Histamine in Lambs with Abomasal Bloat,Haemorrhage and Ulcers

The median concentration of histamine in abomasal fluid of lambs with abomasal haemorrhage and/or ulcers (group 2) was significantly (P < 0.05) higher than the concentrations in lambs presenting abomasal bloat (group 1) and in the healthy and the diseased controls. In group 2, there was also a strong correlation (R² = 0.81) between the histamine concentrations in abomasal tissue and abomasal fluid, although the median value of histamine in the abomasal tissue was not statistically higher in this group than in the others. The urine of lambs in group 2 also had numerically higher median concentration of histamine than the other groups. Five out of eight tested strains of Lactobacillus spp. and one out of two strains of Clostridium sordellii, isolated from abomasal contents of lambs with abomasal disease, were strong producers of histamine. Bacterial production is one possible source for the increased histamine concentrations in lambs suffering from abomasal haemorrhage and/or ulcers.     

Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis

The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis. The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M. bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein. Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase. In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide. Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau. After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate. In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II.     

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.     

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

A multitude of different proteins are synthesized by the mycobacterial cell. It is often valuable to consider these proteins in different broad categories based on common characteristics, such as physicochemical properties, localization in the mycobacterial cell, and active secretion during culture. In turn, these distinct properties are closely related to their functional properties and the tendency to interact with the immune system of the host after infection.MPB70 was initially isolated by Nagai et al. (37). This secreted protein is of particular interest since it is highly species specific (13). It is consistently present in virulent Mycobacterium bovis, whereas it is synthesized and secreted in markedly different amounts by various substrains of avirulent M. bovis BCG (13, 32, 35).We recently studied the closely related MPB83 protein and isolated three peptides derived from it after CNBr cleavage, showing that MPB70 and MPB83 are homologous but clearly distinct proteins and are therefore encoded by different genes (15). The heterogeneity between different substrains of BCG in regard to the synthesis of MPB70 and MPB83 proteins is clearly greater than previously realized (53). Another type of heterogeneity has also previously been identified; 26- and 23-kDa fractions of a protein that was presumed to be MPB70 differed markedly in carbohydrate content (9). The available data indicate that MPB83 occurs in 26- and 23-kDa forms, both glycosylated, whereas MPB70 (at 22 kDa) is nonglycosylated.The term MPB was introduced (37) for the designation of a protein purified from M. bovis BCG, with the number after MPB denoting the relative mobility by electrophoresis on a 7.7% polyacrylamide gel run at pH 9.5. MPT is used for similar designations of proteins purified from M. tuberculosis (38). The designations mpb70 and mpt70 denote the corresponding genes.Cloning of mpb70 (43) revealed the sequence of a polypeptide chain preceded by a signal peptide that is typical of secreted proteins. In contrast, both mpb83 from M. bovis BCG (33, 45) and mpt83 from M. tuberculosis (20) revealed a typical lipoprotein consensus element in the signal sequence. The relative concentrations of the 26- and 23-kDa components of these proteins vary markedly between sonicates and culture fluids of BCG bacilli. In sonicates, the 26-kDa component, consisting of MPB83, dominates. In culture fluids, the reverse is observed, with a markedly higher concentration of the 22- to 23-kDa components in BCG Sweden and BCG Russia (18).The purpose of the present work was to investigate these properties at the level of native proteins in relation to the localization of these and other marker proteins in the mycobacterial cell. Using monoclonal antibody (MAb) MBS43, which reacts with MPB83 but not with MPB70 (53), permitted more precise distinctions between these proteins than previously obtained.     

Increased gammadelta T-cell populations in draining lymph nodes of lambs during the elicitation phase of dinitrochlorobenzene-induced contact hypersensitivity

Ten lambs were sensitised with the hapten DNCB in an acetone/olive oil vehicle. The hapten/vehicle solution was applied onto the skin on the shaved ventral surface of the right ear. Two weeks later these lambs were rechallenged with the DNCB/vehicle solution. Simultaneously, ten non-sensitised lambs were treated with vehicle only, serving as vehicle controls. The 20 lambs were slaughtered 48 h after challenge/vehicle treatment, along with ten untreated animals serving as normal controls. Specimens of draining lymph nodes were collected from the 30 animals. All lambs were between 149 and 187 days old. Lymph node cryosections were stained for several leukocyte markers using monoclonal antibodies with the ABC immunohistochemical method. The stained sections were subsequently assessed in three different cortical compartments in each section, using an image analysis system. The resulting measurements from the three groups were compared. A marked increase of γδ T cells was detected in the DNCB group. The number of CD4+ T helper cells was decreased in the DNCB group compared with the normal control group, but not with the vehicle control group. No differences were revealed for CD8+ T cytotoxic cells or B cells. These findings were interpreted to be the consequences of possible downregulatory mechanisms protecting the lymphoid tissue from hypersensitivity. The prominence of γδ T-cells could indicate that these cells are involved in downregulation.     

Inter-rater reliability of parent and preschool teacher ratings of language in children with autism

Parent reports such as MacArthur-Bates Communicative Development Inventories (CDIs) have been suggested as a measure of language in young children with autism since this group often score below base levels of direct tests. However, questions have been raised concerning the reliability of report-based assessments. Parents and preschool teachers filled out the CDI–Words & Gestures for 55 children diagnosed with autistic disorder. Inter-rater reliability analyses were done for the whole sample and a subgroup of minimally verbal children (n = 28). Further, potential over- or under-estimation, comparing the raters was analyzed. Results suggested excellent to fair inter-rater reliability between parent and preschool teacher. Parents tended to rate the children slightly higher than preschool teachers. However, the differences were small, and most likely due to contextual variations. These findings suggest that parents can be reliable sources of information about language abilities in children with autism. Therefore, when children are difficult to assess through direct tests, parent reports such as the CDI can be a good alternative.