Hemodynamic alterations secondary to an electrical burn in the rat: a pilot study

Rats subjected to a standard electrical burn of 250 volts for 10 seconds receive a severe injury stimulating a pronounced systemic circulatory response. Initial postinjury hyperemia is replaced by a low perfusion state within 24 hours. Our study demonstrates the difficulty in isolating regional microcirculatory alterations under such circumstances. Modification of the burn model or the method of fluid resuscitation may minimize the influence of this dynamic systemic response.     

IL-18 Receptor Expression on Epithelial Cells is Upregulated by TNF Alpha

IL-18 is a multifunctional cytokine that augments both innate and acquired immunity and potentiates Th1 and Th2 reactions. We studied the expression of IL-18 receptor (IL-18R) on renal and respiratory epithelial cell lines. Both cell lines upregulated IL-18R mRNA and IL-18R membrane expression in response to TNF alpha and other proinflammatory cytokines. The function of IL-18R was confirmed by induction of IL-8 release from epithelial cells in response to recombinant IL-18. Epithelial cells may represent an important target for IL-18, mainly under inflammatory conditions associated with TNF alpha release.     

Saliva-mediated aggregation of Enterococcus faecalis transformed with a Streptococcus sanguis gene encoding the SSP-5 surface antigen.

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.     

Cloning and expression of a Streptococcus sanguis surface antigen that interacts with a human salivary agglutinin.

Human saliva contains a high-molecular-weight glycoprotein (agglutinin) which binds to specific streptococci in a calcium-dependent reaction leading to the formation of bacterial aggregates. We report the cloning of a gene encoding a surface antigen from Streptococcus sanguis M5 and show that the expressed protein inhibits agglutinin-mediated aggregation and specifically binds the salivary agglutinin in a calcium-dependent fashion. Clones isolated from the immunological screening of S. sanguis M5 genomic libraries with polyclonal antibodies against whole cells were assayed for the ability to compete with S. sanguis for agglutinin. One clone, pSSP-5, expressed antigens of 165 and 130 kilodaltons (kDa) possessing this activity. A 3-kilobase-pair (kbp) insert fragment from this clone was used to screen a genomic library in lambda EMBL3 which resulted in the isolation of clone SSP-5A. This clone contained an insert of 17 kb and expressed proteins of 170 to 205 kDa that reacted with the anti-S. sanguis antibodies. Subcloning of a 5.3-kbp EcoRI-BamHI fragment from SSP-5A produced pEB-5, which expressed streptococcal components that were indistinguishable from SSP-5A. The streptococcal antigen was purified by gel permeation and ion exchange chromatography and shown to potently compete with S. sanguis M5 cells for agglutinin. The antigen also bound purified salivary agglutinin in the presence of 1 mM CaCl2. This binding was inhibited by EDTA. Both the SSP-5 antigen and a 205-kDa protein in surface protein extracts from S. sanguis M5 cross-reacted with antibodies directed against antigen B from S. mutans and SpaA from S. sobrinus 6715. These results indicate that a 205-kDa surface protein that is antigenically related to SpaA and antigen B is involved in the binding of salivary agglutinin to S. sanguis M5.     

Localizing a putative mutation as the major contributor to the development of sporadic Hirschsprung disease to the RET genomic sequence between the promoter region and exon 2

Hirschsprung disease (HSCR), a congenital disorder characterized by intestinal obstruction due to absence of enteric ganglia along variable lengths of the intestinal tract, occurs both in familial and sporadic cases. RET mutations have been found in approximately 50% of the families, but explains only a minority of sporadic cases. This study aims at investigating a possible role of RET in sporadic HSCR patients. Haplotypes of 13 DNA markers, within and flanking RET, have been determined for 117 sporadic HSCR patients and their parents. Strong association was observed for six markers in the 5' region of RET. The largest distortions in allele transmission were found at the same markers. One single haplotype composed of these six markers was present in 55.6% of patients versus 16.2% of controls. Odds ratios (ORs) revealed a highly increased risk of homozygotes for this haplotype to develop HSCR (OR>20). These results allowed us to conclude that RET plays a crucial role in HSCR even when no RET mutations are found. An unknown functional disease variant(s) with a dosage-dependent effect in HSCR is likely located between the promoter region and exon 2 of RET.     

Different levels of natural antibodies in chickens divergently selected for specific antibody responses

We studied the presence of Natural antibodies in plasma samples from individual birds from selected chicken lines at young and old age. Binding, specificity, and relative affinity to various antigens were determined in plasma from non-immunized female chickens at 5 weeks of age, and in plasma obtained from the same chickens one year later using indirect two-step ELISA. Birds were from three different lines. The lines were divergently selected for either high (H line) or low (L line) antibody titers to Sheep Red Blood Cells at 5 weeks of age, next to a random bred control (C line). Binding of plasma immunoglobulins (Ig) from all three lines was found with chicken-egg-white protein (CEP), ovalbumin (OVA), myoglobin (MYO), thyroglobulin (THYRO), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and transferrin (TRANS). Significantly higher binding to most antigens was found with plasma Ig from adult birds from the H line as compared to plasma Ig from the L line, whereas binding of plasma Ig from C-line birds was in between or similar to the H or L line, respectively. Binding of Ig to all antigens in all three lines was significantly higher in plasma obtained at one year of age as compared to plasma obtained at 5 weeks of age. A competitive ELISA with homologous and heterologous antigens was used for determining specificity of the antigen-binding antibodies. Nai;ve plasma samples were characterized by a broad binding to all antigens tested. Inhibition of binding to specific antigens was possible with a broad range of heterologous antigens, but highest competition of binding was obtained with homologous antigen. Both linear regression analysis of serial dilutions of the plasma Ig binding the antigens, as well as competitive ELISA with homologous antigen indicated that plasma Ig from the H line and plasma Ig from the L line had similar affinity characteristics to the antigens tested with the exception of OVA and KLH. Pooled non-immune plasma from H line birds bound to CEP, OVA, THYRO, TRANS, MYO, KLH, and salt-precipitated extracts and supernatants of extracts from chicken heart, spleen, liver, brain, bursa, thymus, and kidney, respectively, as determined by Western blotting. The increasing presence of antibodies in nai;ve chicken plasma binding heterologous and homologous (tissue) antigens indicates the presence of Natural antibodies in poultry. Apart from age, increasing levels of Natural antibodies may be related with the genetically based magnitude of specific antibody levels in the chicken lines studied.     

Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9

FANCG was the third Faconi anaemia gene identified and proved to be identical to the previously cloned XRCC9 gene. We present the pathogenic mutations and sequence variants we have so far identified in a panel of FA-G patients. Mutation screening was performed by PCR, single strand conformational polymorphism analysis and protein truncation tests. Altogether 18 mutations have been determined in 20 families - 97% of all expected mutant alleles. All mutation types have been found, with the exception of large deletions, the large majority is predicted to lead to shortened proteins. One stop codon mutation, E105X, has been found in several German patients and this founder mutation accounts for 44% of the mutant FANCG alleles in German FA-G patients. Comparison of clinical phenotypes shows that patients homozygous for this mutation have an earlier onset of the haematological disorder than most other FA-G patients. The mouse Fancg sequence was established in order to evaluate missense mutations. A putative missense mutation, L71P, in a possible leucine zipper motif may affect FANCG binding of FANCA and seems to be associated with a milder clinical phenotype.     

Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.     

Comparing single‐target and multitarget approaches for postoperative circulating tumour DNA detection in stage II–III colorectal cancer patients

Circulating tumour DNA (ctDNA) detection for postoperative risk stratification in cancer patients has great clinical potential. However, low ctDNA abundances complicates detection. Multitarget (MT) detection strategies have been developed to increase sensitivity. Yet, empirical evidence supporting performance gains of MT vs. single‐target (ST) strategies in a postoperative setting is limited. We compared ctDNA detection in 379 paired plasma samples from 112 stage II–III colorectal cancer patients by ST digital PCR and MT sequencing of 16 patient‐specific variants. The strategies exhibited good concordance (90%, Cohen''s Kappa 0.79), with highly correlated ctDNA quantifications (Pearson r = 0.985). A difference was observed in ctDNA detection preoperatively (ST 72/92, MT 88/92). However, no difference was observed immediately after surgery in recurrence (ST 11/22, MT 10/22) or nonrecurrence (both 2/34) patients. In serial samples, detection was similar within recurrence (ST 13/16, MT 14/16) and nonrecurrence (ST 3/49, MT 1/49) patients. Both approaches yielded similar lead times to standard‐of‐care radiology (ST 4.0 months, MT 4.1 months). Our findings do not support significant performance gains of the MT strategy over the ST strategy for postoperative ctDNA detection.