Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.

The Anatomic Pathology laboratory adjustments in the era of COVID-19 pandemic: The experience of a laboratory in a Portuguese central hospital.

The coronavirus disease 2019 (COVID-19) pandemic has dramatically changed the world over the past weeks, with already 8,25 million infections and 445,000 deaths worldwide, leading to an unprecedented international global effort to contain the virus and prevent its spread. The emergence of novel respiratory viruses such as the SARS-CoV-2 creates dramatic challenges to the healthcare services, including surgical pathology laboratories, despite their extensive daily experience in dealing with biological and chemical hazards. Here, we cover important aspects on the knowledge on COVID-19 gathered during the first six months of the pandemic and address relevant issues on human biological sample handling in the Anatomic Pathology laboratory in the context of COVID-19 global threat. In addition, we detail our strategy to minimize the risk of contamination upon exposure to the different biological products received in the laboratory, which can be of general interest to other laboratories worldwide. Our approach has enabled a safe work environment for laboratory staff, while ensuring the maintenance of high quality standards of the work performed. In times of uncertainty and given the lack of specific guidelines directed at Anatomic Pathology services to better deal with the global COVID-19 public-health emergency, it is essential to share with the community rigorous methodologies that will enable us to better cope with probable novel waves of COVID-19 infection and other viruses that will possibly arise in the near future.

The skin as a window to the gut: A case of carcinoid syndrome.

Neuroendocrine tumors (NETs) are a group of uncommon neoplasms derived from enterochromaffin or Kulchitsky cells (that secrete serotonin or other molecules into the bloodstream), which can manifest with symptoms of hormonal overproduction, namely carcinoid syndrome (CS). This can be the presenting feature in patients with advanced disease. We report the case of a 66-year-old woman presenting with chronic diarrhea, facial venous telangiectasia and elevated urinary 5-hydrocyindoleacetic acid levels. A 68-Ga DOTATOC PET/CT scan revealed an ileal mass and lesions consistent with liver, ovary and bone metastasis. A liver biopsy confirmed the diagnosis of well-differentiated NET G1. Therapy with somatostatin analogs achieved symptom control, but the liver disease progressed and the patient passed away after 2 years of follow-up. The challenge of diagnosing CS resides in its heterogeneous manifestations, which may range from mild to life-threatening conditions. In this case, the cutaneous findings of venous telangiectasia strongly pointed to the correct diagnosis. Treatment can also be difficult due to refractory symptoms and inevitable progression of disease, highlighting the importance of early detection and thorough disease staging.

Giant Conjunctival Cyst of the Orbit in a Patient with Previous Enucleation.

Introduction: Conjunctival cysts of the orbit development are a rare complication in enucleated patients. They result from the entrapment of conjunctival epithelium within the orbit. Case Presentation: We report the case of a 25-year-old man with a history of enucleation for a retinoblastoma of the right eye who presented with difficulty in fitting his eye prothesis. On his past medical records, there was no reference to the placement of any orbital implant at the time of the surgery. Biomicroscopy of the right eye revealed a thickened bulbar conjunctiva, an inferior symblepharon, and a translucid central area with vascularization. Imaging was remarkable for a cystic cavity filling the whole right orbit. Biopsy revealed the diagnosis of a conjunctival cyst, and drainage was performed, alleviating the patient's symptoms. Conclusion: Orbital conjunctival cysts may pose a clinical problem, and treatment modalities include surgical excision, absolute alcohol injection, and trichloroacetic acid injection.

Enhancing Lung Cancer Care in Portugal: Bridging Gaps for Improved Patient Outcomes.

Lung cancer has the highest incidence and cancer-related mortality worldwide. In Portugal, it ranks as the fourth most common cancer, with nearly 6000 new cases being diagnosed every year. Lung cancer is the main cause of cancer-related death among males and the third cause of cancer-related death in females. Despite the globally accepted guidelines and recommendations for what would be the ideal path for a lung cancer patient, several challenges occur in real clinical management across the world. The recommendations emphasize the importance of adequate screening of high-risk individuals, a precise tumour biopsy, and an accurate final diagnosis to confirm the neoplastic nature of the nodule. A detailed histological classification of the lung tumour type and a comprehensive molecular characterization are of utmost importance for the selection of an efficacious and patient-directed therapeutic approach. However, in the context of the Portuguese clinical organization and the national healthcare system, there are still several gaps in the ideal pathway for a lung cancer patient, involving aspects ranging from the absence of a national lung cancer screening programme through difficulties in histological diagnosis and molecular characterization to challenges in therapeutic approaches. In this manuscript, we address the most relevant weaknesses, presenting several proposals for potential solutions to improve the management of lung cancer patients, helping to decisively improve their overall survival and quality of life.

Characterisation of the protein expression of the emerging immunotherapy targets VISTA, LAG-3 and PRAME in primary uveal melanoma: insights from a southern French patient cohort.

Uveal melanoma (UM) is the most common intraocular tumour in adults, with dismal prognosis once metastases develop, since therapeutic options for the metastatic disease are ineffective. Over the past decade, novel cancer therapies based on immunotherapy have changed the landscape of treatment of different forms of cancer leading to many hopes of improvement in patient overall survival (OS). VISTA, LAG-3 and PRAME are novel promising targets of immunotherapy that have recently gained attention in different solid tumours, but whose relevance in UM remained to be comprehensively evaluated until now. Here, we studied the protein expression of VISTA, LAG-3 and PRAME using immunohistochemistry in representative whole tissue sections from primary UM cases in a cohort of 30 patients from a single centre (Nice University Hospital, Nice, France). The expression of each of these markers was correlated with different clinical and pathological parameters, including onset of metastases and OS. We demonstrated the protein expression of VISTA and LAG-3 in small lymphocytes infiltrating the tumour, while no expression of the proteins was detected in UM cells. For PRAME, nuclear expression was observed in UM cells, but no expression in tumour infiltrating immune cells was identified. Increased levels of VISTA expression in tumour infiltrating lymphocytes (TILs) were associated with nuclear BAP1 expression and better prognosis. Higher levels of LAG-3 in TILs were associated with higher levels of CD8-positive TILs. PRAME nuclear positivity in melanoma cells was associated with epithelioid cell dominant (>90%) UM histological subtype, higher mitotic numbers and a higher percentage of chromosome 8q gain. This study proposes VISTA as a novel relevant immune checkpoint molecule in primary UM and contributes to confirm LAG-3 and PRAME as potentially important immunotherapy targets in the treatment of UM patients, helping to expand the number of immunotherapy candidate molecules that are relevant to modulate in this aggressive cancer.

Basaloid Follicular Hamartoma of the Eyelid: A Case Report and Literature Review about an Unusual Lesion in the Ocular Region.

Basaloid follicular hamartoma (BFH) is a normally benign, uncommon, malformative lesion involving the hair follicles, which usually poses challenges in the differential diagnosis with other benign and malignant tumours, especially basal cell carcinoma, due to significant clinical and morphological overlap. Here, we report the case of a 53-year-old male who presented with a mass in the upper left eyelid evolving for one year. The patient had a previous history of total colectomy and an abdominal desmoid tumour within the context of Familial Adenomatous Polyposis (FAP), with a documented germline mutation in the Adenomatous Polyposis Coli (APC) gene. The eyelid lesion was biopsied and the histological analysis of the three small tissue fragments received revealed fragments with cutaneous-conjunctival lining displaying a subepithelial proliferation of basaloid nests with peripheral palisading, compatible with primitive hair follicles. There were images of anastomosis between different basaloid nests, which had their connection to the epithelial lining preserved. The stroma had high cellularity and sometimes primitive mesenchymal papillae were evident. Pleomorphism was absent, mitotic figures were barely identified, and no necrosis was seen. The basaloid nests did not have epithelial-stromal retraction nor mucin deposits. A diagnosis of BFH was proposed, which was later confirmed after surgical excision of the whole eyelid lesion. No evidence of carcinoma was present. This case illustrates the main features of the rare benign eyelid BFH. The standard medical or surgical approach of these lesions remains to be firmly established. Nearly nine months after surgical excision our patient remains well without signs of disease recurrence.

Immunoglobulin G4 Related-Disease: A Rare Presentation With Secondary Hypereosinophilic Syndrome and Eosinophilic Ascites.

Immunoglobulin G4 related-disease (IgG4-RD) is a multisystemic immune-mediated fibroinflammatory disease, with a strong predilection for salivary and lacrimal glands, pancreas, biliary tree, lungs, kidneys, aorta, and retroperitoneum. In the case of pancreatic involvement, it manifests as autoimmune pancreatitis. Patients with IgG4-RD usually have mild to moderate eosinophilia in the peripheral blood, however, they may present a secondary hypereosinophilic syndrome (HES). Although there are cases described with severe eosinophilia (> 5,000/µL), the 2019 American College of Rheumatology/European League Against Rheumatism classification criteria for IgG4-RD considers peripheral eosinophilia above 3,000/µL as an exclusion criterion, although stating that assessment for the presence of exclusion criteria should be individualized depending on a patient's clinical scenario. Here, we describe the clinical case of a 30-year-old woman who presented with chronic diarrhea and abdominal pain. The diagnostic workup revealed eosinophilic ascites, severe peripheral blood eosinophilia (> 5,000/µL), IgG4 elevation (> 2 × upper normal limit), and also diffuse swelling in the body and pancreatic tail (computed tomography (CT) scan). There was a prompt response to corticosteroid therapy with clinical resolution and continued remission under therapy. The patient was diagnosed with IgG4-RD with secondary HES, explaining the gastrointestinal tract and peritoneum damage in the form of enterocolitis and ascites. Exclusion of alternative diagnosis was made.

Prognostic Biomarkers in Uveal Melanoma: The Status Quo, Recent Advances and Future Directions.

Uveal melanoma (UM) is the most common malignant intraocular tumour in the adult population. It is a rare cancer with an incidence of nearly five cases per million inhabitants per year, which develops from the uncontrolled proliferation of melanocytes in the choroid (≈90%), ciliary body (≈6%) or iris (≈4%). Patients initially present either with symptoms like blurred vision or photopsia, or without symptoms, with the tumour being detected in routine eye exams. Over the course of the disease, metastases, which are initially dormant, develop in nearly 50% of patients, preferentially in the liver. Despite decades of intensive research, the only approach proven to mildly control disease spread are early treatments directed to ablate liver metastases, such as surgical excision or chemoembolization. However, most patients have a limited life expectancy once metastases are detected, since there are limited therapeutic approaches for the metastatic disease, including immunotherapy, which unlike in cutaneous melanoma, has been mostly ineffective for UM patients. Therefore, in order to offer the best care possible to these patients, there is an urgent need to find robust models that can accurately predict the prognosis of UM, as well as therapeutic strategies that effectively block and/or limit the spread of the metastatic disease. Here, we initially summarized the current knowledge about UM by compiling the most relevant epidemiological, clinical, pathological and molecular data. Then, we revisited the most important prognostic factors currently used for the evaluation and follow-up of primary UM cases. Afterwards, we addressed emerging prognostic biomarkers in UM, by comprehensively reviewing gene signatures, immunohistochemistry-based markers and proteomic markers resulting from research studies conducted over the past three years. Finally, we discussed the current hurdles in the field and anticipated the future challenges and novel avenues of research in UM.

Nociceptive, emotional, electrophysiological, and histological characterization of the chronic constriction injury model in female Wistar Han rats.

Chronic neuropathic pain affects 7-10 % of the population and is often accompanied by comorbid emotional disorders, which greatly reduce the quality of life of the patients, impairing physical, cognitive, emotional, and social functioning. Despite the higher prevalence and severity of chronic pain in women, the number of publications using female animals remains scarce. While in the chronic constriction injury (CCI) model the development of mechanical/thermal hyperalgesia, allodynia and spontaneous pain has been shown in both sexes, little is known on CCI-induced emotional impairments and sciatic nerve histopathology in female rats, as well as on the contributions of ovarian hormones to peripheral nerve injury. In this work, young adult rats (Wistar Han) were assigned to one of five groups: gonadally intact females (SHAM/SHAM), ovariectomized females (SHAM/OVX), gonadally intact females with CCI (CCI/SHAM); ovariectomized females with CCI (CCI/OVX) and males with CCI (CCIM). In the postoperative period, CCI animals, both females and males, displayed visible gait abnormalities, limping and guarding the affected hind paw although locomotion was not affected. Neuropathic females developed sustained mechanical allodynia, with CCI/OVX animals displaying symptoms two weeks before CCI/SHAM females. Interestingly, regarding mechanical and cold allodynia, CCI males slowly recovered from week 3 onwards. While CCI induced neither anxiety- nor depressive-like behaviour in females, ovariectomy per se induced anhedonic-like behaviour, regardless of CCI surgery. Histopathological analysis of the sciatic nerve showed CCI induced nerve damage, fibrosis, myelin sheath degradation and inflammation. Single-cell electrophysiological data from the rostral ventromedial medulla (RVM) suggests this area is partly involved in descending facilitation associated with experimental neuropathic pain. Altogether, our findings demonstrate CCI females display distinct sensory, emotional, electrophysiological, and histopathological impairments from males, and that ovariectomy aggravates females' responses to peripheral nerve injury.

Abdominal Actinomycosis misdiagnosed as liposarcoma.

Actinomycosis is an uncommon, endogenous, and chronic infection with varied and nonspecific clinical features such as abdominal, pelvic or cervical masses, ulcerative lesions, abscesses, draining fistula, fibrosis, and constitutional symptoms. The disease ensues when the bacteria disrupt the mucosal barrier, invade, and spread throughout interfascial planes. Currently, the diagnosis of actinomycosis is challenging because of its very low frequency and depending on the clinical presentation it may masquerade malignancies. Therapy consists initially in intravenous penicillin, followed by an oral regimen that may be extended until a year of treatment. A timely diagnosis is crucial to avoid extensive therapeutic attempt as surgery. However, a biopsy or drainage of abscesses and fistula's tract may be required not only as a diagnostic procedure as part of the therapy. We report the case of a 72-year-old woman with an abdominal mass initially misdiagnosed as a liposarcoma. A second biopsy of a skin lesion of the abdominal wall made the diagnosis of actinomycosis, avoiding a major surgical procedure. The patient was treated with a long-term course of antibiotics with favorable outcome. Liposarcoma was ruled out after the patient's full recovery with antibiotics and the misdiagnosis was credit to the overconfidence on the immunohistochemical positivity to MDM2.

A rare cecal subepithelial tumor in a Crohn´s Disease patient.

Appendiceal tumors comprise a variety of histologic types, including appendiceal mucinous neoplasms, which can be grouped as premalignant lesions, tumors of uncertain malignant potential, and malignant lesions. The appendiceal mucinous neoplasms are characterized by mucinous epithelial proliferation with extracellular mucin and pushing tumor margins, commonly an incidental finding during operative exploration. We report the case of a low-grade appendiceal mucinous neoplasm presenting as a subepithelial lesion in Crohn´s Disease patient. The diagnosis was not straightforward, and only surgical resection allowed an accurate diagnosis. Although Inflammatory Bowel Disease is a risk factor for the development of colorectal neoplasms, the absolute risk for appendiceal tumors is uncertain. The frequency of progression to malignancy remains to be determined.

A rare cecal subepithelial tumor in a Crohn´s Disease patient

Appendiceal tumors comprise a variety of histologic types, including appendiceal mucinous neoplasms, which can be grouped as premalignant lesions, tumors of uncertain malignant potential, and malignant lesions. The appendiceal mucinous neoplasms are characterized by mucinous epithelial proliferation with extracellular mucin and pushing tumor margins, commonly an incidental finding during operative exploration. We report the case of a low-grade appendiceal mucinous neoplasm presenting as a subepithelial lesion in Crohn´s Disease patient. The diagnosis was not straightforward, and only surgical resection allowed an accurate diagnosis. Although Inflammatory Bowel Disease is a risk factor for the development of colorectal neoplasms, the absolute risk for appendiceal tumors is uncertain. The frequency of progression to malignancy remains to be determined.

Protein Prenylation Constitutes an Endogenous Brake on Axonal Growth.

Suboptimal axonal regeneration contributes to the consequences of nervous system trauma and neurodegenerative disease, but the intrinsic mechanisms that regulate axon growth remain unclear. We screened 50,400 small molecules for their ability to promote axon outgrowth on inhibitory substrata. The most potent hits were the statins, which stimulated growth of all mouse- and human-patient-derived neurons tested, both in vitro and in vivo, as did combined inhibition of the protein prenylation enzymes farnesyltransferase (PFT) and geranylgeranyl transferase I (PGGT-1). Compensatory sprouting of motor axons may delay clinical onset of amyotrophic lateral sclerosis (ALS). Accordingly, elevated levels of PGGT1B, which would be predicted to reduce sprouting, were found in motor neurons of early- versus late-onset ALS patients postmortem. The mevalonate-prenylation pathway therefore constitutes an endogenous brake on axonal growth, and its inhibition provides a potential therapeutic approach to accelerate neuronal regeneration in humans.

Failure of Y-27632 to improve the culture of adult human adipose-derived stem cells.

Y-27632 is a well-known inhibitor of the Rho-associated coiled kinase (ROCK) and has been shown to significantly improve the culture of a variety of multipotent stem cell types. However, the effects of Y-27632 on the expansion of adult human adipose-derived stem cell (hADSC) cultures remain to be established. Here, we aimed to characterize the effects of Y-27632 on the culture of hADSCs. Adult hADSCs were isolated from subjects submitted to elective plastic surgery procedures and cultivated in vitro under optimized conditions. Our results show that the continuous supplementation of hADSC cultures with Y-27632 led to decreased numbers of cells and decreased global metabolic viability of hADSC cultures when compared with control conditions. This effect appeared to be dependent on the continuous presence of the drug and was shown to be concentration-dependent and significant for 10 µM and 20 µM of Y-27632. Moreover, the Y-27632-induced decrease in hADSC numbers was not linked to a block in global cell proliferation, as cell numbers consistently increased from the moment of plating until passaging. In addition, Y-27632 was not able to increase the number of hADSCs present in culture 24 hours after passaging. Taken together, our results suggest that, in contrast to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance hADSC culture expansion.

Abdominal Actinomycosis misdiagnosed as liposarcoma

Actinomycosis is an uncommon, endogenous, and chronic infection with varied and nonspecific clinical features such as abdominal, pelvic or cervical masses, ulcerative lesions, abscesses, draining fistula, fibrosis, and constitutional symptoms. The disease ensues when the bacteria disrupt the mucosal barrier, invade, and spread throughout interfascial planes. Currently, the diagnosis of actinomycosis is challenging because of its very low frequency and depending on the clinical presentation it may masquerade malignancies. Therapy consists initially in intravenous penicillin, followed by an oral regimen that may be extended until a year of treatment. A timely diagnosis is crucial to avoid extensive therapeutic attempt as surgery. However, a biopsy or drainage of abscesses and fistula's tract may be required not only as a diagnostic procedure as part of the therapy. We report the case of a 72-year-old woman with an abdominal mass initially misdiagnosed as a liposarcoma. A second biopsy of a skin lesion of the abdominal wall made the diagnosis of actinomycosis, avoiding a major surgical procedure. The patient was treated with a long-term course of antibiotics with favorable outcome. Liposarcoma was ruled out after the patient's full recovery with antibiotics and the misdiagnosis was credit to the overconfidence on the immunohistochemical positivity to MDM2.

Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.

Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.