Molecular profiling of sepsis in mice using Fourier Transform Infrared Microspectroscopy

Sepsis is a life threatening condition resulting from a high burden of infection. It is a major health care problem and associated with inflammation, organ dysfunction and significant mortality. However, proper understanding and delineating the changes that occur during this complex condition remains a challenge. A comparative study involving intra-peritoneal injection of BALB/c mice with Salmonella Typhimurium (infection), lipopolysaccharide (endotoxic shock) or thioglycollate (sterile peritonitis) was performed. The changes in organs and sera were profiled using immunological assays and Fourier Transform Infrared (FTIR) micro-spectroscopy. There is a rapid rise in inflammatory cytokines accompanied with lowering of temperature, respiratory rate and glucose amounts in mice injected with S. Typhimurium or lipopolysaccharide. FTIR identifies distinct changes in liver and sera: decrease in glycogen and protein/lipid ratio and increase in DNA and cholesteryl esters. These changes were distinct from the pattern observed in mice treated with thioglycollate and the differences in the data obtained between the three models are discussed. The combination of FTIR spectroscopy and other biomarkers will be valuable in monitoring molecular changes during sepsis. GRAPHICS] Intra-peritoneal infection with high dose of Salmonella Typhimurium leads to rapid increase in inflammatory cytokines, e.g. Tnf alpha (A). FTIR analysis of liver (B) and sera (C) identifies several metabolic changes: glycogen, protein/lipid, cholesteryl esters and DNA.

An evaluation of antigen capture assays for detecting active filarial antigens

Lymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n = 110), microfilaraemic (MF, n = 65), chronic pathology (CP, n = 45) and non-endemic normal (NEN, n = 10) individuals. Of the 230 samples tested, VAHcapture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

Immunological studies on nucleic acids and their components.8.Antibodies specific to a deoxyribotrinucleotide sequence

Bovine serum albumin conjugates of two trinucleotides, dpTpTpA and dTpTpAp, were prepared by linking the trinucleotides through their end phosphates by the ‘carbodiimide method’. Antibodies were raised in rabbits by injecting the trinucleotide-bovine serum albumin conjugates. Analysis by double diffusion in agar gel, quantitative precipitin reaction and its inhibition by haptens showed clearly the presence of antibodies specific to the whole trinucleotide molecule. The titre of antibodies obtained by the trinucleotide-rabbit serum albumin conjugates with their respective antisera was approximately the same, indicating that linking the trinucleotide through either 5′ or 3′ phosphate does not have an appreciable effect on the titre of antibodies. The results also demonstrate that the nucleotide(s) away from the carrier protein is more immunodominant than the one linked directly to the protein.

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

A simple procedure for purification of N-carbamoylputrescine: Application to assays of putrescine transcarbamoylase and agmatine iminohydrolase activities

The observation that N-carbamoylputrescine is quantitatively excluded on O-(carboxymethyl)-cellulose columns with simultaneous retention of putrescine and agmatine has been utilized to develop a sensitive radiometric assay for putrescine transcarbamoylase and a colorimetric assay for agmatine iminohydrolase. A simple procedure for obtaining bulk amounts of pure synthetic N-carbamoylputrescine by separation from putrescine and dicarbamoylputrescine on Dowex 50 (Na+) resin is described.

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.

Clinical and immunological studies on persons exposed toParthenium hysterophorus L

Studies on 300 persons subjected by occupational hazard to the allergenic weed, Parthenium hysterophorus L. for periods ranging from 3 to 12 months revealed that 4% of them developed contact dermatitis of the exposed parts of the body, while 56% of them got sensitized to the weed without apparently exhibiting any dermatitis. None of them suffered from allergic manifestations like rhinitis or bronchial asthma during the period of study which extended for 2 years.

Immunological studies on nucleic-acids and their components .5. Antibodies specific to a deoxyribodinucleotide sequence

Antibodies were raised in rabbits against the bovine serum albumin conjugate of dpApT. Analysis by double diffusion in agar gel and quantitative precipitation test showed the presence of antibodies specific to the hapten in the antisera. Quantitative data on the specificity of the antibodies were obtained by studying the inhibition of the binding of 3H-dpApT to the anti-sera by various nonradioactive mono- and oligonucleotides, using a nitrocellulose membrane binding assay. The antibodies were found to be highly specific for the dinucleotide sequence dpApT. The antibodies were able to bind to synthetic oligonucleotides containing the sequence dpApT and to denatured calf thymus DNA.

Immunological studies on nucleic-acids and their components .6. Antibodies specific to two deoxyribotrinucleotide sequences

Antibodies to the deoxyribotrinucleotides dpApTpA and dpApApT were prepared by injecting the bovine serum albumin conjugates of the respective haptens in rabbits. The specificities of the antibodies were determined by estimating the inhibition of the binding of the tritiated haptens to the immunoglobulins by various nonradioactive mono- and oligonucleotides, using nitrocellulose membrane binding assay. Anti-dpApTpA and anti-dpApApT antisera were found to contain antibodies which were highly specific to the respective hapten sequence.

The influence of esterification of carboxyl groups of ribonuclease-a on its structure and immunological activity

The effect of modification of carboxyl groups of Ribonuclease-Aa on the enzymatic activity and the antigenic structure of the protein has been studied. Modification of four of the eleven free carboxyl groups of the protein by esterification in anhydrous methanol/0.1 M hydrochloric acid resulted in nearly 80% loss in enzymatic activity but had very little influence on the antigenic structure of the protein. Further increases in the modification of the carboxyl groups caused a progressive loss in immunological activity, and the fully methylated RNase-A exhibited nearly 30% immunological activity. Concomitant with this change in the antigenic structure of the protein, the ability of the molecule to complement with RNase-S-protein increased, clearly indicating the unfolding of the peptide "tail" from the remainder of the molecule. The susceptibility to proteolysis, accessibility of methionine residues for orthobenzoquinone reaction and the loss in immunological activity of the more extensively esterified derivatives of RNase-A are suggestive of the more flexible conformation of these derivatives as compared with the compact native conformation. The fact that even the fully methylated RNase-A retains nearly 30% of its immunological activity suggested that the modified protein contained antibody recognizable residual native structure, which presumably accommodates some antigenic determinants.

Perpetuation of immunological memory through common MHC-I binding modes of peptidomimic and antigenic peptides

Understanding the molecular mechanisms of immunological memory assumes importance in vaccine design. We had earlier hypothesized a mechanism for the maintenance of immunological memory through the operation of a network of idiotypic and anti-idiotypic antibodies (Ab2). Peptides derived from an internal image carrying anti-idiotypic antibody are hypothesized to facilitate the perpetuation of antigen specific T cell memory through similarity in peptide-MHC binding as that of the antigenic peptide. In the present work, the existence of such peptidomimics of the antigen in the Ab2 variable region and their similarity of MHC-I binding was examined by bioinformatics approaches. The analysis employing three known viral antigens and one tumor-associated antigen shows that peptidomimics from Ab2 variable regions have structurally similar MHC-I binding patterns as compared to antigenic peptides, indicating a structural basis for memory perpetuation. (C)) 2007 Elsevier Inc. All rights reserved.

Assessment of the mutagenic potential of a fungicide Bavistin using multiple assays

The fungicide Bavistin was assessed for mutagenic potential by various assays. Bavistin was found to be unable to induce gene mutation in Salmonella typhimurium, but it was able to induce transfection inhibition in Mycobacterium smegmatis. Bavistin was able to induce immediate genotoxic effects in plants but these were not carried through in development as in the long term no genotoxic effects were observed by the progeny test. Bavistin did induce micronuclei formation and did cause an increase in the ratio of normochromatic to polychromatic erythrocytes in mice. It was able to induce a very low frequency of sister-chromatid exchange in human lymphocytes and in addition, it was observed that the chemical affected the mitotic index but did not affect the cell cycle duration. Present studies indicate that the pesticide shows a positive response in 4 out of 5 different test systems (Table 8) and most of the observations support that Bavistin is genotoxic.