deCLUTTER2+ - a pipeline to analyze calcium traces in a stem cell model for ventral midbrain patterned astrocytes.

Astrocytes are the most populous cell type of the human central nervous system and are essential for physiological brain function. Increasing evidence suggests multiple roles for astrocytes in Parkinson's disease, nudging a shift in the research focus, which historically pivoted around ventral midbrain dopaminergic neurons (vmDANs). Studying human astrocytes and other cell types in vivo remains challenging. However, in vitro-reprogrammed human stem cell-based models provide a promising alternative. Here, we describe a novel protocol for astrocyte differentiation from human stem cell-derived vmDAN-generating progenitors. This protocol simulates the regionalization, gliogenic switch, radial migration and final differentiation that occur in the developing human brain. We characterized the morphological, molecular and functional features of these ventral midbrain patterned astrocytes with a broad palette of techniques and identified novel candidate midbrain-astrocyte specific markers. In addition, we developed a new pipeline for calcium imaging data analysis called deCLUTTER2+ (deconvolution of Ca2+ fluorescent patterns) that can be used to discover spontaneous or cue-dependent patterns of Ca2+ transients. Altogether, our protocol enables the characterization of the functional properties of human ventral midbrain patterned astrocytes under physiological conditions and in disease.

LRP10 interacts with SORL1 in the intracellular vesicle trafficking pathway in non-neuronal brain cells and localises to Lewy bodies in Parkinson's disease and dementia with Lewy bodies.

Loss-of-function variants in the low-density lipoprotein receptor-related protein 10 (LRP10) gene have been associated with autosomal-dominant Parkinson's disease (PD), PD dementia, and dementia with Lewy bodies (DLB). Moreover, LRP10 variants have been found in individuals diagnosed with progressive supranuclear palsy and amyotrophic lateral sclerosis. Despite this genetic evidence, little is known about the expression and function of LRP10 protein in the human brain under physiological or pathological conditions. To better understand how LRP10 variants lead to neurodegeneration, we first performed an in-depth characterisation of LRP10 expression in post-mortem brains and human-induced pluripotent stem cell (iPSC)-derived astrocytes and neurons from control subjects. In adult human brain, LRP10 is mainly expressed in astrocytes and neurovasculature but undetectable in neurons. Similarly, LRP10 is highly expressed in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons. In astrocytes, LRP10 is present at trans-Golgi network, plasma membrane, retromer, and early endosomes. Interestingly, LRP10 also partially co-localises and interacts with sortilin-related receptor 1 (SORL1). Furthermore, although LRP10 expression and localisation in the substantia nigra of most idiopathic PD and DLB patients and LRP10 variant carriers diagnosed with PD or DLB appeared unchanged compared to control subjects, significantly enlarged LRP10-positive vesicles were detected in a patient carrying the LRP10 p.Arg235Cys variant. Last, LRP10 was detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients and in LRP10 variant carriers. In conclusion, high LRP10 expression in non-neuronal cells and undetectable levels in neurons of control subjects indicate that LRP10-mediated pathogenicity is initiated via cell non-autonomous mechanisms, potentially involving the interaction of LRP10 with SORL1 in vesicle trafficking pathways. Together with the specific pattern of LRP10 incorporation into mature LBs, these data support an important mechanistic role for disturbed vesicle trafficking and loss of LRP10 function in neurodegenerative diseases.

Clinical symptoms of intestinal vascular disorders.

Despite advances made in the diagnostic and therapeutic field, acute intestinal ischemia remains a highly lethal condition. This is related to the variability of symptoms and the absence of typical laboratory alterations in early stage.

[Our experience in the management of patients with Mirizzi syndrome]. / Orlentamenti attuali nel trattamento della sindrome di Mirizzi. Nostra esperienza.

The authors report their experience in the management of patients with Mirizzi Syndrome (MS) admitted, over a period of 15 years, at the General Surgery of Emergency Department of Cardarelli Hospital, Naples, Italy. All patients were admitted and surgically treated in emergency save for one. Out of 12 patients, cholecystectomy was performed in 7 cases. In others 5 patients, with cholecystocholedochal fistula, cholecystectomy with positionig of T-Tube was performed in 4 cases (MS-II); finally, 1 patient with MS type III undewrwent choledochojejunostomy. According to literature, the diagnostic protocol included abdominal ultrasonography and CT scan of the abdomen for all patients; in one case, a cholangio-MRI was performed to clarify the diagnosis. The preoperative diagnosis is essential to reduce risk of iatrogenic injuries. The cholangio-MRI, used to this extent, clarifies the site of obstruction, shows the anatomy of the biliary tree and allows to make all the possible differential diagnoses in order to exclude the presence of biliary tumors before surgery. The intraoperative cholangiography remains mandatory to clarify the anatomy of the biliary tree. In the cases we have treated, ERCP was never performed. We believe that ERCP has limited indications and unsatisfactory outcomes for both diagnosis and treatment of MS. Pathological examination of the fresh-frozen surgical specimens was always performed intraoperatively to exclude the presence of concomitant cancer of the gallbladder. The traditional treatment of patients with MS is surgery, as confirmed by our experience. We perform cholecystectomy for MS type I and cholecystectomy with direct repair of the biliary fistula over aT tube for MS type II. Patients with MS type III usually undergo a tailored operation based on the intraoperative findings, while choledochojejunostomy is mandatory for patients with MS type IV. Laparoscopic surgery is indicated only for MS type I and II. It seems to carry a higher risk for the patient and we do not use this approach in the emergency settings.

[Conservative treatment of the digestive fistulas: personal experience]. / Trattamento conservativo delle fistole digestive: nostra esperienza.

OBJECTIVE: The authors, thanks to experience obtained in an Unit for the treatment of digestive fistulas, discuss the possibility of a conservative treatment for the anastomotic fistulas. MATERIAL AND METHODS: From 2000 to 2003 were treated thirty-five patients with post-anastomotic gastroenteric fistulas marked according to their localization, way end output (51.5% high, 42.8% moderate and 5.7% low). The treatment is based on an aspiration system, sometimes integrated with an irrigation system. A semi-permeable barrier was created over the fistula by vacuum packing a synthetic, hydrophobic, polymer covered with a self-adherent surgical sheet. This system create a vacuum chamber equipped with a subathmospheric pressures between 262.2 and 337.5 mmHg (350-450 mmbar), integrated with a continuous irrigation using antibiotic solutions or 3% lactic acid. RESULTS: The AA. obtained the resolution in 30 patients (85.7%), 3 patients needs the surgery (8.6%), 2 died, one for sepsis and the other one for malnutrition. The mean time for the closure was 45 days (from 20 to 90). A part of digestive external fistulas goes to spontaneous resolution so comes the idea that the creation of particular condition is the basis of their closure.