amastigotes permite la detección de Leishmania en lesiones cutáneas. La detección del DNA de Leishmania por PCR es más sensible, y la PCR se ha utilizado para diagnosticar la leishmaniasis cutánea en humanos utilizando muestras clínicas no invasivas. OBJETIVOS: Estudio I: determinar si el DNA de Leishmania se podría extraer y amplificar de manera eficiente a partir de preparaciones teñidas con Diff-Quik® archivadas de muestras citológicas de lesiones cutáneas caninas. Estudio II: evaluar el valor diagnóstico de una PCR cuantitativa (qPCR) de Leishmania en muestras citológicas teñidas y en impresiones de papel de filtro (FPI) obtenidas de lesiones cutáneas sugestivas de leishmaniasis canina (CanL). ANIMALES: muestras de lesiones cutáneas de 54 perros. MÉTODOS Y MATERIALES: Estudio I: se realizó qPCR para Leishmania en 19 portaobjetos de vidrio (de nueve perros) con amastigotes citológicamente visibles. Quince preparaciones sin amastigotes visibles, obtenidas de 12 perros seronegativos para Leishmania por ELISA, sirvieron como controles. Estudio II: se realizó qPCR para Leishmania en portaobjetos de vidrio y FPI de lesiones cutáneas compatibles con leishmaniasis clínica en 33 perros. RESULTADOS: Estudio I: todas las preparaciones con amastigotes visibles tuvieron qPCR positiva, mientras que todas las preparaciones de control dieron resultados negativos. Estudio II: de 13 perros diagnosticados definitivamente con leishmaniasis clínica, ocho tuvieron amastigotes visibles en la citología, mientras que qPCR para Leishmania fue positiva en 11 portaobjetos de vidrio y 13 FPI. La leishmaniasis se descartó mediante métodos estándar en 20 perros, cuatro de los cuales produjeron qPCR positiva en FPI y/o portaobjetos de vidrio. CONCLUSIONES E IMPORTANCIA CLÍNICA: la qPCR para DNA de Leishmania puede realizarse de forma efectiva a partir de muestras citológicas cutáneas y FPI para diagnosticar la infección por Leishmania en perros con lesiones cutáneas sugestivas de CanL.Leishmanien können in Hautveränderungen durch eine Sichtbarmachung der Amastigoten gefunden werde. Die Feststellung von Leishmania DNA mittels PCR bedeutet eine größere Sensibilität. Außerdem wurde die PCR verwendet, um kutane Leishmaniose bei Menschen mittels nichtinvasiver klinischer Proben zu diagnostizieren.Studie I: festzustellen, ob ausreichend Leishmania DNA aus archivierten Diff-Quick® gefärbten zytologischen Präparaten aus kutanen Veränderungen extrahiert und amplifiziert werden konnte. Studie II: eine Evaluierung der diagnostischen Leistung einer quantitativen Leishmania (q) PCR aus gefärbten Zytologiepräparaten sowie aus Abdrücken auf Filterpapier (FPI), die aus Hautveränderungen stammten, die verdächtig für eine canine Leishmaniose (CanL) waren.Es wurden Proben von Hautveränderungen von 54 Hunden verwendet.Studie I: Es wurde ein Leishmania-qPCR von 19 Objektträgern (von neun Hunden) mit zytologisch sichtbaren Amastigoten durchgeführt. Fünfzehn Objektträger ohne sichtbare Amastigoten, die von 12 Hunden stammten, die mittels ELISA seronegativ auf Leishmania waren, dienten als Kontrollen. Studie II: Ein Leishmania-qPCR wurde von Objektträgern und FPI kutaner Läsionen von 33 Hunden, die mit klinischer Leishmaniose kompatibel waren, durchgeführt.Studie I: alle Objektträger mit sichtbaren Amastigoten zeigten eine positive qPCR, während die Kontrollen ein negatives Ergebnis lieferten. Studie II: von den 13 Hunden, die definitiv mit einer Leishmaniose diagnostiziert worden waren, zeigten acht sichtbare Amastigoten in der Zytologie, während der Leishmania-PCR von 11 Objektträgern und 13 FPI positiv war. Eine Leishmaniose war bei 20 Hunden mit Standardmethoden ausgeschlossen worden, von denen vier eine positive qPCR von FPI und/oder Objektträgern lieferten.Leishmania-DNA kann effektiv mittels qPCR aus kutanen zytologischen Hautproben und aus FPI festgestellt werden, um eine Leishmania Infektion bei Hunden mit Hautveränderungen, die auf CanL hinweisen zu diagnostizieren.背景: 皮膚病変におけるリーシュマニアの検出は、無鞭毛期虫体の視覚化によって可能となる。 PCRによるリーシュマニアDNAの検出はより高い感度を示し、PCRは非侵襲性臨床標本を用いて人の皮膚リーシュマニア症を診断するために使用されてきた。 目的: 研究I:研究Iの目的は、犬の皮膚病変から得られた細胞学的標本のアーカイブされたDiff-Quik®染色スライドからLeishmania DNAを効率的に抽出および増幅できるかどうかを決定することである。研究II:研究IIの目的は、染色した細胞学的標本および犬リーシュマニア症(CanL)を示唆する皮膚病変から得られたfilter paper impressions (FPI)に対するリーシュマニア定量的(q)PCRの診断性能を評価することである。 被験動物: 54頭の犬の皮膚病変からのサンプル。 材料と方法: 研究I:Leishmania-qPCRを、細胞学的に観察可能な無鞭毛期虫体を含む19枚のスライドガラス(9頭の犬から)によって実施した。 ELISAによってリーシュマニアに対して血清陰性の12頭の犬から得られた、目に見える無鞭毛期虫体のない15枚のスライドを対照として用いた。研究II:33頭の犬において、リーシュマニアqPCRを、臨床的にリーシュマニア症と適合性のある皮膚病変からのスライドガラスおよびFPIに対して実施した。 結果: 研究I:目に見える無鞭毛期虫体を含む全てのスライドがqPCR陽性であったのに対し、全対照スライドは陰性結果をもたらした。研究II:臨床リーシュマニア症と確定診断された13頭の犬のうち、8頭が細胞診で目に見える斑点があったのに対し、Leishmania-qPCRは11枚のスライドガラスと13枚のFPIで陽性を示した。リーシュマニア症は、20頭の犬において標準的な方法で除外され、そのうちの4頭はFPIおよび/またはスライドガラス上でqPCR陽性を示した。 結論と臨床的重要性: リーシュマニアDNAは、CanLを示唆する皮膚病変を有する犬におけるリーシュマニア感染を診断するために、皮膚細胞診標本およびFPIからqPCRによって効率的に検出することができる。.背景: 通过发现无鞭毛体可以检测皮肤病变中的利什曼原虫。 PCR检测利什曼原虫DNA具有更高的灵敏度,并且无创临床样本的PCR检测已被用于诊断人类皮肤利什曼病。 目的: 研究I:确认是否能从已被Diff-Quik®染色的犬皮肤病变细胞学载玻片中,有效提取和扩增利什曼原虫DNA。研究II:染色细胞学和滤纸印迹(FPI)均采自疑似犬利什曼病(CanL)的皮肤病变,评估利什曼原虫定量(q)PCR方法对这些样本的诊断效果。 动物: 54只犬的皮肤病变样本。 方法和材料: 研究I:细胞学检查可见无鞭毛体的19张载玻片(来自9只犬),对其进行利什曼原虫-qPCR检测;从 12只犬身上获得的15张载玻片样本作为对照,细胞学检查未见无鞭毛体,经ELISA检测利什曼原虫血清阴性。研究II:33只症状符合利什曼病的犬,用利什曼原虫-qPCR法检测其皮肤病变载玻片和FPI。 结果: 研究I:所有可见无鞭毛体的载玻片,其qPCR检测均为阳性,而所有对照载玻片的检测结果均为阴性。 研究II: 13只确诊利什曼病的患犬中,8只细胞学上可见无鞭毛体,而利什曼原虫-qPCR检测中11张载玻片和13份FPI呈阳性。用标准方法排除利什曼病的20只犬中,经qPCR检测FPI和/或载玻片,有4只呈阳性。 结论和临床价值: 用qPCR技术检测皮肤细胞学和FPI中的利什曼原虫DNA,可有效诊断皮肤病变疑似CanL的利什曼原虫感染。.A detecção de Leishmania em lesões cutâneas é possível pela visualização de amastigotas. A detecção de DNA de Leishmania por PCR possui maior sensibilidade, e a PCR tem sido utilizada para diagnosticar leishmaniose cutânea em humanos utilizando espécimens clínicos não invasivos.Estudo I: determinar se o DNA de Leishmania pode ser eficientemente extraído e amplificado de lâminas coradas em Diff-QuikAmostras cutâneas de lesões de 54 cães. MÉTODOS E MATERIAIS: Estudo I: Leishmania-qPCR foi realizado em 19 lâminas de vidro (de nove cães) com amastigotas citologicamente visíveis. Quinze lâminas sem amastigotas visíveis, obtidas de 12 cães soronegativos para Leishmania por ELISA serviram como controle. Estudo II: Leishmania-qPCR foi realizado em lâminas de vidro e em FPI de lesões cutâneas compatíveis com leishmaniose clínica em 33 cães.Estudo I: todas as amostras com amastigotas visíveis apresentaram qPCR positivo, enquanto todas as lâminas controle tiveram resultados negativos. Estudo II: dos 13 cães diagnosticados definitivamente com leishmaniose clínica, oito apresentaram amastigotas visíveis na citologia, enquanto Leishmania-qPCR foi positivo em 11 lâminas de vidro e 13 FPI. Descartou-se a leishmaniose pelos métodos convencionais em 20 cães, dos quais quatro apresentaram qPCR positiva na FPI e/ou nas lâminas de vidro. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: O DNA de Leishmania pode ser eficientemente detectado por qPCR de espécimens citológicos cutâneos e FPI para diagnosticar uma infecção por Leishmania em cães com lesões cutâneas sugestivas de CanL.

Crime scene investigators and laboratory analysts use chemical tests to detect and differentiate body fluids. Testing often requires a sample of the stain, and the chemicals may cause degradation of the fluid or interfere with subsequent tests. Colorimetric chemical tests do not differentiate between different types of the same fluid, such as venous and menstrual blood, and there is no presumptive test available to simultaneously differentiate several body fluids. In this study, we recorded ATR FT-IR spectra of venous and menstrual blood, semen, saliva, and breastmilk. Neat and simulated casework body fluid samples were analyzed on cotton, nylon, wood, paper, and glass substrates. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences. Venous and menstrual blood is differentiated by the peak at 1039 cm−1 attributed to phosphoric acid found in menstrual blood. Peak intensity is influenced by the porosity and weave of the substrate fabric.

Significance: Understanding how the brain recovers from cerebral tissue and vascular damage after an ischemic event can help develop new therapeutic strategies for the treatment of stroke. Aim: We investigated cerebral tissue repair and microvasculature regeneration and function after a targeted ischemic stroke. Approach: Following photothrombosis occlusion of microvasculature, chronic optical coherence tomography (OCT)-based angiography was used to track ischemic tissue repair and microvasculature regeneration at three different cortical depths and up to 28 days in awake animals. Capillary network orientation analysis was performed to study the structural pattern of newly formed microvasculature. Based on the time-resolved OCT-angiography, we also investigated capillary stalling, which is likely related to ischemic stroke-induced inflammation. Results: Deeper cerebral tissue was found to have a larger ischemic area than shallower regions at any time point during the course of poststroke recovery, which suggests that cerebral tissue located deep in the cortex is more vulnerable. Regenerated microvasculature had a highly organized pattern at all cortical depths with a higher degree of structural reorganization in deeper regions. Additionally, capillary stalling event analysis revealed that cerebral ischemia augmented stalling events considerably. Conclusion: Longitudinal OCT angiography reveals that regenerated capillary network has a highly directional pattern and an increased density and incidence of capillary stalling event.

We study a problem scenario of super-resolution (SR) algorithms in the context of whole slide imaging (WSI), a popular imaging modality in digital pathology. Instead of just one pair of high- and low-resolution images, which is typically the setup in which SR algorithms are designed, we are given multiple intermediate resolutions of the same image as well. The question remains how to best utilize such data to make the transformation learning problem inherent to SR more tractable and address the unique challenges that arises in this biomedical application. We propose a recurrent convolutional neural network model, to generate SR images from such multi-resolution WSI datasets. Specifically, we show that having such intermediate resolutions is highly effective in making the learning problem easily trainable and address large resolution difference in the low and high-resolution images common in WSI, even without the availability of a large size training data. Experimental results show state-of-the-art performance on three WSI histopathology cancer datasets, across a number of metrics.

To the Editor: Breast cancer is a frequently diagnosed solid tumor in women worldwide (1). MicroRNAs (miRNAs), small noncoding RNAs that regulate protein expression, are potential biomarkers in the diagnosis, prognosis, and prediction of response to treatment in breast cancer (2). We recently reported a paper-based method to extract miRNAs from plasma and circulating tumor cells (3). Here, we applied this method to plasma of patients with breast cancer and age- and gender-matched healthy controls, after treating the plasma with a commercial exosome isolation kit employing a nonspecific precipitation step; we compared the miRNA expression profiles from the plasma with and without the use of the precipitation method. After informed consent and institutional approval, we obtained peripheral blood samples from 9 patients with breast cancer and 9 healthy controls. Plasma was isolated by centrifugation at 3000 g for 10 min and stored at −80 °C. All plasma samples were visually inspected to exclude any discernible hemolysis. Then, 60 μL of Total Exosome Isolation reagent …

The combination of ultra-sensitive assay techniques and recent improvements in the instrumentation used to collect microdroplets of lung fluid (MLF) from exhaled breath has enabled the development of non-invasive lung disease diagnostics that are based on MLF analysis. In one example of this approach, electrospun nylon filters were used to collect MLFs from patients with pulmonary tuberculosis. The filters were washed to obtain liquid probes, which were then tested for human immunoglobulin A (h-IgA) and fractions of h-IgA specific to ESAT-6 and Psts-1, two antigens secreted by Mycobacterium tuberculosis. Probes collected for 10 min contained 100–1500 fg of h-IgA and, in patients with pulmonary tuberculosis, a portion of these h-IgA molecules showed specificity to the secreted antigens. Separate MLFs and their dry residues were successfully collected using an electrostatic collector and impactor developed especially for this purpose. Visualization of MLF dry residues by atomic force microscopy made it possible to estimate the lipid content in each MLF and revealed mucin molecules in some MLFs. This exciting new approach will likely make it possible to detect biomarkers in individual MLFs. MLFs emerging from an infection site (‘hot’ microdroplets) are expected to be enriched with infection biomarkers. This paper discusses possible experimental approaches to detecting biomarkers in single MLFs, as well as certain technological problems that need to be resolved in order to develop new non-invasive diagnostics based on analysing biomarkers in separate MLFs.

We explore cortical microvasculature changes during the progression of atherosclerosis using young and old transgenic atherosclerotic (ATX) mice with thinned-skull cranial window. In awake animals, exploiting intrinsic signal optical imaging, Doppler optical coherence tomography, and two-photon microscopy, we investigate how the progression of atherosclerotic disease affects the morphology and function of cortical microvasculature as well as baseline cerebral tissue oxygenation. Results show that aged ATX mice exhibited weaker hemodynamic response in the somatosensory cortex to whisker stimulation and that the diameter of their descending arterioles and associated mean blood flow decreased significantly compared with the young ATX group. Data from two-photon phosphorescence lifetime microscopy indicate that old ATX mice had lower and more heterogeneous partial pressure of oxygen (PO2) in cortical tissue than young ATX mice. In addition, hypoxic micropockets in cortical tissue were found in old, but not young, ATX mice. Capillary red blood cell (RBC) flux, RBC velocity, RBC velocity heterogeneity, hematocrit, and diameter were also measured using line scans with two-photon fluorescence microscopy. When compared with the young group, RBC flux, velocity, and hematocrit decreased and RBC velocity heterogeneity increased in old ATX mice, presumably due to disturbed blood supply from arterioles that were affected by atherosclerosis. Finally, dilation of capillaries in old ATX mice was observed, which suggests that capillaries play an active role in compensating for an oxygen deficit in brain tissue.

Intraoperative margin assessment is imperative to cancer cure but is a continued challenge to successful surgery. Breast conserving surgery is a relevant example, where a cosmetically improved outcome is gained over mastectomy, but re-excision is required in >25% of cases due to positive or closely involved margins. Clinical translation of margin assessment modalities that must directly contact the patient or required administered contrast agents are time consuming and costly to move from bench to bedside. Tumor resections provide a unique surgical opportunity to deploy margin assessment technologies including contrast agents on the resected tissues, substantially shortening the path to the clinic. However, staining of resected tissues is plagued by nonspecific uptake. A ratiometric imaging approach where matched targeted and untargeted probes are used for staining has demonstrated substantially improved biomarker quantification over staining with conventional targeted contrast agents alone. Our group has developed an antibody-based ratiometric imaging technology using fluorescently labeled, spectrally distinct targeted and untargeted antibody probes termed dual-stain difference specimen imaging (DDSI). Herein, the targeted biomarker expression level and pattern are evaluated for their effects on DDSI diagnostic potential. Epidermal growth factor receptor expression level was correlated to DDSI diagnostic potential, which was found to be robust to spatial pattern expression variation. These results highlight the utility of DDSI for accurate margin assessment of freshly resected tumor specimens.

The objective of our study is to develop a multimodality approach by combining magnetic resonance imaging (MRI) and optical imaging methods to assess acute murine colitis at the macro- and microscopic level. In vivo MRI is used to measure the cross-sectional areas of colons at the macroscopic level. Dual-color confocal laser endomicroscopy (CLE) allows in vivo examination of the fluorescently labeled epithelial cells and microvessels in the mucosa with a spatial resolution of ∼1.4 μm during ongoing endoscopy. To further validate the structural changes of the colons in three-dimensions, ex vivo light-sheet fluorescence microscopy (LSFM) is applied for in-toto imaging of cleared colon sections. MRI, LSFM, and CLE findings are significantly correlated with histological scoring (p

This study assessed validity, reliability, and feasibility of a filter paper blood spot insulin-like growth factor 1 (IGF-1) assay for retinopathy of prematurity (ROP) research. Blood samples were collected from 45 healthy children. Half of each sample was spun to obtain serum; half was applied to filter cards and stored for varying times and at different temperatures. IGF-1 assays were performed using a commercially available kit. Intraclass correlation between blood spot and serum IGF-1 values was high (0.97) for validity, and the mean differences were low for test-retest reliability. Time (up to 25 days) and temperature (4° C to 37° C) had no significant effect on sample stability. Feasibility was further assessed in a second cohort study of 74 premature infants being screened for ROP. A total of 817 filter card samples were successfully collected and transported to a central lab, where IGF-1 assays were successfully performed.

Background Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. Methods We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), β-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. Results We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. Conclusions We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.

Toll-like receptors (TLRs) are members of the pattern recognition receptor family that detect components of foreign pathogens or endogenous molecules released in response to injury. Recent studies demonstrate that TLRs also have a functional role in regulating neuronal proliferation in the developing brain. This study investigated cellular expression of TLR3 using immunohistochemistry on human brain tissue. The tissue sections analysed contained anterior and lateral periventricular white matter from the frontal and parietal lobes in post-mortem neonatal cases with a postmenstrual age range of 23.6-31.4 weeks. In addition to preterm brains without overt pathology (control), preterm pathology cases with evidence of white matter injuries (WMI) were also examined. In order to identify TLR-positive cells, we utilized standard double-labelling immunofluorescence co-labelling techniques and confocal microscopy to compare co-expression of TLR3 with a neuronal marker (NeuN) or with glial markers (GFAP for astrocytes, Iba-1 for microglia and Olig2 for oligodendrocytes). We observed an increase in the neuronal (28 vs. 17%) and astroglial (38 vs. 21%) populations in the WMI group compared to controls in the anterior regions of the periventricular white matter in the frontal lobe. The increase in neurons and astrocytes in the WMI cases was associated with an increase in TLR3 immunoreactivity. This expression was significantly increased in the astroglia. The morphology of the TLR3 signal in the control cases was globular and restricted to the perinuclear region of the neurons and astrocytes, whilst in the cases of WMI, both neuronal, axonal and astroglial TLR3 expression was more diffuse (i.e., a different intracellular distribution) and could be detected along the extensions of the processes. This study demonstrates for the first time that neurons and glial cells in human neonatal periventricular white matter express TLR3 during development. The patterns of TLR3 expression were altered in the presence of WMI, which might influence normal developmental processes within the immature brain. Identifying changes in TLR3 expression during fetal development may be key to understanding the reduced volumes of grey matter and impaired cortical development seen in preterm infants.

Animal models are of great importance for medical research. They have enabled analysis of the aetiology and pathogenesis of complex congenital malformations and have also led to major advances in the surgical and therapeutic management of these conditions. Animal models allow us to comprehend the morphological and molecular basis of disease and consequently to discover novel approaches for both surgical and medical therapy. The anthracycline antibiotic adriamycin was incidentally found to have teratogenic effects on rats, producing a range of defects remarkably similar to the VACTERL association of congenital anomalies in humans, providing a reproducible animal model of this condition. VACTERL association is a spectrum of birth defects which includes vertebral, anal, cardiovascular, tracheo-oesophageal, renal and limb anomalies. In recent years, adriamycin rodent models of VACTERL have provided valuable insights into the pathogenesis of this complex association, particularly in relation to tracheo-oesophageal malformations. The adriamycin rat model and adriamycin mouse model are now well established in the investigation of the morphology of faulty organogenesis and the regulation of gene expression in tracheo-oesophageal anomalies.

A paper-based upconversion fluorescence resonance energy transfer assay device is proposed for sensitive detection of CEA. The device is fabricated on a normal filter paper with simple nano-printing method. Upconversion nanoparticles tagged with specific antibodies are printed to the test zones on the test paper, followed by the introduction of assay antigen. Upconversion fluorescence measurements are directly conducted on the test zones after the antigen-to-antibody reactions. Furthermore, a multi-channel test paper for simultaneous detection of multiple cancer biomarkers was established by the same method and obtained positive results. The device showed high anti-interfere, stability, reproducible and low detection limit (0.89 ng/mL), moreover it is very easy to fabricate and operate, which is a promising prospect for a clinical point-of-care test.

Cutaneous leishmaniasis (CL) is a major vectorborne disease in Panama. In this study, the diagnostic performance and usefulness of two DNA extraction procedures from skin scraping samples collected on FTA filter paper for subsequent PCR diagnosis of CL was evaluated. A positive CL laboratory diagnosis was based on a positive parasitological test (Giemsa-stained smears or in vitro culture) and/or positive PCR test performed from skin scrapings collected in TE buffer (PCR-TE). Of 100 patients with skin lesions suggestive of CL, 82 (82%) were confirmed as CL positive. The sensitivity was calculated for each of the PCR approaches from samples collected on filter paper. The highest sensitivity was achieved by PCR-FTA processed by Chelex 100 (PCR-Chelex) (0.94). PCR-FTA extracted using the FTA purification reagent presented a lower sensitivity (0.60). Good concordance between routine PCR-TE and PCR-Chelex was observed (percent agreement=0.88, κ index=0.65). In conclusion, use of FTA filter paper for skin scraping collection combined with PCR is a reliable and convenient method for CL diagnosis in Panama, with comparable performance to the routine PCR method and with improved sensitivity compared with those of conventional parasitological methods.

Background: N-terminally truncated and modified pyroglutamate-3 amyloid-β protein (pE3-Aβ) is present in most, if not all, cerebral plaque and vascular amyloid deposits in human Alzheimer’s disease (AD). pE3-Aβ deposition is also found in AD-like transgenic (tg) mouse brain, albeit in lesser quantities than general Aβ. pE3-Aβ resists degradation, is neurotoxic, and may act as a seed for Aβ aggregation. Objective: We sought to determine if pE3-Aβ removal by passive immunization with a highly specific monoclonal antibody (mAb) impacts pathogenesis in a mouse model of Alzheimer’s amyloidosis. Methods: APPswe/PS1ΔE9 tg mice were given weekly intraperitoneal injections of a new anti-pE3-Aβ mAb (mAb07/1) or PBS from 5.8 to 13.8 months of age (prevention) or from 23 to 24.7 months of age (therapeutic). Multiple forms of cerebral Aβ were quantified pathologically and biochemically. Gliosis and microhemorrhage were examined. Results: Chronic passive immunization with an anti-pE3-Aβ mAb significantly reduced total plaque deposition and appeared to lower gliosis in the hippocampus and cerebellum in both the prevention and therapeutic studies. Insoluble Aβ levels in hemibrain homogenates were not significantly different between immunized and control mice. Microhemorrhage was not observed with anti-pE3-Aβ immunotherapy. Conclusions: Selective removal of pE3-Aβ lowered general Aβ plaque deposition suggesting a pro-aggregation or seeding role for pE3-Aβ.

Background/Aims: There is increased amyloid-β protein precursor (AβPP) expression and amyloid-β protein (Aβ) production in the brain shortly following cerebral ischemic stroke in rodent models. It has been postulated that this may seed amyloid deposition in the brain. On the other hand, it remains unclear how cerebral ischemia affects preexisting Aβ deposits in the brain. Here we determine the consequences of focal ischemic stroke on existing Aβ pathology in Tg-SwDI transgenic mice. Methods: At 12 months of age, Tg-SwDI mice were subjected to photo-induced focal cerebral ischemia in one hemisphere. One, 7, or 21 days after lesioning, the amount of deposited Aβ in the ischemic and control hemispheres was measured using ELISA. Image analysis was used to visualize deposited Aβ and the presence of microglia/macrophages. Results: After 7 days, and further after 21 days, there was a dramatic reduction in the amount of deposited Aβ and increased presence of microglia/macrophages in the ischemic hemisphere of the mice. Conclusions: Focal cerebral ischemia leads to clearance of deposited Aβ in Tg-SwDI mice starting at 7 days with almost complete removal in the ischemic area by 21 days. The delayed clearance of Aβ following focal cerebral ischemia may involve the infiltration of activated neuroinflammatory cells.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2flox/flox mice. Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric and open forked incisors. Microradiographs revealed broken incisor tips and dental pulp chamber exposure. The enamel layer of incisors and molars was thin with hypomineralization. Scanning electron microscopy analysis showed that the enamel surface was rough with chipping and the enamel lacked a typical prismatic architecture. These results demonstrate that Bmp2 is essential for enamel formation.

Pathological examination has been the gold standard for diagnosis in cancer and its role has also included the elucidation of etiology, pathogenesis, clinicopathological correlation, and prognostication. The advent of newer technologies and the realization that breast cancer is heterogeneous has shifted the focus to prognostication, with increased attention being paid to the identification of morphological features and immunohistochemical markers of prognostic relevance. However, despite the massive efforts invested in the identification of immunohistochemical biomarkers in breast cancer the majority have not proven to be of value in multivariate analyses and only estrogen receptor, progesterone receptor, and Her2/neu expression have remained essential components of pathological examination. These 3 markers were initially employed for prognostication but their role in treatment also rendered them of predictive value. Newer molecular methods, especially high-throughput technologies, have shown that even morphologically similar subtypes of breast cancer can show molecular heterogeneity; moreover, infiltrating ductal carcinoma can be separated into at least 4 molecular subtypes designated luminal (ER+, PR+, and Her2/neu–), Her2 overexpressing (ER–, PR–, and Her2/neu+), basal-like (ER–, PR–, Her2/neu–, and CK5/6+, EGFR+), and normal breast-like (ER–, PR–, and Her2/neu–), each with different clinical outcomes. The importance of proliferative gene expression in these subtypes has been demonstrated and surrogate immunohistochemical markers include ER, PR, Her2/neu, and Ki67 for the more expensive molecular tests. Molecular technologies, importantly, have not only provided further insights into the heterogeneity of breast cancer but have also opened new avenues for treatment through the identification of signaling molecules important in the proliferation and survival of the neoplastic cells. The treatment of cancer thus shifts from the conventional approach of ‘one size fits all’ to one of personalized treatment tailored to the specific characteristics of the tumor. Pathologists continue to play their traditional role in diagnosis but, as purveyors of the excised tissue, pathologists now have the additional role of identifying biomarkers responsive to therapeutic manipulation, thus playing an inextricable role as diagnostic oncologists in the management of breast cancer.

Objective: This study explores the potential use of high-resolution magic angle spinning proton magnetic resonance spectroscopy as an ancillary diagnostic technique for papillary thyroid carcinoma in thyroid fine-needle aspiration biopsies. The method has already been shown to be effective in the classification of various other nonthyroid cancers. Study Design: Twenty-six samples (13 paired cytologic and histologic samples) from patients being evaluated for thyroid abnormalities at the Massachusetts General Hospital were spectroscopically analyzed, and included: papillary thyroid carcinomas (n = 4), follicular adenomas (n = 4), and normal thyroid samples (n = 5). Metabolic profiles were statistically generated based on the spectroscopy results, which were then correlated with the final cytologic and histologic diagnoses from the same samples to determine the diagnostic capacity of the profiles. Results: Principal component analysis of the tissue samples revealed statistically significant correlations among principal components and various cytologic and histologic features. Canonical score 1, calculated with principal components in correlation with analyzed pathologies, revealed the ability of the metabolic profile to differentiate all three examined histologic tissue groups (ANOVA, p < 0.0001). Applying coefficients of principal components and canonical scores obtained with tissue samples directly onto spectral results of cytology samples, the calculated canonical score 1 also revealed similar trends among the three fine-needle aspiration biopsy groups. In particular, the papillary thyroid carcinoma group exhibited significant differences from both the adenomatous and normal cytology groups (p < 0.0170). Conclusions: The results indicate the potential of high-resolution magic angle spinning proton magnetic resonance spectroscopy as an ancillary marker for papillary thyroid carcinoma in fine-needle aspiration biopsy specimens.

Pitt-Hopkins syndrome (PTHS, MIM #610954) is characterized by severe intellectual disability, typical facial features and tendency to epilepsy, panting-and-holding breathing anomaly, stereotypic movements, constipation, and high myopia. Growth is normal or only mildly retarded, but half of the patients have postnatal microcephaly. Remarkably, congenital malformations are practically nonexistent. The cause of PTHS is de novo haploinsufficiency of the TCF4 gene (MIM *602272) at 18q21.2. Altogether 78 PTHS patients with abnormalities of the TCF4 gene have been published since 2007 when the etiology of PTHS was revealed. In addition, 27 patients with 18q deletion encompassing the TCF4 gene but without given PTHS diagnosis have been published, and thus, the number of reported patients with a TCF4 abnormality exceeds 100. The mutational spectrum includes large chromosomal deletions encompassing the whole TCF4 gene, partial gene deletions, frameshift (including premature stop codon), nonsense, splice site, and missense mutations. So far, almost all patients have a private mutation and only 2 recurrent mutations are known. There is no evident genotype-phenotype correlation. No familial cases have been reported. Diagnosis of PTHS is based on the molecular confirmation of the characteristic clinical features. Recently, a Pitt-Hopkins-like phenotype has been assigned to autosomal recessive mutations of the CNTNAP2 gene at 7q33q36 and the NRXN1 gene at 2p16.3.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder affecting almost all organs. It has wider phenotypic variation than often appreciated, with less than half showing the combination of characteristic facial angiofibromas, epilepsy, and mental retardation. Renal angiomyolipomata or cysts are found in 90% and renal failure was historically a common mode of adult death from the disease. Pulmonary lymphangioleiomyomatosis is restricted to females. Angiomyolipomata or cystic disease, or both, may cause renal failure. Angiomyolipomata may also haemorrhage, especially from larger lesions. Manifestations of brain involvement substantially complicate management of many patients with TSC. The causative genes TSC1 and TSC2 encode tuberin and hamartin which are involved in control of the mammalian target of rapamycin pathway. Inhibitors of that pathway, such as sirolimus and everolimus, are therefore logical approaches to therapy and have been shown to be effective in reducing angiomyolipomata volume. It remains to be seen whether they can protect renal function.

An emerging group of human genetic diseases termed ‘ciliopathies’ are caused by dysfunction of two functionally and physically associated organelles, the centrosome and cilium. These organelles are central to perception of the physical environment through detection of a diverse variety of extracellular signals such as growth factors, chemicals, light and fluid flow. Many of the described ciliopathies display multi-organ involvement, with renal and retina being the most commonly affected. Nephronophthisis is a recessive disorder of the kidney that is the leading cause of end-stage renal failure in children. Through positional cloning, many of the causative mutations have been mapped to genes involved in centrosome and cilia function. In this review, we discuss the identified causative mutations that give rise to nephronophthisis and how these are related to the disease etiology in both the kidney and other organs.

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

This study aims to determine if light scatter parameters measured with spatial frequency domain imaging (SFDI) can accurately predict stromal, epithelial, and adipose fractions in freshly resected, unstained human breast specimens. An explicit model was developed to predict stromal, epithelial, and adipose fractions as a function of light scattering parameters, which was validated against a quantitative analysis of digitized histology slides for N = 31 specimens using leave-one-out cross-fold validation. Specimen mean stromal, epithelial, and adipose volume fractions predicted from light scattering parameters strongly correlated with those calculated from digitized histology slides (r = 0.90, 0.77, and 0.91, respectively, p-value