Development of an automated manufacturing process for large-scale production of autologous T cell therapies.

Engineered T cell therapies have shown significant clinical success. However, current manufacturing capabilities present a challenge in bringing these therapies to patients. Furthermore, the cost of development and manufacturing is still extremely high due to complexity of the manufacturing process. Increased automation can improve quality and reproducibility while reducing costs through minimizing hands-on operator time, allowing parallel manufacture of multiple products, and reducing the complexity of technology transfer. In this article, we describe the results of a strategic alliance between GSK and Miltenyi Biotec to develop a closed, automated manufacturing process using the CliniMACS Prodigy for autologous T cell therapy products that can deliver a high number of cells suitable for treating solid tumor indications and compatible with cryopreserved apheresis and drug product. We demonstrate the ability of the T cell Transduction - Large Scale process to deliver a significantly higher cell number than the existing process, achieving 1.5 × 1010 cells after 12 days of expansion, without affecting other product attributes. We demonstrate successful technology transfer of this robust process into three manufacturing facilities.

Observation of hydrogen trapping at dislocations, grain boundaries, and precipitates.

Hydrogen embrittlement of high-strength steel is an obstacle for using these steels in sustainable energy production. Hydrogen embrittlement involves hydrogen-defect interactions at multiple-length scales. However, the challenge of measuring the precise location of hydrogen atoms limits our understanding. Thermal desorption spectroscopy can identify hydrogen retention or trapping, but data cannot be easily linked to the relative contributions of different microstructural features. We used cryo-transfer atom probe tomography to observe hydrogen at specific microstructural features in steels. Direct observation of hydrogen at carbon-rich dislocations and grain boundaries provides validation for embrittlement models. Hydrogen observed at an incoherent interface between niobium carbides and the surrounding steel provides direct evidence that these incoherent boundaries can act as trapping sites. This information is vital for designing embrittlement-resistant steels.

The Laplace Project: An integrated suite for preparing and transferring atom probe samples under cryogenic and UHV conditions.

We present sample transfer instrumentation and integrated protocols for the preparation and atom probe characterization of environmentally-sensitive materials. Ultra-high vacuum cryogenic suitcases allow specimen transfer between preparation, processing and several imaging platforms without exposure to atmospheric contamination. For expedient transfers, we installed a fast-docking station equipped with a cryogenic pump upon three systems; two atom probes, a scanning electron microscope / Xe-plasma focused ion beam and a N2-atmosphere glovebox. We also installed a plasma FIB with a solid-state cooling stage to reduce beam damage and contamination, through reducing chemical activity and with the cryogenic components as passive cryogenic traps. We demonstrate the efficacy of the new laboratory protocols by the successful preparation and transfer of two highly contamination- and temperature-sensitive samples-water and ice. Analysing pure magnesium atom probe data, we show that surface oxidation can be effectively suppressed using an entirely cryogenic protocol (during specimen preparation and during transfer). Starting with the cryogenically-cooled plasma FIB, we also prepared and transferred frozen ice samples while avoiding significant melting or sublimation, suggesting that we may be able to measure the nanostructure of other normally-liquid or soft materials. Isolated cryogenic protocols within the N2 glove box demonstrate the absence of ice condensation suggesting that environmental control can commence from fabrication until atom probe analysis.

The yield of endoscopic investigation for unintentional weight loss.

AIM: The aim of this study was to assess the yield of endoscopic evaluation in isolated unintentional weight loss (UWL) patients compared with patients with weight loss and additional symptoms or signs. PATIENTS AND METHODS: A retrospective review of all patients who underwent an endoscopic evaluation for the investigation of UWL at Soroka University Medical Center between 2006 and 2012. Data on clinical indication, endoscopic, and laboratory finding were retrieved. Severe inflammation, ulcers, achalasia, and neoplasias were considered clinically significant endoscopic findings (CSEF) that could explain weight loss. Detection rates of CSEF were compared between endoscopic studies for which UWL was the sole indication (group 1) and those performed for UWL and at least one other indication (group 2). RESULTS: During the study period, 1843 patients with UWL were evaluated with 2098 endoscopic procedures. Of these, 1540 underwent esophagogastroduodenoscopy (EGD) and 558 underwent colonoscopy. EGD was performed in 229 (14.8%) patients in group 1 (mean age: 60.9±16.4, 43.3% men), and in 1311 (85.2%) patients in group 2 (mean age: 60.5±18.5, 45% men). Pathological endoscopic findings were identified in 712 (46%) EGDs. Of these, 155 (10%) studies detected significant outcomes: six (3.9%) in group 1 and 149 (96.1%) in group 2. Of the 558 colonoscopies performed, 105 (18.8%) were performed in group 1 (mean age: 61.7±17.5, 43% men) and 453 (82.2%) in group 2 patients (mean age: 62.9±14.6, 49% men). Abnormal findings were found in 190 (33.8%) of the procedures. CSEF were found in 34 (6%) patients: two in group 1 and 32 in group 2. CONCLUSION: The diagnostic yield of endoscopy for investigation of patients with UWL is non-negligible, and should be considered as part of its baseline evaluation, especially in older individuals and those who present with other gastrointestinal manifestations.

Whole-Genome Sequencing of Mycobacterium tuberculosis Provides Insight into the Evolution and Genetic Composition of Drug-Resistant Tuberculosis in Belarus.

The emergence and spread of drug-resistant Mycobacterium tuberculosis (DR-TB) are critical global health issues. Eastern Europe has some of the highest incidences of DR-TB, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. To better understand the genetic composition and evolution of MDR- and XDR-TB in the region, we sequenced and analyzed the genomes of 138 M. tuberculosis isolates from 97 patients sampled between 2010 and 2013 in Minsk, Belarus. MDR and XDR-TB isolates were significantly more likely to belong to the Beijing lineage than to the Euro-American lineage, and known resistance-conferring loci accounted for the majority of phenotypic resistance to first- and second-line drugs in MDR and XDR-TB. Using a phylogenomic approach, we estimated that the majority of MDR-TB was due to the recent transmission of already-resistant M. tuberculosis strains rather than repeated de novo evolution of resistance within patients, while XDR-TB was acquired through both routes. Longitudinal sampling of M. tuberculosis from 34 patients with treatment failure showed that most strains persisted genetically unchanged during treatment or acquired resistance to fluoroquinolones. HIV+ patients were significantly more likely to have multiple infections over time than HIV- patients, highlighting a specific need for careful infection control in these patients. These data provide a better understanding of the genomic composition, transmission, and evolution of MDR- and XDR-TB in Belarus and will enable improved diagnostics, treatment protocols, and prognostic decision-making.

Effect of psychosocial stressors on patients with Crohn's disease: threatening life experiences and family relations.

BACKGROUND AND AIMS: Threatening life experiences and adverse family relations are major psychosocial stressors affecting mental and physical health in chronic illnesses, but their influence in Crohn's disease (CD) is unclear. We assessed whether these stressors would predict the psychological and medical condition of CD patients. METHODS: Consecutive adult CD patients completed a series of instruments including demography, Patient Harvey-Bradshaw Index (P-HBI), Short Inflammatory Bowel Disease Questionnaire (SIBDQ), short-form survey instrument (SF-36), brief symptom inventory (BSI), family assessment device (FAD), and list of threatening life experiences (LTE). Associations of FAD and LTE with P-HBI, SIBDQ, SF-36, and BSI were examined by multiple linear and quantile regression analyses. RESULTS: The cohort included 391 patients, mean age 38.38±13.95 years, 59.6% women, with intermediate economic status. The median scores were as follows: P-HBI 4 (2-8), FAD 1.67 (1.3-2.1), LTE 1 (0-3), SF-36 physical health 43.75 (33.7-51.0), SF-36 mental health 42.99 (34.1-51.9), and BSI-Global Severity Index 0.81 (0.4-1.4). The SIBDQ was 47.27±13.9. LTE was associated with increased P-HBI in all quantiles and FAD in the 50% quantile. FAD and LTE were associated with reduced SIBDQ (P<0.001). Higher LTE was associated with lower SF-36 physical and mental health (P<0.001); FAD was associated with reduced mental health (P<0.001). FAD and LTE were associated positively with GSI in all quantiles; age was associated negatively. CONCLUSION: CD patients with more threatening life experiences and adverse family relations were less healthy both physically and mentally. Physicians offering patients sociopsychological therapy should relate to threatening life experiences and family relations.

PET/CT imaging correlates with treatment outcome in patients with multidrug-resistant tuberculosis.

Definitive clinical trials of new chemotherapies for treating tuberculosis (TB) require following subjects until at least 6 months after treatment discontinuation to assess for durable cure, making these trials expensive and lengthy. Surrogate endpoints relating to treatment failure and relapse are currently limited to sputum microbiology, which has limited sensitivity and specificity. We prospectively assessed radiographic changes using 2-deoxy-2-[(18)F]-fluoro-D-glucose (FDG) positron emission tomography/computed tomography (PET/CT) at 2 and 6 months (CT only) in a cohort of subjects with multidrug-resistant TB, who were treated with second-line TB therapy for 2 years and then followed for an additional 6 months. CT scans were read semiquantitatively by radiologists and were computationally evaluated using custom software to provide volumetric assessment of TB-associated abnormalities. CT scans at 6 months (but not 2 months) assessed by radiologist readers were predictive of outcomes, and changes in computed abnormal volumes were predictive of drug response at both time points. Quantitative changes in FDG uptake 2 months after starting treatment were associated with long-term outcomes. In this cohort, some radiologic markers were more sensitive than conventional sputum microbiology in distinguishing successful from unsuccessful treatment. These results support the potential of imaging scans as possible surrogate endpoints in clinical trials of new TB drug regimens. Larger cohorts confirming these results are needed.

Autoimmune hepatitis in southern Israel: a 15-year multicenter study.

OBJECTIVE: In this study we aimed to assess the incidence, prevalence and clinical outcomes of patients with autoimmune hepatitis (AIH) in southern Israel. METHODS: Case-finding methods and population-based administrative data were used to evaluate the epidemiology and prognostic factors of AIH from 1995 to 2010. RESULTS: During the study period, the average annual prevalence and incidence of AIH in southern Israel were 11.0/100000 and 0.67/100000, respectively. We identified 100 AIH cases with a mean age of 47.9 years, including 95 women and five men. Type 1 AIH was found in 77 cases, and liver fibrosis and cirrhosis were found in 73.4% and 22.3% of all patients who underwent liver biopsy. In all, 98 patients were treated with a combination of steroids and azathioprine or steroids alone (prednisone and azathioprine in 71, budesonide and azathioprine in 11, prednisone or budesonide alone in six and ten, respectively). Complete remission was recorded in 56 patients, whereas partial response or failure of treatment was noted in 42 patients. In multivariate analysis the independent predictors of remission were the degree of liver fibrosis (mild vs bridging fibrosis (F3) and cirrhosis [F4]) (P=0.003) and level of albumin (P=0.031). The estimated 1-year and 10-year survival for AIH were 96.5% and 89.7%, respectively. CONCLUSIONS: The prevalence of AIH in Israel is quite similar to that of other European Caucasian populations, with a relatively long-term good prognosis, despite a low rate of response to immunosuppressive therapy.

Differential virulence and disease progression following Mycobacterium tuberculosis complex infection of the common marmoset (Callithrix jacchus).

Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades.

Primary biliary cirrhosis in Southern Israel: a 20 year follow up study.

BACKGROUND: The epidemiology of primary biliary cirrhosis (PBC) in Israel is unknown. We aimed to determine the epidemiology, long-term survival and outcomes of PBC in Southern Israel from 1990 to 2010. METHODS: Case-finding methods and population-based administrative data were used to estimate and evaluate the incidence, prevalence and prognostic factors of outcome in our PBC cohort. RESULTS: 138 cases of PBC were identified. The average annual prevalence of PBC was 255 cases per million. The overall age/sex-adjusted annual incidence of PBC was 10 cases per million from 1990 through 1999 and 20 cases per million from 2000 to 2010. Among 138 incident cases with a total follow-up of 960 persons-years from diagnosis, 30 patients (21.7%) died. Survival in PBC patients was significantly lower than that of the age/sex-matched Israeli population. Mortality was significantly increased in patients with an initial MELD score greater than 8 (P<0.001), with portal hypertension (P<0.001), and in non-responders to ursodeoxycholic acid (UDCA) therapy according to Barcelona criteria (P=0.005). Out of 138 patients, 95 patients (68.0%) responded to UDCA therapy according to Barcelona and Paris criteria. None of the responders died during the follow-up period as opposed to 30 out of 43 (69.8%) of non-responders. In multivariate analysis the factors associated with response to UDCA were: albumin levels above 3.5 g/dL (P<0.001) and lower degree of fibrosis per liver biopsy (P=0.003). CONCLUSIONS: This study addresses the increasing burden of PBC in Israel and confirms the importance of some clinical and therapeutic factors as predictors of long-term prognosis.