Effect of heat-stable and heat-labile enterotoxins of Escherichia coli on intestinal brush border membrane enzymes of mice

The activities of intestinal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, lactase, sucrase, gamma-glutamyl transpeptidase and leucine aminopeptidase were determined in intestinal homogenates and purified BBMs from control, heat-stable and heat-labile enterotoxin treated mice. The activities of all the enzymes except lactase were decreased significantly (p less than 0.01) in homogenates while increased significantly (p less than 0.001) in BBMs of experimental groups as compared to controls. Calmodulin activities were increased significantly (p less than 0.01) as compared to control in heat-stable enterotoxin treated mice but remained unaltered in heat-labile enterotoxin treated mice. DNA contents of intestinal homogenates were decreased in experimental groups demonstrating the decrease in cell number in these groups. The altered BBM enzyme activities could not be attributed to changes in calmodulin activities. The increase in enzyme activities in BBMs may reflect a compensatory phenomenon in the remaining cells.     

BNIP3 Regulates AT101 [(-)-Gossypol] Induced Death in Malignant Peripheral Nerve Sheath Tumor Cells

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive Schwann cell-derived sarcomas and are the leading cause of mortality in patients with neurofibromatosis type 1 (NF1). Current treatment modalities have been largely ineffective, resulting in a high rate of MPNST recurrence and poor five-year patient survival. This necessitates the exploration of alternative chemotherapeutic options for MPNST patients. This study sought to assess the cytotoxic effect of the BH3-mimetic AT101 [(-)-gossypol] on MPNST cells in vitro and to identify key regulators of AT101-induced MPNST cell death. We found that AT101 caused caspase-independent, non-apoptotic MPNST cell death, which was accompanied by autophagy and was mediated through HIF-1α induced expression of the atypical BH3-only protein BNIP3. These effects were mediated by intracellular iron chelation, a previously unreported mechanism of AT101 cytotoxicity.     

Ethnic‐Specific Differences in Abdominal Subcutaneous Adipose Tissue Compartments

South Asians have a higher prevalence of cardiovascular disease (CVD) than Europeans. Studies have identified distinct subcompartments of subcutaneous adipose tissue (SAT) that provide insight into the relationship between abdominal obesity and metabolic risk factors in different ethnic groups. Our objective was to determine the relationship between SAT compartments and fat‐free mass (FFM) between South Asian and European cohorts, and between men and women. Healthy Europeans and South Asians (n = 408) were assessed for FFM via dual energy X‐ray absorptiometry, and SAT areas by computed tomography (CT). SAT was subdivided into superficial subcutaneous abdominal adipose tissue (SSAT) and deep subcutaneous abdominal adipose tissue (DSAT). Linear regression analyses were performed using DSAT and SSAT as separate dependent variables and FFM and ethnicity as primary independent variables adjusting for age, gender, income, education, and smoking status. Results showed that South Asian men had significantly higher amounts of DSAT (median 187.65 cm² vs. 145.15 cm², P < 0.001), SSAT (median 92.0 cm² vs. 76.1 cm², P = 0.046), and body fat mass (BFM) (25.1 kg vs. 22.6 kg, P = 0.049) than European men. In a fully adjusted model, South Asians showed significantly greater DSAT at any FFM than Europeans. Women had more SSAT at any given FFM than men and less DSAT at any given FFM than men, irrespective of ethnic background. In conclusion, South Asians had more DSAT than Europeans and men had relatively more DSAT than women. These data suggest that specific fat depots are influenced by ethnicity and gender; therefore, could provide insight into the relationship between ethnicity, gender and subsequent risk for CVD.     

Reconstitution of CD4 T Cells in Bronchoalveolar Lavage Fluid after Initiation of Highly Active Antiretroviral Therapy

The massive depletion of gastrointestinal-tract CD4 T cells is a hallmark of the acute phase of HIV infection. In contrast, the depletion of the lower-respiratory-tract mucosal CD4 T cells as measured in bronchoalveolar lavage (BAL) fluid is more moderate and similar to the depletion of CD4 T cells observed in peripheral blood (PB). To understand better the dynamics of disease pathogenesis and the potential for the reconstitution of CD4 T cells in the lung and PB following the administration of effective antiretroviral therapy, we studied cell-associated viral loads, CD4 T-cell frequencies, and phenotypic and functional profiles of antigen-specific CD4 T cells from BAL fluid and blood before and after the initiation of highly active antiretroviral therapy (HAART). The major findings to emerge were the following: (i) BAL CD4 T cells are not massively depleted or preferentially infected by HIV compared to levels for PB; (ii) BAL CD4 T cells reconstitute after the initiation of HAART, and their infection frequencies decrease; (iii) BAL CD4 T-cell reconstitution appears to occur via the local proliferation of resident BAL CD4 T cells rather than redistribution; and (iv) BAL CD4 T cells are more polyfunctional than CD4 T cells in blood, and their functional profile is relatively unchanged after the initiation of HAART. Taken together, these data suggest mechanisms for mucosal CD4 T-cell depletion and interventions that might aid in the reconstitution of mucosal CD4 T cells.The assessment of the degree of memory CD4 T-cell depletion at mucosal sites during human immunodeficiency virus (HIV) infection is perhaps the most comprehensive way to estimate the impact of HIV on the T-cell pool. As such, the massive depletion of gastrointestinal CD4 T cells is a hallmark of HIV and simian immunodeficiency virus (SIV) infection (5, 12, 17, 19, 20, 30). This depletion occurs during the acute phase of infection and is maintained throughout the chronic phase. Mechanisms underlying this depletion have been shown to include the direct consequence of target cell infection (4, 19) and virus-induced Fas-mediated apoptosis (17). However, while it is clear that the substantial depletion of CD4 T cells occurs in the gastrointestinal (GI) tract and vaginal mucosa (31) of SIV-infected macaques and HIV-infected individuals (5, 12, 20, 30), similar depletion does not manifest at all mucosal sites, particularly the lung, in human studies (4).Highly active antiretroviral therapy (HAART) has significantly improved the prognosis of HIV-infected individuals (15, 16). Individuals who initiate HAART before their CD4 T-cell counts in peripheral blood (PB) fall below 350 cells/μl have significantly improved survival compared to that of individuals who initiate HAART with CD4 T-cell counts less than 350 cells/μl (15). Several studies also have shown that when HAART is initiated after CD4 T-cell counts fall below 350 cells/μl, the reconstitution of CD4 T cells in the GI tract is very poor, even after years of therapy (10, 12, 21). However, HIV-infected individuals treated with HAART during the early phase of infection may reconstitute CD4 T cells in the GI tract (18, 21). In contrast to the GI tract, little is known regarding CD4 T-cell reconstitution in the lung compartment during the course of HIV treatment. Nevertheless, the timing of HAART initiation after infection appears to be an important predictor of successful mucosal T-cell reconstitution.The massive depletion of CD4 T cells during the acute phase of infection does not occur at all mucosal sites, as CD4 T cells in bronchoalveolar lavage (BAL) are relatively spared and are slowly depleted during the chronic phase of infection (4). Despite this preservation of lung CD4 T cells, diminished BAL T-cell immune responses to certain pathogens have been reported in HIV-infected subjects (14). Given that many patients worldwide have access to and will receive antiretroviral therapy, the study of mucosal responses longitudinally during the course of treatment is likely to enhance our understanding of immune restoration. In addition, the early cellular events following HAART initiation are likely to skew the immune system toward both protective (i.e., immunosurveillance) and pathological (i.e., immune reconstitution inflammatory syndrome) responses. In this context, the study of the human pulmonary immune response remains an important aspect of HIV infection and treatment. To examine the dynamics of lung CD4 T-cell reconstitution, we studied the treatment of naïve HIV-infected individuals longitudinally during their course of HAART. We sampled peripheral blood and BAL T cells prior to, at 1 month, and after 1 year of HAART. From each subject and within each compartment, we examined the proliferative and functional capacity of stimulated CD4 and CD8 T cells.     

Ctp1 and the MRN-Complex Are Required for Endonucleolytic Rec12 Removal with Release of a Single Class of Oligonucleotides in Fission Yeast

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5′ ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Δ and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.     

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos^{1, 2}, PAmCherry³, Kaede^4-7, pKindling⁸, and KikGR^{9, 10} have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking¹¹. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems^{7, 12-14}. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation¹⁵, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol¹⁶ to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. Note: The reagents used in this protocol can be found in the table appended at the end of the article.     

Rapid optimization and prototyping for therapeutic antibody-like molecules

Multispecific antibody-like molecules have the potential to advance the standard-of-care in many human diseases. The design of therapeutic molecules in this class, however, has proven to be difficult and, despite significant successes in preclinical research, only one trivalent antibody, catumaxomab, has demonstrated clinical utility. The challenge originates from the complexity of the design space where multiple parameters such as affinity, avidity, effector functions, and pharmaceutical properties need to be engineered in concurrent fashion to achieve the desired therapeutic efficacy. Here, we present a rapid prototyping approach that allows us to successfully optimize these parameters within one campaign cycle that includes modular design, yeast display of structure focused antibody libraries and high throughput biophysical profiling. We delineate this approach by presenting a design case study of MM-141, a tetravalent bispecific antibody targeting two compensatory signaling growth factor receptors: insulin-like growth factor 1 receptor (IGF-1R) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3). A MM-141 proof-of-concept (POC) parent molecule did not meet initial design criteria due to modest bioactivity and poor stability properties. Using a combination of yeast display, structured-guided antibody design and library-scale thermal challenge assay, we discovered a diverse set of stable and active anti-IGF-1R and anti-ErbB3 single-chain variable fragments (scFvs). These optimized modules were reformatted to create a diverse set of full-length tetravalent bispecific antibodies. These re-engineered molecules achieved complete blockade of growth factor induced pro-survival signaling, were stable in serum, and had adequate activity and pharmaceutical properties for clinical development. We believe this approach can be readily applied to the optimization of other classes of bispecific or even multispecific antibody-like molecules.     

Possible Associations of NTRK2 Polymorphisms with Antidepressant Treatment Outcome: Findings from an Extended Tag SNP Approach

Background

Data from clinical studies and results from animal models suggest an involvement of the neurotrophin system in the pathology of depression and antidepressant treatment response. Genetic variations within the genes coding for the brain-derived neurotrophic factor (BDNF) and its key receptor Trkb (NTRK2) may therefore influence the response to antidepressant treatment.

Methods

We performed a single and multi-marker association study with antidepressant treatment outcome in 398 depressed Caucasian inpatients participating in the Munich Antidepressant Response Signature (MARS) project. Two Caucasian replication samples (N = 249 and N = 247) were investigated, resulting in a total number of 894 patients. 18 tagging SNPs in the BDNF gene region and 64 tagging SNPs in the NTRK2 gene region were genotyped in the discovery sample; 16 nominally associated SNPs were tested in two replication samples.

Results

In the discovery analysis, 7 BDNF SNPs and 9 NTRK2 SNPs were nominally associated with treatment response. Three NTRK2 SNPs (rs10868223, rs1659412 and rs11140778) also showed associations in at least one replication sample and in the combined sample with the same direction of effects (P_corr = .018, P_corr = .015 and P_corr = .004, respectively). We observed an across-gene BDNF-NTRK2 SNP interaction for rs4923468 and rs1387926. No robust interaction of associated SNPs was found in an analysis of BDNF serum protein levels as a predictor for treatment outcome in a subset of 93 patients.

Conclusions/Limitations

Although not all associations in the discovery analysis could be unambiguously replicated, the findings of the present study identified single nucleotide variations in the BDNF and NTRK2 genes that might be involved in antidepressant treatment outcome and that have not been previously reported in this context. These new variants need further validation in future association studies.     

Protector turns predator

Therapy-induced autophagy is recognized as a critical determinant of treatment outcome in cancer patients, primarily as a factor underlying drug resistance. However, recent investigations point toward a context-dependent, death-inducing role for autophagy, the mechanism of which remains largely unknown. Our recent study provides evidence that autophagy can directly mediate cell killing in multiple tumor cell types by facilitating degradation of KRAS/K-Ras, a key survival protein. These findings have broad implications for strategies employing autophagy modulation to target tumor cells.