3-Dimensional Immunostaining and Automated Deep-Learning Based Analysis of Nerve Degeneration.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.

Understanding Associative Learning Through Higher-Order Conditioning.

Associative learning is often considered to require the physical presence of stimuli in the environment in order for them to be linked. This, however, is not a necessary condition for learning. Indeed, associative relationships can form between events that are never directly paired. That is, associative learning can occur by integrating information across different phases of training. Higher-order conditioning provides evidence for such learning through two deceptively similar designs - sensory preconditioning and second-order conditioning. In this review, we detail the procedures and factors that influence learning in these designs, describe the associative relationships that can be acquired, and argue for the importance of this knowledge in studying brain function.

Dopamine transients do not act as model-free prediction errors during associative learning.

Dopamine neurons are proposed to signal the reward prediction error in model-free reinforcement learning algorithms. This term represents the unpredicted or 'excess' value of the rewarding event, value that is then added to the intrinsic value of any antecedent cues, contexts or events. To support this proposal, proponents cite evidence that artificially-induced dopamine transients cause lasting changes in behavior. Yet these studies do not generally assess learning under conditions where an endogenous prediction error would occur. Here, to address this, we conducted three experiments where we optogenetically activated dopamine neurons while rats were learning associative relationships, both with and without reward. In each experiment, the antecedent cues failed to acquire value and instead entered into associations with the later events, whether valueless cues or valued rewards. These results show that in learning situations appropriate for the appearance of a prediction error, dopamine transients support associative, rather than model-free, learning.

Causal evidence supporting the proposal that dopamine transients function as temporal difference prediction errors.

Reward-evoked dopamine transients are well established as prediction errors. However, the central tenet of temporal difference accounts-that similar transients evoked by reward-predictive cues also function as errors-remains untested. In the present communication we addressed this by showing that optogenetically shunting dopamine activity at the start of a reward-predicting cue prevents second-order conditioning without affecting blocking. These results indicate that cue-evoked transients function as temporal-difference prediction errors rather than reward predictions.

[Evolution of polyvalent intravenous immunoglobulins (IVIg) prescription, at the Assistance Publique - Hopitaux de Paris between 1988 and 1997. Results of the plan of action set up in order to regulate the prescription]. / Evolution des prescriptions des immunoglobulines intraveineuses polyvalentes (IgIV) à l'Assistance Publique -Hôpitaux de Paris entre 1988 et 1997.

In 1991 the public hospitals in Paris set up a plan to regulate the prescription of IVIg. The plan includes an expert committee and reliable data collection. The expert committee has a threefold mission: i) perform an annual up-date of IVIg classification using three categories: accepted indications (group I), currently deabated indications (group II), and unwarranted indications (group III); ii) develop guidelines for improved therapeutic strategies; iii) stimulate research. Data on use of IVIg are collected in 16 pilot hospitals. These data designate IVIg prescriptions by indication. Data are centralized by the CEDIT which publishes an annual report. Between 1988 and 1991, prescription of IVIg increased at an average annual rate of 33%. Between 1991 and 1996, the amount of IVIg used leveled off: approximately 330 kilograms/year, excluding research protocols. In 1997 there was a decline to 299 kilograms accounting for a total expenditure of 44 million French francs (US$ 6.7M). In 1997, group I prescriptions represented 80% of all IVIg prescriptions, group II 9.8% and group III 9.1%. Comparison of medical practice with a scientificaly recognised reference made it possible for AP-HP to set up an effective regulation of IVIg prescriptions. The longevity of this evaluation work is by itself a success.

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay.

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.

Biliary symptoms and complications more than 8 years after endoscopic sphincterotomy for choledocholithiasis.

BACKGROUND & AIMS: Although long-term results of endoscopic sphincterotomy (ES) have been poorly estimated, extended indications of ES have been proposed, especially in young patients. The aim of this study was to assess late biliary complications of ES. METHODS: Between 1981 and 1986, 169 patients younger than age 70 (55+/-11.8 years; range, 24-70 years; male-female sex ration, 0.55) underwent ES for choledocholithiasis. One hundred fifteen patients (68%) underwent cholecystectomy. Long-term data were obtained retrospectively from the patients and general practitioners. RESULTS: Information was obtained for 156 patients, 2 of whom died within 1 month (one ES-related death). The mean follow-up for 154 patients was 9.6+/-3.3 years (range 8-13 years); 138 patients had no biliary symptoms. During follow-up, 16 patients experienced biliary symptoms; 2 of these patients underwent elective cholecystectomy, 3 had malignant strictures, 1 had a complicated cirrhosis, and 1 had a benign stricture related to the previous cholecystectomy. Nine patients developed potentially ES-related biliary symptoms. Second endoscopic exploration showed papillary stenosis in 3 patients (with stones in 2 patients) and recurrent bile duct stones in 3 others. Two patients had sine materia cholangitis, and 1 patient developed liver abscesses. CONCLUSIONS: Long-term ES-related complications seem to be rare, ES could reasonably be included in management strategies of choledocholithiasis, even in young patients.

Bile levels of carcino-embryonic antigen in patients with hepatopancreatobiliary disease.

OBJECTIVE: To evaluate the value of biliary carcino-embryonic antigen (CEA) in the differential diagnosis of malignant and benign hepatopancreatobiliary disease. PATIENTS: One hundred patients were prospectively studied. Benign diseases were present in 39% of the patients while 61% had malignant diseases. METHODS: Samples of serum were taken from all patients just before endoscopic retrograde cholangiopancreatography (ERCP) and samples of biliary CEA were obtained during ERCP. RESULTS: The sensitivity of serum CEA and carbohydrate antigen 19-9 (CA 19-9) in detecting malignancy were 50% and 92%, respectively, while the respective specificities were 95% and 72%. The mean biliary CEA level of the benign group was significantly different from that of the malignant group (35.7 +/- 8.7 ng/ml vs 268 +/- 85.5 ng/ml), but there was considerable overlap between the two groups. With a cut-off level of 20 ng/ml, the sensitivity and specificity were 84% and 64% respectively. The mean bilirubinaemia value was significantly higher in malignant disease than in benign disease (57.4 +/- 13.9 mumol/l vs 235 +/- 19.8 mumol/l). Multidimensional analysis indicated that only bilirubinaemia (P < 109-3)) was independently predictive of malignant disease. CONCLUSION: Biliary CEA assessment seems useless in distinguished between benign and malignant causes of cholestasis.

Endoscopic sphincterotomy in patients with liver cirrhosis.

BACKGROUND: Nonsurgical alternatives in biliary diseases have not been studied in large series of patients with cirrhosis. Our aim was to determine the indications and results of endoscopic treatment in this subset of patients. METHODS: Fifty-two patients (36 men, 16 women-Child-Pugh Class A, 18; B, 22; C, 12) aged 63 +/- 18 years underwent endoscopic sphincterotomy (ES) between 1988 and 1993. Antibiotic prophylaxis was routinely carried out and coagulopathy was corrected before ES when required. The data were collected retrospectively up to 30 days after ES. RESULTS: ES succeeded in 98% of the patients (12 cases of needle-knife papillotomy). Twenty-nine patients (55.7%) had choledocholithiasis, 18 had biliary strictures (12 malignant), and 5 had pancreatic or other biliary diseases. Five days after ES, morbidity was 13.5% and mortality was 7.7%. At 1 month, morbidity was 22.9% and mortality 12.5%. Only endoscopic procedures and Ineffective drainage were seen to be significant risk factors or morbidity. The results of the subgroup of cirrhotic patients with choledocholithiasis (n = 29) were compared with those of matched noncirrhotic patients (n = 58). The cirrhotic and noncirrhotic patients treated for choledocholithiasis showed similar results for stone clearance, morbidity, and mortality. CONCLUSION: ES is a safe and effective procedure for treating choledocholithiasis in cirrhotic patients. ES can therefore be considered as an alternative to surgery in Child class A and B patients and must be preferred for Child class C patients with life-threatening biliary complications.

Prospective controlled study of endoscopic ultrasonography and endoscopic retrograde cholangiography in patients with suspected common-bileduct lithiasis.

BACKGROUND: Endoscopic sphincterotomy is sometimes done unnecessarily in patients with suspected choledocholithiasis. Our aims were to assess the diagnostic accuracy of endoscopic ultrasonography and endoscopic retrograde cholangiography (ERC) and to find out whether endoscopic ultrasonography may help to prevent unnecessary sphincterotomy or surgical explorations. METHODS: We recruited 119 patients aged 70.4 (SD 16.1) years with strongly suspected choledocholithiasis who presented to our endoscopy unit between January, 1994, and January, 1995. During the same spell of sedation or within 2 h of each other, endoscopic ultrasonography and ERC were carried out by investigators unaware of the patient's history. Endoscopic sphincterotomy with instrumental exploration was then done as the gold standard for the presence or the absence of stones. FINDINGS: 78 (66%) patients had choledocholithiasis; 17 (14%) had other bileduct diseases; 24 (20%) had a clear bileduct or did not require an invasive endoscopic procedure. The sensitivity of endoscopic ultrasonography was 93%, specificity 97%, positive predictive value 98%, and negative predictive value 88%. The corresponding values for ERC were 89%, 100%, 100%, and 83%. There were five false-negative cases by endoscopic ultrasonography (of which three were also negative with ERC) and one false-positive. The morbidity rate was 4.1%. INTERPRETATION: We conclude that endoscopic ultrasonography is at least as sensitive as ERC. Endoscopic ultrasonography may prevent inappropriate invasive explorations of the common bileduct.

Cholangiographic features in fibrosis and cirrhosis of the liver. Radiological-pathological correlation.

The cholangiographic features of intrahepatic bile ducts associated with cirrhosis or fibrosis are not well known. In order to achieve a radiological-pathological correlation, we studied nine livers with fibrosis or cirrhosis excised at autopsy. Cholangiograms were obtained within 24 hr after death from the nonfixed liver and multiple tissues samples were taken for histologic examination. Radiological data were interpreted by two independent investigators blinded to the clinical and histological findings. Cirrhosis (alcoholic in 4, posthepatitis in two) was observed in six livers, fibrosis (alcoholic in 2, posthepatitis in one) in three. No liver with fibrosis had cholangiographic abnormalities. In contrast, cholangiography of all livers with cirrhosis was abnormal. Abnormalities were a diminished arborization, a decrease of the distal opacification, an irregularity of caliber, and a tortuous course of the bile ducts. Histological study showed that the irregular and tortuous course were due to compression of the bile ducts by regenerative nodules. Furthermore, a thick fibrosis was organized around the bile ducts. In conclusion, fibrosis alone was not associated with cholangiographic abnormalities. In cirrhotic livers, intrahepatic bile ducts showed an irregular and tortuous course, a diminished arborization and a decrease of the distal opacification. These abnormalities were secondary to the presence of regenerative nodules and fibrosis organized around the bile ducts.

Diagnostic ERCP in the era of laparoscopic surgery.

BACKGROUND/AIMS: The purpose of this paper is to reassess the place and risks of ERCP in a diagnostic view. METHODS: Analysis of 196 non-operative ERCP performed in 196 patients aged 57 +/- 20 yrs. ERCP was performed for: unexplained cholestasis = 98; non tumoral pancreatic diseases = 43; suspected neoplasm of the pancreatic/biliary tract = 13; cholangitis = 12; unexplained abdominal pain = 30. Precut papillotomy was performed in 40 cases (20.4%). 10.7% were cirrhotics; 3.1% were gastrectomized. 164/196 received peri-operative broad-spectrum antibiotics. RESULTS: 108 had normal ERCP (group I); 74 had abnormal ERCP (group II); 12 had undetermined diagnosis after ERCP (group III). Strictly ERCP-induced mortality was nil; 2 patients died a few days after ERCP from: hemorrhage after transhepatic drainage (1); continuing severe cholangitis after failed CBD cannulation (1). Morbidity was: acute pancreatitis = 6 (3%) and fever = 4 (2%). No complication followed precut papillotomy. The 6 pancreatitis recovered within 48 h to 5 days. Fevers alleviated with antibiotics within 12 to 48 h. Pancreatitis occurred in 5/6 after normal ERCP; fevers followed pathologic ERCP in 3/4 (NS). The high rate of precuts in this series did not increase morbidity. CONCLUSION: ERCP-related morbidity was 5.1 % and ERCP accurately diagnosed or unequivocally eliminated biliary-pancreatic disease in 92.9%. These results suggest that ERCP remains a useful and safe diagnostic tool.

Endoscopic sphincterotomy for suspected choledocholithiasis in patients with and without stones.

OBJECTIVE: To characterize a subgroup of patients treated with endoscopic sphincterotomy (ES) for a suspected common bile duct lithiasis (CBDL) that was not confirmed and to compare it with the subgroup with confirmed CBDL. METHODS AND RESULTS: Over 18 months, ES was successful in 245 consecutive patients (age 23-97, mean 75 yr, SE 17 yr) with suspected CBDL; 159 patients had CBDL (group 1), and 86 did not (group 2), as confirmed by CBD exploration. Fifty-nine percent of the patients in group 1 and 76.7% of the patients in group 2 had gallbladder in situ. Both groups were different for age (p < 0.001), prevalence of chronic alcoholism (p < 0.001), gallbladder in situ (p < 0.01), and gallbladder stones (p < 0.05). Patients from group 1 had two or more presenting symptoms suggestive of CBDL more often than patients from group 2 (p < 0.05), and pancreatitis was a more frequent presenting manifestation in group 2 (p < 0.0001). Overall morbidity and mortality were not different between groups, but acute cholecystitis developed in six patients from group 2 and in one patient from group 1 (p < 0.01). In a univariate analysis, only elevated alkaline phosphatase and a dilated common bile duct were positively discriminant for the diagnosis of CBDL; chronic alcoholism was negatively discriminant for the diagnosis of CBDL. In a multivariate analysis, only chronic alcoholism and a dilated bile duct were found to be independently discriminant. CONCLUSION: The risk of ES-related complications in the group without CBDL suggests that the selection of patients should be improved by a better use of preoperative criteria.

Survival after a first episode of spontaneous bacterial peritonitis. Prognosis of potential candidates for orthotopic liver transplantation.

To determine the potential role of orthotopic liver transplantation (OLT) in cirrhotic patients surviving a first episode of spontaneous bacterial peritonitis (SBP), medical records of 79 patients presenting with a first episode of SBP were reviewed. Of these patients, 37 were selected as potential candidates for OLT using the following criteria: absence of hepatocellular carcinoma; no severe organ failure other than the liver; age < or = 66 years; and survival after SBP > 60 days. Survival time was calculated from the day of SBP diagnosis. Prognostic value of clinical, biological and bacteriological data recorded at the time of SBP was determined using univariate and multivariate analysis (Cox's regression model). Survival rate of the potential candidates for OLT at 3 months, 1 year and 2 years was 94, 46 and 30% respectively. Serum creatinine value (P = 0.001) and Pugh score (P = 0.005) were independently correlated with death. The 1 year survival rate was 80% for the 11 patients with a Pugh score < 10, and 26% for the 26 patients with a Pugh score > or = 10. Our results suggest that after SBP, OLT should be considered in patients with severe liver disease. Survival of patients with a moderate liver disease (i.e. Pugh score < 10) might be relatively high.

[Managing of an innovation in health care: the case of polyvalent intravenous immunoglobulins at the Assistance-Publique-Hôpitaux de Paris]. / La maîtrise de la diffusion d'une innovation en Santé: le cas des immunoglobulines intraveineuses polyvalentes à l'Assistance Publique-Hôpitaux de Paris.

Managing new innovations in medicine is a particularly timely subject. There is an abundant history concerning over expectations resulting from the development of new treatments or diagnostic procedures, some shown to be less effective than promised, others even found to be dangerous. A new aspect to the question is the importance of economic pressures which require rational investment decisions when diffusing innovating technologies. In 1991, the Commission for the evaluation and diffusion of innovating technologies (CEDIT) at the University Hospitals of Paris (Assistance Publique-Hôpitaux de Paris) developed a programme aimed at better managing the distribution and use of polyvalent intravenous immunoglobulins (IgIV), a new promising therapeutic tool with both a high cost and a certain number of risks. The programme was designed to assist prescribers in elaborating better therapeutic strategies and to help hospital managers rationalize expenditures for IgIV. The results of this experience are presented here together with certain conclusions concerning the way management decisions can be applied to the diffusion of an innovation in health care.