Benchmarking of a Bayesian single cell RNAseq differential gene expression test for dose–response study designs

The application of single-cell RNA sequencing (scRNAseq) for the evaluation of chemicals, drugs, and food contaminants presents the opportunity to consider cellular heterogeneity in pharmacological and toxicological responses. Current differential gene expression analysis (DGEA) methods focus primarily on two group comparisons, not multi-group dose–response study designs used in safety assessments. To benchmark DGEA methods for dose–response scRNAseq experiments, we proposed a multiplicity corrected Bayesian testing approach and compare it against 8 other methods including two frequentist fit-for-purpose tests using simulated and experimental data. Our Bayesian test method outperformed all other tests for a broad range of accuracy metrics including control of false positive error rates. Most notable, the fit-for-purpose and standard multiple group DGEA methods were superior to the two group scRNAseq methods for dose–response study designs. Collectively, our benchmarking of DGEA methods demonstrates the importance in considering study design when determining the most appropriate test methods.     

Effect of immunization with lipid associated polysaccharide antigen and anti CD-2 antibodies on class II MHC expression and cellular immune response in BALB/C mice infected with Leishmania donovani

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.     

Antithrombin binding of low molecular weight heparins and inhibition of factor Xa

Fluorescence and stopped flow methods were used to compare clinically used heparins with regard to their ability to bind to antithrombin and to accelerate the inactivation of factor Xa. Titration of antithrombin with both low molecular weight heparin (LMWH) (enoxaparin, fragmin and ardeparin) and unfractionated heparin (UFH) produced an equivalent fluorescence increase and indicates similar affinity of all heparin preparations to antithrombin. However, relative to UFH enoxaparin, the LMWH with the smallest average molecular mass, contained only 12% material with high affinity for antithrombin. The rate of factor Xa inhibition by antithrombin increased with the concentration of the examined heparins to the same limiting value, but the concentration required for maximal acceleration depended on the preparation. According to these data the high affinity fraction of the heparin preparations increased the intrinsic fluorescence and inhibitory activity equally without additional effects by variations in chain length and chemical composition. In contrast, in the presence of Ca UFH accelerated the inhibition of factor Xa by antithrombin 10-fold more efficiently than comparable concentrations of the high affinity fractions of enoxaparin and fragmin. The bell-shaped dependence of this accelerating effect suggests simultaneous binding of both proteins to heparin. In conclusion, under physiologic conditions the anti-factor Xa activity of heparin results from a composite effect of chain length and the content of material with high affinity to antithrombin. Thus, the reduced antithrombotic activity of LMWH relative to UFH results from a smaller content of high affinity material and the absence of a stimulating effect of calcium.     

Suppressor cell activity of intestinal mucosal leucocytes from buffalo (Bubalus bubalis arni)

Suppressor activity of buffalo intestinal intraepithelial leucocytes and lamina propria leucocytes was induced by Concanavalin A, and was assayed against the mitogenic response of autologous and allogenic leucocytes to mitogens. Appreciable suppression was observed with 25 micrograms ConA/ml on the proliferative activity of the responder cells cocultured at a ratio of < 2:1 (suppressor:responder cell). Mitomycin C treatment of intestinal leucocytes did not totally vanish the viability and functionality of leucocytes.     

A minimized Fc binding peptide from protein A induces immunocyte proliferation and evokes Th1-type response in mice

It is now well established that PA is a potent biological response modifier, showing simultaneously antitumor, antitoxic, anticarcinogenic, antifungal, antiparasitic and immunomodulatory properties. Since PA is a foreign protein, it is quite logical to assume that it may be cleaved into smaller peptide fragments in vivo which may be responsible for biological activities of whole PA molecule. The present study was undertaken to dissect out the structural entities of PA responsible for its biological properties. Protein A (PA) of Staphylococcus aureus has a unique property of binding with immunoglobulins. On the basis of molecular modeling and energy minimization studies a 20-mer tryptic fragment (theoretical) was predicted to retain IgG binding capacity which has been verified by immunoblot. This peptide sequence was selected to carry out experimental studies to show its functional mimicry of PA. We observed in the sera of 20-mer peptide treated mice that the concentrations of IFNgamma, TNFalpha and IL1alpha increase to a peak level by 4 h; on the other hand, there was a decrease in IL4, IL6 and IL10 concentrations at the same time (4 h). The ratio of IFNgamma to IL4 showed Th1 type of response with the peptide as well as with that of PA. The nitric oxide concentration in sera also increases and the peak increase was in 6 h with both the peptide and PA. Cell cycle analysis using FACS shows that 20 micrograms dose of peptide was non-toxic to thymocytes and spleenocytes; on the other hand, it was immunoproliferative, shifting the thymocytes and spleenocytes from G0/G1 to S phase of the cell cycle. Further studies are in progress to evaluate other biological properties of the peptide, to evaluate if this peptide could be used as a substitute of PA to mimic at least some of its biological activities.     

The role of insulin as an antithrombotic humoral factor

Insulin is well known for its essential role in carbohydrate metabolism: insulin deficiency results in the development of diabetes mellitus. It has been known for many years that people with diabetes mellitus are predisposed to develop thrombotic diseases including myocardial infarction. It was thought that the thrombus formation was the consequence of impaired carbohydrate metabolism. In recent years, it has become apparent that insulin is capable of ameliorating several pathophysiological events, leading to the inhibition and dissolution of the formed thrombus in the system. These insulin-induced events include inhibition of platelet aggregation by prompting the synthesis of NO in platelet and prostacyclin in endothelial cells. Furthermore, insulin upregulates prostacyclin receptors and downregulates alpha(2) adrenergic receptor in platelets, thereby amplifying the inhibition of platelet aggregation. Insulin also releases tissue plasminogen activator, a potent thrombolytic enzyme, from the platelet membrane which dissolves the formed thrombus leading to the resumption of normal blood circulation. In effect, insulin could be an essential tool in the control of thrombotic disorders.     

Relationships between stem cells and cancer stem cells

Stem cells have been shown to exist in a variety of tissues. Recent studies have characterized stem cell gene expression patterns, phenotypes, and potential therapeutic uses. One of the most important properties of stem cells is that of self renewal. This raises the possibility that some of the clinical properties of human tumors may be due to transformed stem cells. Similar signaling pathways may regulate self renewal in normal and transformed stem cells. These rare transformed stem cells may drive the process of tumorigenesis due to their potential for self renewal. There are important ramifications for clinical cancer treatment if the growth of solid tumors is at least partially dependent on a cancer stem cell population. In the cancer stem cell model, tumor recurrence may be due to the non-targeted stem cell compartment repopulating the tumor. If cancer stem cells can be prospectively identified and isolated, it should be possible to identify therapies that will selectively target these cells.