External validation of the 2-year mortality prediction tool in hemodialysis patients developed using a Bayesian network.

Background: In recent years, a number of predictive models have appeared to predict the risk of medium-term mortality in hemodialysis patients, but only one, limited to patients aged over 70 years, has undergone sufficiently powerful external validation. Recently, using a national learning database and an innovative approach based on Bayesian networks and 14 carefully selected predictors, we have developed a clinical prediction tool to predict all-cause mortality at 2 years in all incident hemodialysis patients. In order to generalize the results of this tool and propose its use in routine clinical practice, we carried out an external validation using an independent external validation database. Methods: A regional, multicenter, observational, retrospective cohort study was conducted to externally validate the tool for predicting 2-year all-cause mortality in incident and prevalent hemodialysis patients. This study recruited a total of 142 incident and 697 prevalent adult hemodialysis patients followed up in one of the eight Association pour l'Utilisation du Rein Artificiel dans la région Lyonnaise (AURAL) Alsace dialysis centers. Results: In incident patients, the 2-year all-cause mortality prediction tool had an area under the receiver curve (AUC-ROC) of 0.73, an accuracy of 65%, a sensitivity of 71% and a specificity of 63%. In prevalent patients, the performance for the external validation were similar in terms of AUC-ROC, accuracy and specificity, but was lower in term of sensitivity. Conclusion: The tool for predicting all-cause mortality at 2 years, developed using a Bayesian network and 14 routinely available explanatory variables, obtained satisfactory external validation in incident patients, but sensitivity was insufficient in prevalent patients.

A Preliminary Study Assessing a Transfer Learning Approach to Intestinal Image Analysis to Help Determine Treatment Response in Canine Protein-Losing Enteropathy.

Dogs with protein-losing enteropathy (PLE) caused by inflammatory enteritis, intestinal lymphangiectasia, or both, have a guarded prognosis, with death occurring as a result of the disease in approximately 50% of cases. Although dietary therapy alone is significantly associated with a positive outcome, there is limited ability to differentiate between food-responsive (FR) PLE and immunosuppressant-responsive (IR) PLE at diagnosis in dogs. Our objective was to determine if a transfer learning computational approach to image classification on duodenal biopsy specimens collected at diagnosis was able to differentiate FR-PLE from IR-PLE. This was a retrospective study using paraffin-embedded formalin-fixed duodenal biopsy specimens collected during upper gastrointestinal tract endoscopy as part of the diagnostic investigations from 17 client-owned dogs with PLE due to inflammatory enteritis at a referral teaching hospital that were subsequently classified based on treatment response into FR-PLE (n = 7) or IR-PLE (n = 10) after 4 months of follow-up. A machine-based algorithm was used on lower magnification and higher resolution images of endoscopic duodenal biopsy specimens. Using the pre-trained Convolutional Neural Network model with a 70/30 training/test ratio for images, the model was able to differentiate endoscopic duodenal biopsy images from dogs with FR-PLE and IR-PLE with an accuracy of 83.78%. Our study represents an important first step toward the use of machine learning in improving the decision-making process for clinicians with regard to the initial treatment of canine PLE.

MUM-1 in canine lymphoma: A pilot study.

Expression of interferon regulatory factor 4 (IRF4)/multiple myeloma oncogene-1 (MUM1) in peripheral T-cell lymphoma (PTCL) in people is associated with a poorer survival outcome compared to cases of PTCL lacking MUM1 expression. The aim of this study was to determine whether MUM1 is expressed in canine peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). For comparison, the presence of MUM1 antigen was also investigated in canine diffuse large B cell lymphoma (DLBCL). Nine cases of PTCL-NOS and 9 cases of DLBCL diagnosed by a commercial veterinary diagnostic laboratory were selected. Positive immunohistochemical labeling for MUM1 was observed in PTCL-NOS (2 out of 9 cases) and DLBCL (3 out of 9 cases). These findings suggest that a subset of neoplastic T and B lymphocytes can express MUM1. The role of MUM1 in the biological behavior and outcome of canine lymphoma (CL) requires further investigation on a larger number of cases.

Canine Clitoral Carcinoma: A Clinical, Cytologic, Histopathologic, Immunohistochemical, and Ultrastructural Study.

Vaginal and vulvar tumors are uncommon in dogs. Knowledge of canine primary clitoral neoplasia is restricted to a few case reports, and only carcinomas have been reported. Cytologic and histologic features reported in the literature seem to overlap with those of canine apocrine gland anal sac adenocarcinoma (AGASA). Clinical features also recall those of canine AGASA, such as locoregional metastases and hypercalcemia of malignancy (HM). In this study, 6 cases of primary canine clitoral carcinomas (CCCs), with and without HM, were investigated by means of cytology, histopathology, electron microscopy, and immunohistochemistry for neuroendocrine markers including chromogranin A (CGA), synaptophysin (SYN), neuron-specific enolase (NSE), and S-100. In all 6 tumors, cytologic findings were consistent with malignant epithelial neoplasia of apocrine gland origin. The tumors examined were classified into 3 different histological patterns representing different degrees of differentiation: tubular, solid, and rosette type. Both CGA and SYN were mildly expressed in 2 of 6 tumors, while NSE was consistently expressed in all 6 cases. None of the tumors were S-100 positive. Transmission electron microscopy revealed electron-dense cytoplasmic granules compatible with neuroendocrine granules in all 6 cases. CCCs presented clinicopathologic features resembling AGASAs with neuroendocrine characteristics, and 2 of 6 neoplasms were considered as carcinomas with neuroendocrine differentiation and were positive for 3 neuroendocrine markers. CCCs can often present with HM, and long-term outcome is likely poor. Our study concludes that CCC seems to be a rare tumor, but it might be underestimated because of the overlapping features with AGASA. Further studies should aim to define the true incidence of this disease.

Safety of parenteral nutrition in newborns: Results from a nationwide prospective cohort study.

BACKGROUND & AIMS: Limited or delayed availability of parenteral nutrition (PN) solutions, as well as difficulties in ordering are often identified as reasons for non-compliance with international guidelines in newborns. This study aims at assessing the modality of use and safety of premixed standardized PN solutions in a nationwide prospective cohort of newborns treated in clinical practice. METHODS: Two premixed fixed formulations with respective osmolarity of 715 and 790 mOsm/L specifically designed for neonates were made available throughout the country for clinical use from birth onwards. Descriptive data and modality of use were prospectively collected in a case report form, whereas all related and unrelated adverse events were recorded on a separate adverse event form. RESULTS: A total of 14,167 infants were prospectively included and 16,640 parenteral nutrition periods were analyzed. Mean age was 33 weeks of gestation, and mean weight was 2086 g. The majority of infants (81%) started the parenteral nutrition the first day of life or the day after. The route of parenteral nutrition delivery was peripheral in 47% of the parenteral nutrition periods. During the whole study, a total of 72 adverse events occurring in 68 infants were reported. Of these adverse events, 59 (0.37% of the nutrition periods), among which 19 serious adverse events, were reported as related to the parenteral nutrition solutions. The events related to parenteral nutrition solutions were general disorders and administration site conditions (n = 42 including 9 cases of cutaneous necrosis), and nutrition and metabolism disorders (n = 17). There was no case of thrombophlebitis. Six of the 19 serious events related to the parenteral nutrition solutions (32%) were due to the misuse of the infusion bag. CONCLUSIONS: These data support the concept that ready-to-use parenteral nutrition formulations can safely provide parenteral nutrition from birth onwards. They further support that parenteral solutions with an osmolarity up to 800 mOsm/L are well-tolerated when infused on a peripheral vein. Considering the potential risk of errors and misuses, this study also highlights the need for nutrition practice care guidelines for neonates and for regular campaigns providing information and strategies for a safe use of parenteral nutrition solutions.

Imaging the living inner ear using intravital confocal microscopy.

Confocal laser scanning microscopy permits detailed visualization of structures deep within thick fluorescently labeled specimen. This makes it possible to investigate living cells inside intact tissue without prior chemical sample fixation and sectioning. Isolated guinea pig temporal bones have previously been used for confocal experiments in vitro, but tissue deterioration limits their use to a few hours after the death of the animal. In order to preserve the cochlea in an optimal functional and physiological condition, we have developed an in vivo model based on a confocal microscopy approach. Using a ventral surgical approach, the inner ear is exposed in deeply anaesthetized, tracheotomized, living guinea pigs. To label the inner ear structures, scala tympani is perfused via an opening in the basal turn, delivering tissue culture medium with fluorescent vital dyes (RH 795 and calcein AM). An apical opening is made in the bony shell of cochlea to enable visualization using a custom-built objective lens. Intravital confocal microscopy, with preserved blood and nerve supply, may offer an important tool for studying auditory physiology and the pathology of hearing loss. After acoustic overstimulation, shortening and swelling of the sensory hair cells were observed.

Hepatitis C in 6,865 patients 65 yr or older: a severe and neglected curable disease?

BACKGROUND: Few data are available on chronic hepatitis C (CHC) in elderly patients. The aim of this study was to compare the features and severity of CHC and the efficacy/safety of antiviral therapy in patients<65, between 65 and 80, and >80 yr old, and to determine the usefulness of biochemical markers (Fibrotest-Fibrosure/ActiTest [FT-AT]) in aged patients. METHODS: This was a retrospective study with two groups of patients: Group 1: prospective cohort including all hepatitis C virus patients from our institution (N=4,182); Group 2: all consecutive patients who had FT-AT performed in France between 2002 and 2004 (N=33,738). RESULTS: A total of 6,865 patients>or=65 yr old was included (Group 1=881, Group 2=5,984). Group 1: patients>or=65 had a longer duration of and a higher age at infection, more genotype 1, and a history of transfusion (p<0.001). Among the 2,169 patients who underwent liver biopsy, bridging fibrosis (F2,F3,F4) was more frequent in patients>or=65 yr old, regardless of the duration of infection. In multivariate analysis, ages at biopsy and at infection were associated with F2,F3,F4. Discovery of CHC by a complication was more frequent in patients>or=65 yr (p<0.001). One hundred seventy patients>or=65 yr received antiviral therapy. A sustained virologic response was obtained in 45% of patients>or=65 yr treated with pegylated interferon/ribavirin. Group 2: At FT, 58% of patients>80 yr, 37% of patients between 65 and 80 yr, and 14% of patients<65 yr (p<0.001) had cirrhosis. Patients>80 yr (43%) with cirrhosis had nonelevated alanine amino transferase (ALT), compared with 31% of patients<65 yr (p<0.001). CONCLUSION: In patients>or=65 yr, CHC is more severe and presents with lower ALT than in younger patients. Treatment is effective. Biochemical markers seem particularly useful as a noninvasive alternative to liver biopsy in this population.

Subcutaneous microchip-associated tumours in B6C3F1 mice: a retrospective study to attempt to determine their histogenesis.

Fifty-two subcutaneous tumours associated with microchip were collected from three carcinogenicity B6C3F1 mice studies. Two of these 52 tumours were adenocarcinoma of the mammary gland located on the dorsal region forming around the chip. All the other 50 were mesenchymal in origin and were difficult to classify on morphological grounds with haematoxylin-eosin. These sarcomas were investigated using a panel of immunohistochemistry and special stains consisting of desmin, smooth muscle actin (SMA), myogenin, S100, mouse macrophages, phosphotungstic acid haematoxylin (PTAH) and Masson's trichrome (MT). All the sarcomas displayed the same histochemical characteristics and a close immunophenotype, characterized by desmin +/-, SMA+, myogenin--, S100--, mouse macrophages + and PTAH-. These tumours thus appear to have similar histologic-type lineage and designation as sarcomas not otherwise specified (NOS) with a large myofibroblastic component appears today to be more appropriate and it is likely to clarify them in the future with the emergence of new markers.

Long-term therapy with lamivudine in renal transplant recipients with chronic hepatitis B.

BACKGROUND: Lamivudine is a potent inhibitor of hepatitis B virus (HBV) replication. As other available HBV therapies, its efficacy is hampered by relapse after discontinuation and by the risk of viral breakthrough. A recent study suggests that pre-emptive lamivudine therapy improves survival in HBV renal transplants, but few data are available regarding its long-term use in this population. The clinical features, course and viral mutations associated with the emergence of viral resistance in this population have not been well studied. METHODS: We followed 14 consecutive renal transplant patients treated with lamivudine for chronic hepatitis B. Breakthrough was defined as the reappearance of HBV DNA by hybridization. In patients with breakthrough, lamivudine was always continued and patients were followed up monthly. Mutations associated with viral resistance were determined by sequencing the polymerase encoding gene at the beginning of treatment and at the time of breakthrough. RESULTS: The median duration of treatment was 64.5 months. Resistance to lamivudine appeared in eight patients (57%) after a median duration of treatment of 15 (9-24) months. During a 51 month follow-up after breakthrough, only three of eight patients had a flare-up with alanine aminotransferase levels more than 5 ULN, and no hepatic decompensation was observed. Analysis of HBV sequencing after breakthrough revealed specific resistance mutations in both the B and C domains of the polymerase (rtL180M/M204V, n = 5; rtM204I, n = 2). CONCLUSION: Lamivudine is a safe and effective treatment of active hepatitis B in renal transplant patients. Resistance to treatment is frequent but seems to have little clinical impact over the considered period. In our experience, the YMDD mutation accounts for most cases of virological escape in patients with good compliance.

Prospective analysis of discordant results between biochemical markers and biopsy in patients with chronic hepatitis C.

BACKGROUND: The FibroTest and ActiTest are noninvasive biochemical markers of liver injury that are intended for use as alternatives to liver biopsy in patients with chronic hepatitis C. The aims of this study were to assess the quality of biopsy and the prevalence of discordances between biopsy and markers, to identify factors associated with discordances, and to attribute these discordances to either markers or biopsy failure. METHODS: Fibrosis stage and activity grade were prospectively assessed on the same day by a liver biopsy and by markers. On the basis of risk factors for failure and independent endpoints, discordance was classified as being attributable to biopsy or to markers. RESULTS: Only 74 of 537 patients (14%) had a biopsy size > or =25 mm. Discordance was observed in 154 of 537 patients (29%), including 16% for fibrosis staging and 17% for activity grading. Steatosis, an inflammatory profile, and biopsy size were associated with discordance. Discordance was attributable to failure of markers in 13 patients (2.4%) and to biopsy failure in 97 (18%; P <0.001 vs Fibrotest and Actitest), and was nonattributable in 44 patients (8.2%). The most frequent failures attributable to markers were false negatives (1.3%) attributable to inflammation. The most frequent failures attributable to biopsy were false negatives of activity grading (10.1%) and of fibrosis staging (4.5%), both associated with smaller biopsy size and steatosis. False positives of fibrosis staging (3.5%) were associated with fragmented biopsies. CONCLUSION: In this series, the size of liver biopsy is adequate in only a minor proportion (approximately 14%) of patients with chronic hepatitis C. When biopsy and marker results are discordant, a reason can be identified in more than two-thirds of cases and, in those cases, biopsy failure is >7 times more common than diagnostic failure of markers.

Image-adaptive deconvolution for three-dimensional deep biological imaging.

Deconvolution algorithms are widely used in conventional fluorescence microscopy, but they remain difficult to apply to deep imaging systems such as confocal and two-photon microscopy, due to the practical difficulty of measuring the system's point spread function (PSF), especially in biological experiments. Since a separate PSF measurement performed under the design optical conditions of the microscope cannot reproduce the true experimental conditions prevailing in situ, the most natural approach to solve the problem is to extract the PSF from the images themselves. We investigate here the approach of cropping an approximate PSF directly from the images, by exploiting the presence of small structures within the samples under study. This approach turns out to be practical in many cases, allowing significantly better restorations than with a design PSF obtained by imaging fluorescent beads in gel. We demonstrate the advantages of this approach with a number of deconvolution experiments performed both on artificially blurred and noisy test images, and on real confocal images taken within an in vitro preparation of the mouse hearing organ.

A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Exploring the living cochlea using confocal microscopy.

To obtain a more integrated view of the cellular behaviour of the cochlea it is essential to observe not only wider regions of the exposed turns but also to visualize structures below the reticular lamina. Using confocal microscopy and in vitro preparations of guinea pig and mouse inner ears, cellular structures within the intact organ of Corti can be visualized at high resolution. The approach thus offers a means to investigate detailed cellular events, e.g. structural reorganization following acoustic overstimulation. Confocal microscope images, although sharper than images acquired using regular light microscopy, are still subject to problems related to light scattering within the optical system and low signal-to-noise ratio. Significant image restoration can, however, be obtained by applying a combination of wavelet denoising techniques and deconvolution algorithms. Future work will focus both on more dynamical cellular events and on new in vivo models where the inner ear is visualized at a better functional state.