High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.

Assessment of epithelial barrier function is critically important for studying healthy and diseased biological models. Here we introduce an instrument that measures transepithelial electrical resistance (TEER) of perfused epithelial tubes in the microfluidic OrganoPlate platform. The tubules are grown in microfluidic channels directly against an extracellular matrix, obviating the need for artificial filter membranes. We present TEER measurements on Caco-2 intestinal and renal proximal tubule epithelium. Forty tubules on one single plate were interrogated in less than a minute. We show that TEER measurement is significantly more sensitive than a fluorescent reporter leakage assay in response to staurosporine. We demonstrate a 40-channel time-lapse data acquisition over a 25 hour time period under flow conditions. We furthermore observed a 50% reduction in Caco-2 TEER values following exposure to a cocktail of inflammatory cytokines. To our best knowledge, this is the first instrument of its kind that allows routine TEER studies in perfused organ-on-a-chip systems without interference by artificial filter membranes. We believe the apparatus will contribute to accelerating routine adoption of perfused organ-on-a-chip systems in academic research and in industrial drug development.

Comparison of 18F-FDG and 67Ga-citrate in sarcoidosis imaging.

AIM: (67)Ga citrate has been used long and successfully to diagnose and stage sarcoidosis. (18)F-fluorodeoxyglucose ((18)F-FDG) has been suggested as a positron emission tomography (PET) tracer for sarcoidosis imaging. This study aimed to analyze possible advantages of (18)F-FDG-PET over (67)Ga citrate scintigraphy during the primary assessment of patients with sarcoidosis. PATIENTS AND METHODS: Twenty-four patients (11 men, 13 women, aged 52 years +/-12.4) with histologically proven sarcoidosis were investigated with (18)F-FDG and (67)Ga citrate. Equipment included a full-ring PET scanner (ECAT EXACT HR+, Siemens/CTI, Knoxville TN, USA) and a double-headed gamma camera (ECAM, Siemens, Illinois, USA) for scintigraphy. The mean time difference between the two studies was 6.5 days (range: 5-8 days). RESULTS: There was a significant difference in the detection of pulmonary and nonpulmonary sarcoidosis lesions between planar (67)Ga citrate scans and (18)F-FDG-PET images (<0.0021). A total of 64 lesions were detected with (67)Ga citrate scans in the thorax and elsewhere with a mean of 2.6 lesions (4%) per patient, while 85 lesions were found with (18)F-FDG-PET, with a mean of 3.5 lesions (4.1%) per patient. There was complete agreement between (18)F-FDG and (67)Ga citrate in thoracic manifestations in four (16.6%) patients, and in non-thoracic manifestations in five (20.8%) patients. The interobserver variability showed a kappa value of 0.79. CONCLUSION: (67)Ga citrate and (18)F-FDG are useful tracers for diagnostic evaluation of thoracic sarcoidosis. (18)F-FDG seems to be more suitable for imaging the mediastinum, the bi-hilar lymph nodes, the posterior regions of the lungs and non-thoracic lesions. Further prospective studies are needed to clarify the role of both tracers in early diagnosis and staging of sarcoidosis, and to resolve questions concerning medical treatment and follow-up.

Combining Extracellular miRNA Determination with Microfluidic 3D Cell Cultures for the Assessment of Nephrotoxicity: a Proof of Concept Study.

Drug-induced kidney injury is often observed in the clinics and can lead to long-term organ failure. In this work, we evaluated a novel in vitro system that aims at detecting whether compounds can cause renal proximal tubule damage in man. For this, we implemented organotypic cultures of human conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1) in a three-channel OrganoPlate under microfluidic conditions. Cells were exposed to four known nephrotoxicants (cisplatin, tenofovir, cyclosporine A, and tobramycin). The effect on cell viability and NAG release into the medium was determined. A novel panel of four miRNAs (mir-21, mir-29a, mir-34a, and mir-192) was selected as potential biomarkers of proximal tubule damage. After nephrotoxicant treatment, miRNA levels in culture medium were earlier indicators than cell viability (WST-8 assay) and outperformed NAG for proximal tubule damage. In particular, mir-29a, mir-34a, and mir-192 were highly reproducible between experiments and across compounds, whereas mir-21 showed more variability. Moreover, similar data were obtained in two different laboratories, underlining the reproducibility and technical transferability of the results, a key requirement for the implementation of novel biomarkers. In conclusion, the selected miRNAs behaved like sensitive biomarkers of damage to tubular epithelial cells caused by several nephrotoxicity mechanisms. This biomarker panel, in combination with the 3D cultures of ciPTEC-OAT1 in the OrganoPlate, represents a novel tool for in vitro nephrotoxicity detection. These results pave the way for the application of miRNAs in longitudinal, time-course in vitro toxicity studies.

Bile salt regulation of fatty acid absorption and esterification in rat everted jejunal sacs in vitro and into thoracic duct lymph in vivo.

This study was performed to investigate whether the malabsorption of fat in the blind loop syndrome is due to the presence of free bile acids or to a deficiency of conjugated bile salts produced by bacterial degradation of normal bile salts, as well as to learn something of the mechanisms by which bile salts might regulate fat absorption. In the everted gut sac of the rat in vitro, conjugated bile salts were necessary for maximal rates of fatty acid esterification to triglycerides, whereas free bile acids inhibited this process even in the presence of physiologically normal or higher concentrations of conjugated bile salts. In contrast, in the living animal the addition of similar or higher concentrations of free bile acids to infusions of fatty acids in taurocholate micellar solutions produced no reduction in the amount of fatty acid absorbed into lymph or the amount of fatty acid esterified into lymph triglyceride. Both in vitro and in the living animal, reduction in the conjugated bile salt concentration reduced both the rate of fatty acid uptake by the intestine and the esterification into triglycerides. It is concluded that the steatorrhea of the blind loop syndrome or other conditions in which upper intestinal stasis allows bacterial proliferation is not due to presence of increased gut luminal concentrations of free bile acids, but rather is a consequence of lowered concentrations of conjugated bile salts.

96 perfusable blood vessels to study vascular permeability in vitro.

Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.

Cholesteryl ester transfer protein TaqIB variant, high-density lipoprotein cholesterol levels, cardiovascular risk, and efficacy of pravastatin treatment: individual patient meta-analysis of 13,677 subjects.

BACKGROUND: Several studies have reported that the cholesteryl ester transfer protein (CETP) TaqIB gene polymorphism is associated with HDL cholesterol (HDL-C) levels and the risk of coronary artery disease (CAD), but the results are inconsistent. In addition, an interaction has been implicated between this genetic variant and pravastatin treatment, but this has not been confirmed. METHODS AND RESULTS: A meta-analysis was performed on individual patient data from 7 large, population-based studies (each >500 individuals) and 3 randomized, placebo-controlled, pravastatin trials. Linear and logistic regression models were used to assess the relation between TaqIB genotype and HDL-C levels and CAD risk. After adjustment for study, age, sex, smoking, body mass index (BMI), diabetes, LDL-C, use of alcohol, and prevalence of CAD, TaqIB genotype exhibited a highly significant association with HDL-C levels, such that B2B2 individuals had 0.11 mmol/L (0.10 to 0.12, P<0.0001) higher HDL-C levels than did B1B1 individuals. Second, after adjustment for study, sex, age, smoking, BMI, diabetes, systolic blood pressure, LDL-C, and use of alcohol, TaqIB genotype was significantly associated with the risk of CAD (odds ratio=0.78 [0.66 to 0.93]) in B2B2 individuals compared with B1B1 individuals (P for linearity=0.008). Additional adjustment for HDL-C levels rendered a loss of statistical significance (P=0.4). Last, no pharmacogenetic interaction between TaqIB genotype and pravastatin treatment could be demonstrated. CONCLUSIONS: The CETP TaqIB variant is firmly associated with HDL-C plasma levels and as a result, with the risk of CAD. Importantly, this CETP variant does not influence the response to pravastatin therapy.

Phase III study of weekly high-dose infusional fluorouracil plus folinic acid with or without irinotecan in patients with metastatic colorectal cancer: European Organisation for Research and Treatment of Cancer Gastrointestinal Group Study 40986.

PURPOSE: To demonstrate that adding irinotecan to a standard weekly schedule of high-dose, infusional fluorouracil (FU) and leucovorin (folinic acid [FA]) can prolong progression-free survival (PFS). PATIENTS AND METHODS: Four hundred thirty patients with measurable or assessable metastatic colorectal cancer were randomly assigned to receive either FA 500 mg/m(2) as a 2-hour infusion and FU 2.6 g/m(2) by intravenous 24-hour infusion, both administered weekly for 6 weeks, followed by a 2-week rest (Arbeitsgemeinschaft für Internistische Onkologie [AIO] arm, n = 216), or a similar schedule but with FU 2.3 or 2.0 g/m(2) preceded by irinotecan 80 mg/m(2) administered over 30 minutes (experimental group, n = 214). RESULTS: The median PFS time in the experimental group was 8.5 months (95% CI, 7.6 to 9.9 months) compared with 6.4 months (95% CI, 5.3 to 7.2 months) in the AIO arm (P < .0001). The median overall survival time was increased from 16.9 to 20.1 months (P = .2779). The objective response rate was 62.2% (95% CI, 55.0% to 69.5%) in the experimental group and 34.4% (95% CI, 27.5% to 41.3%) in the AIO arm (P < .0001). CONCLUSION: The addition of irinotecan to the standard AIO FU/FA regimen was associated with a highly significant improvement in PFS and response rate and was well tolerated. The results of this study confirm that irinotecan in combination with high-dose infusional FU/FA is a reference first-line treatment.

No Evidence of Dengue Virus Infections in Several Species of Bats Captured in Central and Southern Mexico.

Bats are reservoirs for viruses with zoonotic potential in the Americas, and scattered evidence exists suggesting that bats may act as reservoirs for dengue virus (DENV). To explore further the role of bats as part of DENV sylvatic cycles, 240 bats of 18 species were captured in 2 states of Mexico with contrasting ecological characteristics but concurrent DENV activity in humans. RT-PCR analysis of RNA extracted from liver or spleen tissue from de bats failed to show evidence for the presence of DENV nucleic acids in these organs. In addition, plasma assayed by plaque reduction neutralization test showed no evidence of neutralizing anti-DENV antibodies. These results suggest that American bats may not be reservoirs or amplification host for DENV infection.

First-line therapy of advanced chronic lymphocytic leukemia.

There is general agreement that patients with advanced chronic lymphocytic leukemia (CLL) should be treated if they develop anemia or thrombocytopenia. The combination of chlorambucil (CLB) and prednisone is often used for first-line therapy of these patients, but compared to monotherapy with CLB, no difference in survival could be demonstrated. Steroids should be generally reserved, therefore, for the management of complications such as hemolytic anemia and thrombocytopenia or other autoimmune manifestations. CLB can still be considered standard therapy for advanced CLL, since polychemotherapy protocols as well as newer agents such as fludarabine have failed to show an improvement in survival compared to CLB. However, the results regarding response and survival of the CLB-treated patients seem to depend on dosage intensity and treatment duration. Biological response modifiers such as interferons, interleukins, and monoclonal antibodies have not improved responses or remission duration. Because experiences with CLL patients are limited, the indications and procedure of bone marrow transplantation are not yet clear. However, since results of current treatment protocols are unsatisfactory, regardless of age, patients should be involved in clinical studies that address the question whether high-dose CLB, fludarabine or the combination of fludarabine with other active agents can improve patients' outcome. In addition, autologous and allogeneic bone marrow transplantation as a consolidation therapy is under study and might be a step towards a potential cure of this disease.

Morphological and biochemical characterization of Procambarus clarki blood cells.

Morphological and cytochemical analysis of Procambarus clarki hemocytes demonstrated three cell types: hyaline, semigranular, and granular. Hyaline cells showed a higher nuclear/cytoplasmic ratio with few small electron-dense granules in the cytoplasm. Semigranular cells presented numerous round or oval eosinophilic granules (0.40-0.78 micron). Granular cell contained large eosinophilic granules (1.79-3.05 microns). Ultrastructurally, all cells showed microtubules near the borders, a poorly developed Golgi complex, and secretory-type electron-dense particles. No mitotic figures were seen. Cell monolayers showed three morphologically distinct cell types (composed of flattened and well-spread cells) depending on the presence and size of granules (hyaline, semigranular, and granular). No sex-related differences could be documented in cell features or proportions. Cytochemical studies showed that the three cell types were positive for acid phosphatase. Granular and semigranular cells were also positive for nonspecific esterase. Phenoloxidase activity was localized only in granular and semigranular hemocytes, and peroxidase activity was observed only in the granular hemocytes. These results may suggest that the semigranular and granular hemocytes participate in the prophenoloxidase system and also in phagocytic or cytotoxic function.

Prophenoloxidase system activation in the crayfish Procambarus clarki.

The prophenoloxidase system (proPO) was studied in primary cultures of hemocytes of the crayfish Procambarus clarki. Both zymosan and lipopolysaccharide (LPS) separately induced rapid degranulation and lysis of semigranular hemocytes, with concurrent release of proPO. ProPO could be demonstrated in the hemocyte lysate supernatant (HLS) obtained by a freeze/thaw method, and was specifically activated by LPS and zymosan. Phenoloxidase activity was blocked by serine protease inhibitors, such as soybean trypsin inhibitor (STI), leupeptin, and phenylmethyl-sulphonylfluoride (PMSF), and substantially increased by cysteine protease inhibitors (N-methylmaleimide, N-ethylmaleimide, and iodoacetamide). This enhancement was observed only when the proPO system was activated. Incubation without activators or preincubation with STI prevented the induced enhancement. Electrophoretic analyses of HLS treated with zymosan or LPS showed that three bands at 41, 39, and 37 kDa were specifically modified when the system was activated. These results suggest that a serine protease is involved in the activation of the proPO system in P. clarki, and a mechanism susceptible to cysteine protease inhibitors could be related to its regulation.

Acute trichloroethylene poisoning with additional ingestion of ethanol--concentrations of trichloroethylene and its metabolites during hyperventilation therapy.

One hour after suicidal ingestion of about 150 g of trichloroethylene, a 32-year-old male was admitted to hospital. On admission, the patient's state of consciousness deteriorated from somnolence to coma. Based on blood level data, an absorbed trichloroethylene dose of at least 35 g was estimated. Additionally, ethanol, which is a strong inhibitor of trichloroethylene metabolism, had been ingested. With respect to the high dose of trichloroethylene, hyperventilation therapy was performed for 28 h. Concentrations of trichloroethylene and its metabolites in blood and urine were determined by gas chromatography. Due to hyperventilation and inhibition of trichloroethylene metabolism, not more than 30% of the absorbed dose was metabolized and excreted via kidneys. Under normal respiratory conditions and in the absence of ethanol, this fraction amounts to about 75%. Obviously, hyperventilation and ethanol-induced inhibition of metabolism led to considerably enforced pulmonary elimination of the absorbed trichloroethylene.

Adjuvant therapy for gastric adenocarcinoma with the apoptosis-inducing human monoclonal antibody SC-1: first clinical and histopathological results.

In a first clinical trial with the apoptosis-inducing human antibody SC-1 eight patients with poorly differentiated stomach adenocarcinoma of diffuse-type received 20 or 30 mg of purified SC-1 antibody intravenously, followed 24 or 48 h later by gastrectomy and lymphadenectomy. In seven cases a significant induction of apoptotic activity was measured in primary tumors as compared with earlier biopsy material and in five patients a significant regression of tumor mass could be determined histopathologically. No toxic crossreactivity was observed with normal tissue or organs of patients.

Morphological and cytochemical characterization of female Anopheles albimanus (Diptera: Culicidae) hemocytes.

Hemocytes of 2- to 3-d-old female Anopheles albimanus Wiedemann are described by morphology, cytochemistry, and functional criteria. Supplemented Grace's insect medium in a modified Foley's perfusion method was used to obtain hemolymph from An. albimanus. Morphological analysis indicated 3 types of hemocytes were present, prohemocytes, plasmatocytes, and granular cells. Prohemocytes were small round cells with a high nuclear/cytoplasmic ratio. Plasmatocytes were the most abundant cell types in the hemolymph, and appeared as small to large and spindle-shaped cells with round or elongate nucleus, variable number of vacuoles, small granules, and pseudopodia. Granular cells were small to large and round with a large number of cytoplasmic granules, vacuoles, and numerous filopodia. Ultrastructurally, prohemocytes were undifferentiated with abundant free ribosomes and with few small electron-dense granules. Plasmatocytes were rich in mitochondria, rough endoplasmic reticulum, free ribosomes, small electron-dense granules, numerous peripheral vacuoles and with an important organelle polarization. Granular cells contained numerous large electron-dense granular inclusions and vacuoles. Cytochemical studies showed that plasmatocytes and granular cells have cationic bactericidal proteins. Only granular cells showed phenoloxidase and probably lysosomal activities. In vitro functional studies demonstrated that both plasmatocytes and granular cells were able to attach to glass slides, and only plasmatocyte had phagocytic activity and motility. These results characterize the hemocytes of An. albimanus and suggest that plasmatocytes and granular cells may have a role in defense responses to foreign organisms.

[New therapeutic options in chemotherapy of advanced colorectal cancer]. / Neue Therapieoptionen in der Chemotherapie des fortgeschrittenen kolorektalen Karzinoms.

BACKGROUND: Chemotherapeutic options in the treatment of advanced colorectal cancer have markedly improved during the last years. This is partly due to the high-dose 5-FU regimen, but also to the development of new cytotoxic agents and drug combinations. RESULTS: High-dose 5-FU, irinotecan, and oxaliplatin seem to be superior to low-dose 5-FU in terms of response rate and disease control. Combination of irinotecan with 5-FU/FA showed significant longer overall survival rates compared to 5-FU/FA alone in both published phase III trials. Today most patients are treated by a sequential therapeutic concept using the newer drugs mainly for second or third line therapy. However, there are reasons for the use of more intensive chemotherapy combinations in first line treatment. Combination of oxaliplatin with 5-FU/FA, that failed improvement of overall survival compared to 5-FU/FA alone, could downstage previously unresectable liver metastases for potentially curative surgery in some patients. Oral fluoropyrimidines mark another progress in the treatment of advanced colorectal cancer. They seem to be comparable to low-dose 5-FU/FA and could ease chemotherapy. PERSPECTIVE: Prospective randomized phase III trials must confirm the best chemotherapy and the best strategy for the different subgroup of patients.

Mitotic catastrophe triggered in human cancer cells by the viral protein apoptin.

Mitotic catastrophe is an oncosuppressive mechanism that senses mitotic failure leading to cell death or senescence. As such, it protects against aneuploidy and genetic instability, and its induction in cancer cells by exogenous agents is currently seen as a promising therapeutic end point. Apoptin, a small protein from Chicken Anemia Virus (CAV), is known for its ability to selectively induce cell death in human tumor cells. Here, we show that apoptin triggers p53-independent abnormal spindle formation in osteosarcoma cells. Approximately 50% of apoptin-positive cells displayed non-bipolar spindles, a 10-fold increase as compared to control cells. Besides, tumor cells expressing apoptin are greatly limited in their progress through anaphase and telophase, and a significant drop in mitotic cells past the meta-to-anaphase transition is observed. Time-lapse microscopy showed that mitotic osteosarcoma cells expressing apoptin displayed aberrant mitotic figures and/or had a prolonged cycling time during mitosis. Importantly, all dividing cells expressing apoptin eventually underwent cell death either during mitosis or during the following interphase. We infer that apoptin can efficiently trigger cell death in dividing human tumor cells through induction of mitotic catastrophe. However, the killing activity of apoptin is not only confined to dividing cells, as the CAV-derived protein is also able to trigger caspase-3 activation and apoptosis in non-mitotic cancer cells.

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing.

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A-B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A-B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.