Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. SUMMARY: Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

Iron Deficiency, Zinc, Magnesium, Vitamin Deficiencies in Crohn's Disease: Substitute or Not?

Inflammatory bowel disease (IBD) is characterized by inflammatory reactions, complications, extraintestinal manifestations and a loss of intestinal functions, for example, failures of absorption and secretion. According to intestinal dysfunction, a wide array of pathogenetic pathways is existing leading to iron deficiency and numerous vitamins as well as trace element deficiencies. Complications, symptoms and signs of those deficiencies are common in IBD with varying degrees of clinical significance. This review focuses on selected micronutrients including iron, zinc, magnesium and some vitamins. Epidemiology with respect to IBD, pathophysiology, diagnosis and clinical aspects are addressed. Finally, some suggestions for treatment of deficient situations are discussed. In conclusion, some micronutrients have significant impact on complications and quality of life in IBD. Deficiencies may even influence the course of the disease. Those deficiencies should be thoroughly supplemented.

Clostridium difficile associated risk of death score (CARDS): a novel severity score to predict mortality among hospitalised patients with C. difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) is a public health threat and associated with significant mortality. However, there is a paucity of objectively derived CDI severity scoring systems to predict mortality. AIM: To develop a novel CDI risk score to predict mortality entitled: Clostridium difficile associated risk of death score (CARDS). METHODS: We obtained data from the United States 2011 Nationwide Inpatient Sample (NIS) database. All CDI-associated hospitalisations were identified using discharge codes (ICD-9-CM, 008.45). Multivariate logistic regression was utilised to identify independent predictors of mortality. Clostridium difficile associated risk of death score was calculated by assigning a numeric weight to each parameter based on their odds ratio in the final logistic model. Predictive properties of model discrimination were assessed using the c-statistic and validated in an independent sample using the 2010 NIS database. RESULTS: We identified 77 776 hospitalisations, yielding an estimate of 374 747 cases with an associated diagnosis of CDI in the US, 8% of whom died in the hospital. The eight severity score predictors were identified on multivariate analysis: age, cardiopulmonary disease, malignancy, diabetes, inflammatory bowel disease, acute renal failure, liver disease and ICU admission, with weights ranging from -1 (for diabetes) to 5 (for ICU admission). The overall risk score in the cohort ranged from 0 to 18. Mortality increased significantly as CARDS increased. CDI-associated mortality was 1.2% with a CARDS of 0 compared to 100% with CARDS of 18. The model performed equally well in our validation cohort. CONCLUSION: Clostridium difficile associated risk of death score is a promising simple severity score to predict mortality among those hospitalised with C. difficile infection.

Higher Adalimumab Drug Levels are Associated with Mucosal Healing in Patients with Crohn's Disease.

BACKGROUND AND AIMS: The current approach to managing the loss of response to anti-tumour necrosis factor (TNF) agents is generally empirical. Prior studies have suggested that adalimumab levels of >4.9 µg/mL are required to achieve clinical remission. Our aim was to identify an optimal adalimumab level to achieve endoscopic healing in Crohn's disease (CD). METHODS: A cohort of 60 CD patients treated with adalimumab between 2005 and 2013 were reviewed for the study. Demographic and clinical information was obtained from chart review and patient interview. Disease activity was determined using the Harvey-Bradshaw index (HBI), ileocolonoscopy reports and C-reactive protein (CRP) levels. Clinical remission was defined as HBI <5. Endoscopic remission/mucosal healing (MH) was defined as the absence of any ulceration in all ileocolonic segments. Trough adalimumab levels and adalimumab antibody levels were tested using a liquid-phase mobility shift assay. RESULTS: Lower median CRP was significantly associated with MH 1.2mg/dl vs no MH 14.4mg/dl (p = 6.93×10(-6)). Higher adalimumab trough level was significantly associated with MH (median 14.7 µg/mL in those with MH vs 3.4 µg/mL in those without, p = 6.25×10(-5)). Higher adalimumab trough level was also significantly associated with the combined outcome of clinical and endoscopic remission (median 13.0 vs 4.8 µg/mL, p = 5.36×10(-3)). A cut-off of 8.14 µg/ml best discriminated subjects with MH from those without MH, with sensitivity and specificity of 91.4 and 76.0%, respectively (positive and negative predictive values 84.2 and 86.4%, respectively). CONCLUSIONS: Higher adalimumab levels were significantly associated with MH. This study suggests that attaining MH alone or a combined outcome of clinical and endoscopic remission is more likely to occur in those patients who achieve an adalimumab trough level of at least 8.14 µg/mL.

An RNA interference screen identifies new avenues for nephroprotection.

Acute kidney injury is a major public health problem, which is commonly caused by renal ischemia and is associated with a high risk of mortality and long-term disability. Efforts to develop a treatment for this condition have met with very limited success. We used an RNA interference screen to identify genes (BCL2L14, BLOC1S2, C2ORF42, CPT1A, FBP1, GCNT3, RHOB, SCIN, TACR1, and TNFAIP6) whose suppression improves survival of kidney epithelial cells in in vitro models of oxygen and glucose deprivation. Some of the genes also modulate the toxicity of cisplatin, an anticancer agent whose use is currently limited by nephrotoxicity. Furthermore, pharmacological inhibition of TACR1 product NK1R was protective in a model of mouse renal ischemia, attesting to the in vivo relevance of our findings. These data shed new light on the mechanisms of stress response in mammalian cells, and open new avenues to reduce the morbidity and mortality associated with renal injury.

Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease. Various disease-related and patient-related factors have been shown to influence the course of the disease. The aim of this study was to identify novel biomarkers of significant clinical relevance. Pretreatment CD19-separated lymphocytes (n=237; discovery set) and peripheral blood mononuclear cells (n=92; validation set) from the REACH trial, a randomized phase III trial in relapsed CLL comparing rituximab plus fludarabine plus cyclophosphamide with fludarabine plus cyclophosphamide alone, underwent gene expression profiling. By using Cox regression survival analysis on the discovery set, we identified inositol polyphosphate-5-phosphatase F (INPP5F) as a prognostic factor for progression-free survival (P<0.001; hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.35-1.98) and overall survival (P<0.001; HR, 1.47; 95% CI, 1.18-1.84), regardless of adjusting for known prognostic factors. These findings were confirmed on the validation set, suggesting that INPP5F may serve as a novel, easy-to-assess future prognostic biomarker for fludarabine-based therapy in CLL.

SU-E-T-275: Dose Verification in a Small Animal Image-Guided Radiation Therapy X-Ray Machine: A Dose Comparison between TG-61 Based Look-Up Table and MOSFET Method for Various Collimator Sizes.

PURPOSE: To verify the accuracy of TG-61 based dosimetry with MOSFET technology using a tissue-equivalent mouse phantom. METHODS: Accuracy of mouse dose between a TG-61 based look-up table was verified with MOSFET technology. The look-up table followed a TG-61 based commissioning and used a solid water block and radiochromic film. A tissue-equivalent mouse phantom (2 cm diameter, 8 cm length) was used for the MOSFET method. Detectors were placed in the phantom at the head and center of the body. MOSFETs were calibrated in air with an ion chamber and f-factor was applied to derive the dose to tissue. In CBCT mode, the phantom was positioned such that the system isocenter coincided with the center of the MOSFET with the active volume perpendicular to the beam. The absorbed dose was measured three times for seven different collimators, respectively. The exposure parameters were 225 kVp, 13 mA, and an exposure time of 20 s. RESULTS: For a 10 mm, 15 mm, and 20 mm circular collimator, the dose measured by the phantom was 4.3%, 2.7%, and 6% lower than TG-61 based measurements, respectively. For a 10 × 10 mm, 20 × 20 mm, and 40 × 40 mm collimator, the dose difference was 4.7%, 7.7%, and 2.9%, respectively. CONCLUSIONS: The MOSFET data was systematically lower than the commissioning data. The dose difference is due to the increased scatter radiation in the solid water block versus the dimension of the mouse phantom leading to an overestimation of the actual dose in the solid water block. The MOSFET method with the use of a tissue- equivalent mouse phantom provides less labor intensive geometry-specific dosimetry and accuracy with better dose tolerances of up to ± 2.7%.

Colon cancer screening in 2010: an up-date.

Colorectal cancer is among the most common cancers worldwide. The prognosis for limited disease is excellent; however, it becomes poor for more advanced disease. The majority of colorectal cancers arise from premalignant adenomatous polyps. This makes the detection of polyps and early carcinoma an attractive screening strategy. This article will review the current tests available for screening for colorectal cancer. These include stool based tests (guaiac-based fecal occult blood tests, immunochemical fecal tests, stool DNA panel), radiologic tests (double contrast barium enema and computed tomography colonography), and endoscopy (flexible sigmoidoscopy and colonoscopy). The current use of these tests in population-based screening programs and the most recent screening guidelines from the largest advisory groups in North America and Europe will be discussed.

The utilization of parenteral nutrition during the in-patient management of inflammatory bowel disease in the United States: a national survey.

BACKGROUND: Parenteral nutrition has a limited role in the in-patient management of inflammatory bowel disease. AIM: To determine nationwide patterns of in-patient parenteral nutrition utilization and its demographic determinants and impact on outcomes. METHODS: We identified inflammatory bowel disease discharges in the Nationwide Inpatient Sample between 1998 and 2003 and determined rates of parenteral nutrition utilization among US census regions, in-hospital mortality and hospital resource utilization. RESULTS: The parenteral nutrition utilization rate among hospitalized inflammatory bowel disease patients was 6%. Only 64% of Crohn's disease and 55% of ulcerative colitis discharges who received parenteral nutrition had malnutrition, fistulizing or obstructive Crohn's disease, or surgery as an indication. The adjusted odds ratio of receiving parenteral nutrition were 0.36 (95% CI: 0.26-0.51) for the mid-west, 0.47 (0.37-0.56) for the south and 0.70 (0.56-0.89) for the west, compared to the north-east. Use of parenteral nutrition was associated with higher in-hospital mortality (OR 2.5; 95% CI: 1.93-3.24), length of stay (13.7 vs. 5.7 days, P < 0.001) and hospital charges ($51,729 vs. $19,563, P < 0.001). CONCLUSIONS: In-patient utilization of parenteral nutrition for inflammatory bowel disease varies markedly by census region, expends significant resources, and leads to potentially significant adverse outcomes. These findings underscore the need for guidelines for judicious parenteral nutrition use in inflammatory bowel disease.

Renin and angiotensinogen expression and functions in growth and apoptosis of human glioblastoma.

The expression and function in growth and apoptosis of the renin-angiotensin system (RAS) was evaluated in human glioblastoma. Renin and angiotensinogen (AGT) mRNAs and proteins were found by in situ hybridisation and immunohistochemistry in glioblastoma cells. Angiotensinogen was present in glioblastoma cystic fluids. Thus, human glioblastoma cells produce renin and AGT and secrete AGT. Human glioblastoma and glioblastoma cells expressed renin, AGT, renin receptor, AT(2) and/or AT(1) mRNAs and proteins determined by RT-PCR and/or Western blotting, respectively. The function of the RAS in glioblastoma was studied using human glioblastoma cells in culture. Angiotensinogen, des(Ang I)AGT, tetradecapaptide renin substrate (AGT1-14), Ang I, Ang II or Ang III, added to glioblastoma cells in culture, did not modulate their proliferation, survival or death. Angiotensin-converting enzyme inhibitors did not diminish glioblastoma cell proliferation. However, the addition of selective synthetic renin inhibitors to glioblastoma cells decreased DNA synthesis and viable tumour cell number, and induced apoptosis. This effect was not counterbalanced by concomitant addition of Ang II. In conclusion, the complete RAS is expressed by human glioblastomas and glioblastoma cells in culture. Inhibition of renin in glioblastoma cells may be a potential approach to control glioblastoma cell proliferation and survival, and glioblastoma progression in combination therapy.

[American pulmonary histoplasmosis. Prospective study with 232 soldiers after a 2-year assignment in Guiana]. / Histoplasmose pulmonaire americaine. Etude prospective chez 232 militaires ayant effectue un sejour de 2 ans en Guyane.

The purpose of this report is to describe the results of a prospective study on pulmonary histoplasmosis in French Guiana. Chest radiographs were performed in 232 French legionnaires returning from a two-year assignment in French Guiana. Further examinations were performed in a total of 8 subjects in whom chest radiographs demonstrated the presence of nodules in the lungs. No evidence of cancer or tuberculosis was found. Findings confirmed histoplasmomas in two cases and demonstrated probable histoplasmosis nodules in 6 cases including three involving calcified lesions. Five of these eight patients had been in high-risk rain forest environments. Pulmonary histoplasmosis should be considered as a possible diagnosis in subjects returning from endemic zones. Confirmation depends on a spectrum of findings. Calcified nodules require only radiographic surveillance with follow-up at six months. Non-calcified nodules require further investigation including CT-scan, bronchoscopy, and serological tests. Surgical biopsy may be necessary to achieve exact histological and mycological identification of the lesion and is recommended in smokers.

Cytopathology of insular carcinoma of the thyroid.

Four pure insular carcinomas (IC) and one IC with focal anaplastic carcinoma (AC) of the thyroid with cytologic evaluation by fine-needle aspiration (FNA) were reviewed. The needle aspirates from the four pure ICs revealed abundant monomorphic follicular cells present singly, in small, loose aggregates, and in cohesive trabecular and acinar clusters. Tumor cells showed fragile, ill-defined, granular cytoplasm and oval nuclei with conspicuous or inconspicuous nucleoli. The case of IC with focal AC yielded, in addition to the follicular cells as seen in the FNA of the 4 cases of pure IC, large pleomorphic malignant cells with prominent nucleoli that were characteristic for an AC, giant-cell type. No intact insulae of tumor cells were identified in any of the 5 cases. Thus, a thyroid IC may be suspected if abundant cohesive and dyshesive monomorphic follicular cells are present in the tumor FNA. However, a firm diagnosis of thyroid IC can only be made by histologic examination of the excised tumor.

Epithelioid leiomyosarcoma of the uterus with oncocytic change.

We report on a rare case of epithelioid leiomyosarcoma of the uterus with oncocytic change and aggressive clinical behavior. The tumor arose in the left lateral wall of the uterus and measured 22 cm in greatest dimension. Histologically, it consisted of solid sheets of round and polygonal cells displaying a homogeneously eosinophilic cytoplasm and pleomorphic nuclei with prominent nucleoli. Six mitotic figures were found per 10 high-power fields in the most mitotically active areas, and focal tumor cell necrosis was present elsewhere. Under electron microscopy, numerous normal-appearing mitochondria and occasional bundles of microfilaments with focal densities were noted in the tumor cell cytoplasm, and poorly-formed external basal lamina and occasional pinocytic vesicles were found on the cell membrane of some tumor cells.

Cytopathology of papillary oncocytic carcinoma of the thyroid in fine-needle aspiration biopsy.

The fine-needle aspirates from two histologically, immunohistochemically, and ultrastructurally confirmed encapsulated papillary oncocytic carcinoma of the thyroid revealed large and small papillary tumor tissue fragments consisting of fibrovascular cores covered with polygonal cells showing abundant and granular cytoplasm and small, oval nuclei. Abundant single and loosely clustered tumor cells with similar cytologic features were also present. Nuclear pleomorphism, irregular nuclear contours, nuclear grooves, and intranuclear cytoplasmic inclusions were not observed in any tumor cells. The two patients were alive and well and showed no evidence of tumor recurrence and/or metastatic disease 6 and 8 years after their thyroid surgeries, respectively.

Cytodiagnosis of bronchogenic carcinoma and neuroendocrine tumor of the lung by transthoracic fine-needle aspiration.

To evaluate our experience with the cytodiagnosis of primary lung cancers by transthoracic fine-needle aspiration (TFNA), 106 bronchogenic carcinomas (BC) and 6 neuroendocrine tumors of the lung (NTL) with adequate needle aspirates were reviewed. The cytodiagnostic accuracy rates of BCs were 75.5%, 72%, 100%, 53%, and 50% for bronchogenic adenocarcinomas, squamous-cell carcinomas, small-cell carcinomas, large-cell carcinomas, and mixed carcinomas, respectively. Of the 6 NTLs, 4 typical carcinoid tumors (CT) were correctly diagnosed, 1 atypical CT was wrongly identified as small-cell carcinoma, and 1 large-cell NTL was mistaken for an adenocarcinoma.