Isolation of Candida famata from a patient with acute zonal occult outer retinopathy

The etiology of a number of retinopathies, such as acute zonal occult outer retinopathy (AZOOR), remains undetermined, Candida famata was isolated from conjunctival exudates of a patient diagnosed with AZOOR. This yeast was very abundant, particularly in the more affected eye, while no other pathogens or fungal species were in evidence. Immunological tests revealed the presence of antigen-specific T lymphocytes by using C. famata as a challenge. Moreover, enzyme-linked immunosorbent assay analysis showed the presence of specific antibodies against this yeast in the patient's blood. Delayed hypersensitivity by use of a skin test was also positive. Finally, antifungal treatments led to improvements in several clinical symptoms, including funduscopic analysis. However, despite prolonged treatment with fluconazole and itraconazole, C. famata still appeared in the conjunctival exudates. The new antifungal voriconazole may represent a better choice for treatment.Organización Nacional de Ciegos Españoles (ONCE) for help and financial support. The institutional grant to Centro de Biología Molecular from Fundación Ramón ArecesPeer Reviewe

Isolation of Candida famata from a Patient with Acute Zonal Occult Outer Retinopathy

The etiology of a number of retinopathies, such as acute zonal occult outer retinopathy (AZOOR), remains undetermined. Candida famata was isolated from conjunctival exudates of a patient diagnosed with AZOOR. This yeast was very abundant, particularly in the more affected eye, while no other pathogens or fungal species were in evidence. Immunological tests revealed the presence of antigen-specific T lymphocytes by using C. famata as a challenge. Moreover, enzyme-linked immunosorbent assay analysis showed the presence of specific antibodies against this yeast in the patient's blood. Delayed hypersensitivity by use of a skin test was also positive. Finally, antifungal treatments led to improvements in several clinical symptoms, including funduscopic analysis. However, despite prolonged treatment with fluconazole and itraconazole, C. famata still appeared in the conjunctival exudates. The new antifungal voriconazole may represent a better choice for treatment

Murine models of HRAS-mediated cutaneous skeletal hypophosphatemia syndrome suggest bone as the FGF23 excess source

Cutaneous skeletal hypophosphatemia syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and FGF23-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels. This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation–bearing bone or skin lesions. Toward this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia, and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23

Genetically engineered rat gliomas: PDGF-driven tumor initiation and progression in tv-a transgenic rats recreate key features of human brain cancer.

Previously rodent preclinical research in gliomas frequently involved implantation of cell lines such as C6 and 9L into the rat brain. More recently, mouse models have taken over, the genetic manipulability of the mouse allowing the creation of genetically accurate models outweighed the disadvantage of its smaller brain size that limited time allowed for tumor progression. Here we illustrate a method that allows glioma formation in the rat using the replication competent avian-like sarcoma (RCAS) virus / tumor virus receptor-A (tv-a) transgenic system of post-natal cell type-specific gene transfer. The RCAS/tv-a model has emerged as a particularly versatile and accurate modeling technology by enabling spatial, temporal, and cell type-specific control of individual gene transformations and providing de novo formed glial tumors with distinct molecular subtypes mirroring human GBM. Nestin promoter-driven tv-a (Ntv-a) transgenic Sprague-Dawley rat founder lines were created and RCAS PDGFA and p53 shRNA constructs were used to initiate intracranial brain tumor formation. Tumor formation and progression were confirmed and visualized by magnetic resonance imaging (MRI) and spectroscopy. The tumors were analyzed using histopathological and immunofluorescent techniques. All experimental animals developed large, heterogeneous brain tumors that closely resembled human GBM. Median survival was 92 days from tumor initiation and 62 days from the first point of tumor visualization on MRI. Each tumor-bearing animal showed time dependent evidence of malignant progression to high-grade glioma by MRI and neurological examination. Post-mortem tumor analysis demonstrated the presence of several key characteristics of human GBM, including high levels of tumor cell proliferation, pseudopalisading necrosis, microvascular proliferation, invasion of tumor cells into surrounding tissues, peri-tumoral reactive astrogliosis, lymphocyte infiltration, presence of numerous tumor-associated microglia- and bone marrow-derived macrophages, and the formation of stem-like cell niches within the tumor. This transgenic rat model may enable detailed interspecies comparisons of fundamental cancer pathways and clinically relevant experimental imaging procedures and interventions that are limited by the smaller size of the mouse brain

RANKL inhibition reduces lesional cellularity and Gαs variant expression and enables osteogenic maturation in fibrous dysplasia

Abstract Fibrous dysplasia (FD) is a rare, disabling skeletal disease for which there are no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated the mechanisms underlying RANKL inhibition in FD tissue and its likely indirect effects on osteoprogenitors by evaluating human FD tissue pre- and post-treatment in a phase 2 clinical trial of denosumab (NCT03571191) and in murine in vivo and ex vivo preclinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic Gαs variant in FD lesions after RANKL inhibition. RNA sequencing of human and mouse tissue supported these findings. The interaction between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, which indicated that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclasts. The results from this study demonstrated that, in addition to its expected antiosteoclastic effect, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased bone formation within lesions. These findings highlight the unappreciated role of cellular crosstalk between osteoclasts and preosteoblasts/osteoblasts as a driver of FD pathology and demonstrate a novel mechanism of action of denosumab in the treatment of bone disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT0357119

MRI and MRS analyses of brain tumor progression.

<p><b>(A)</b> Early and late MRI time points using T2- and T1-weighted imaging. The early MRI findings are consistent with low-grade glioma, including T2 hyperintensity with discrete, homogeneous features. T1 imaging with (T1C) and without (T1) contrast enhancing dye shows little to no enhancement in the early timepoint image. The later imaging reveals a marked transformation in the same animal to a large, heterogeneous tumor with significant mass effect and midline shift, as well as early signs of obstructive hydrocephalus. This is confirmed by T1C imaging, which reveals marked and heterogeneous enhancement within the tumor. <b>(B)</b> The MRS comparing early and later brain tumor regions versus spectra from the contralateral cerebral hemispheres provides additional detail regarding malignant progression with evidence of increased cellular proliferation (red box: higher Cho/Cr) and expansion of non-neuronal tumor elements (blue box: decreased NAA).</p

Immunofluorescent labeling of brain tumors reveals dense and diverse monocytic tumor infiltration.

<p><b>(A)</b> Labeling of tumor-associated macrophages and microglia (Iba-1+) showed dense infiltration within the tumor and a high degree of cellular activation (ED-1+). <b>(B)</b> Further delineation of microglia (P2Y12+)- and <b>(C)</b> bone marrow-derived monocytes (CD49d+) revealed separate contributions from each of these unique cell populations and lineages to the tumor ecosystem. Of note, co-labeling with Iba-1 and each P2Y12 and CD49d showed some overlap (white arrows) but also some distinct, Iba-1 only positive cells.</p

Immunohistochemical analysis of <i>Ntv-a</i> rat glioma.

<p>Immunohistochemical labeling of PDGFA/p53 shRNA-driven brain tumors reveals features in common with human GBM. Immunohistochemical (IHC) labeling (brown) for <b>(A)</b> Ki-67, demonstrating the high degree of proliferation in the tumor compared to the adjacent brain, and nests of proliferative cells in the invasive rim. <b>(B)</b> GFAP staining revealed variable staining with the greatest degree at the invasive tumor rim and regions of the tumor core. Notably, a significant portion of the tumor had minimal GFAP signal, reinforcing the proneural features of the tumor. <b>(C)</b> SMA (blood vessel marker) labeling detailed the extensive neo-vascularization within the tumor. <b>(D)</b> Olig2, considered a marker of glioma stemness and glial lineage, was strongly positive throughout the tumor. <b>(E)</b> Nestin labeling revealed persistent activation of neural stem-like progenitor cells throughout the tumor but not within the rest of the brain. <b>(F)</b> CD44 was broadly expressed through the brain pockets of strong positivity at the invasive areas near the tumor margins and associated white matter tracts. (Scale bar = 200 μm).</p