Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines

Background

Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG.

Methods/Principal Findings

We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that “rBCG prime - DNA boost” strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)β and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFβ in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.     

Use of equivalent loss models under Section 316(b) of the Clean Water Act

Equivalent loss models encompass a variety of life table-based approaches that can be used to convert age- and life stage-specific estimates of entrainment and impingement loss to a common, easily understood currency. This common currency can be expressed in terms of numbers of individuals, yield to the fishery, or biomass to the ecosystem. These models have at least two key uses in the Section 316(b) assessment process: screening for adverse environmental impact (AEI) and determination of environmental benefits associated with intake alternatives. This paper reviews the various forms of equivalent loss models, their data input requirements, and their assumptions and limitations. In addition, it describes how these models can be used as a second-level screening tool as part of the assessment of the potential for AEI. Given their relative simplicity and ease of use, equivalent loss models should prove to be an important tool in the arsenal of impact assessment methods for Section 316(b).     

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8 μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180 μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120 min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90 min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a K _D and K _i values of 2.36 × 10⁻⁹ and 4.8 × 10⁻⁸ M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.     

Exploring the interaction of some N-benzyloxycarbonyl-L-phenyl alanyl-L-alanine ketones and bovine spleen cathepsin B by molecular modeling and binding free energy calculation

A semi-empirical method for estimation of binding free energy, recently proposed by Aqvist and coworkers, has been effectively tested in several protein-ligand binding cases. We have applied this linear interaction energy method to predict the binding of some N-benzyloxycarbonyl-L-phenyl alanyl-L-alanine ketones with bovine cathepsin B and computed the respective absolute binding constants from averages of molecular dynamics simulations. It is found that the computer simulation results agree well with available experimental data and make it possible to understand better the origin of tight binding and inhibitor specificity of cathepsin B.     

Fractal dimension in endometrial carcinoma

OBJECTIVE: To mathematically assess in a pilot study, endometrial glandular margin irregularity in simple hyperplasia, complex atypical hyperplasia and well-differentiated endometrial carcinoma with the help of box counting of fractal dimension and to discriminate these lesions on the basis of box counting of fractal dimension of the gland. STUDY DESIGN: Ten cases each of endometrial simple hyperplasia (without atypia), complex hyperplasia with atypia and endometrial carcinoma (well-differentiated, endometrioid) were assessed in the study. Five fields at 20 x magnification from each case were randomly selected, and the glands were outlined with the help of a pointer. Using the box counting method, the fractal dimension of each case was measured. RESULTS: Mean fractal dimension in simple hyperplasia, complex atypical hyperplasia and endometrial carcinoma was, 0.899 +/- 0.13, 0.932 +/- 0.042 and 0.939 +/- 0.02, respectively. Statistical analysis showed that the fractal dimension of glands of simple hyperplasia were significantly different from that of complex atypical hyperplasia and endometrial carcinoma (P = .041 and .013, respectively, ANOVA). However, there was no significant difference in fractal dimension between glands of complex hyperplasia and of endometrial carcinoma (P = .659, ANOVA). CONCLUSION: This study provides mathematical (objective) assessment of the measurement of glandular margin irregularities in simple hyperplasia, complex atypical hyperplasia and endometrial carcinoma. Fractal dimension of gland margin may have diagnostic potential in the future.     

Synthesis and structure-activity relationship studies of cinnamic acid-based novel thiazolidinedione antihyperglycemic agents

A number of 2,4-thiazolidinedione derivatives of -phenyl substituted cinnamic acid were synthesized and studied for their PPAR agonist activity. The E-isomer of cinnamic acid, 11, showed moderate PPAR transactivation. The corresponding Z-isomer, 23, and double bond reduced derivative, 15, were found to be much less potent. Although the E-isomer showed a moderate PPAR gamma transactivation, it demonstrated a strong glucose-lowering effect in a genetic rodent model of diabetes. Results of pharmacokinetic, metabolism and permeability studies are consistent with 11 being an active prodrug with an active metabolite, 14, that has similar glucose lowering and PPAR gamma agonist properties.     

Uncertainty and conservatism in assessing environmental impact under paragraph 316(b): lessons from the Hudson River case

Initially, regulation of cooling water intakes under paragraph 316(b) was extremely conservative due to the rapid increase predicted for generating capacity, and to the uncertainty associated with our knowledge of the effects of entrainment and impingement. The uncertainty arose from four main sources: estimation of direct plant effects; understanding of population regulatory processes; measurement of population parameters; and predictability of future conditions. Over the last quarter-century, the uncertainty from the first three sources has been substantially reduced, and analytical techniques exist to deal with the fourth. In addition, the dire predictions initially made for some water bodies have not been realized, demonstrating that populations can successfully withstand power plant impacts. This reduced uncertainty has resulted in less conservative regulation in some, but not all venues. New York appears to be taking a more conservative approach to cooling water intakes. The conservative approach is not based on regulations, but in a philosophy that power plant mortality is an illegitimate use of the aquatic resources. This philosophy may simplify permitting decisions, but it does not further the development of a science-based definition of adverse environmental impact.     

Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.