Rapid PCR Detection of Helicobacter pylori-Associated Virulence and Resistance Genes Directly from Gastric Biopsy Material

We have developed a PCR-based method to detect macrolide resistance and the virulence gene cagA in Helicobacter pylori within 24 h, thereby improving the lengthy process of culture-based approaches. Total DNA was prepared directly from stomach biopsy specimens. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.     

Molecular basis for serological cross-reactivity between enteroviruses.

The recognition sites for human antibodies which are cross-reactive between different types of enteroviruses were determined and characterized. Serum samples obtained from 58 patients with culture-confirmed enteroviral infections were analyzed in enzyme immunoassays against two sets of overlapping synthetic peptides covering residues 31 to 96 of poliovirus 1 VP1 (Mahoney strain) and residues 31 to 148 of coxsackievirus B1 VP1 (position based on alignment with poliovirus 1 VP1, Mahoney strain). A major antigenic region eliciting cross-reactive antibodies could be located to residues 37 to 51 of VP1. Furthermore, a single peptide covering residues 42 to 55 almost completely inhibited the binding of human antibodies to heat-inactivated enteroviruses, indicating that residues 42 to 55 of VP1 contain a major region eliciting cross-reactive antibodies. By using peptide analogs in which each residue within positions 42 to 55 of VP1 was sequentially substituted by Ala or Gly, we were able to determine the most essential residues for human antibody binding in 38 of the convalescent-phase patient serum samples. In a majority of the serum samples, the most essential residues for antibody binding were found to be Pro-42, Ala-43, Leu-44, Thr-45, Ala-46, Glu-48, Thr-49, and Gly-50. All of these residues are conserved, according to known enterovirus sequences, with the divergent echovirus 22 excepted. In conclusion, we could demonstrate that the essential residues for binding of cross-reactive antibodies are well conserved within the enterovirus family. These findings provide a molecular basis for the observed antibody cross-reactivity within the enterovirus group.     

Cytotoxicity in vitro of preleukaemic lymphoid cells on syngeneic monolayers of embryo or thymus cells

Lymphoid cells from preleukaemic AKR mice were cytotoxic for monolayers of syngeneic embryo and thymus target cells in tissue culture. This reactivity was detectable with cells from mice aged 3–36 weeks but was not present with cells from younger mice. Cytotoxic reactions to syngeneic embryo tissues were also seen with lymphoid cells from high leukaemia strain C3H mice carrying Moloney virus but not with lymphoid cells from normal low leukaemic strain C3H/Bi or DBA/2 mice. Lymphoid or lymphoma cells from leukaemic AKR mice showed reduced reactivity. Phytohaemagglutinin was not necessary for the reaction of preleukaemic AKR cells against AKR monolayers and cytotoxicity was inhibited by preincubation of target cells with an antiserum directed against AKR G+ cells.

The reactivity of preleukaemic AKR and C3H lymphoid cells against syngeneic monolayers may represent some type of allogeneic inhibition due to acquired antigenic differences between aggressor and target cell but the data fit best an interpretation that some lymphoid cells in preleukaemic AKR and C3H mice acquire immunological reactivity to virus-induced G+ or M+ antigens exhibited by the target cells.

     

Granulocyte Stimulating Factor in Patients on Peritoneal Dialysis and LPS Stimulated Peripheral Blood Mononuclear Cells

Dialysate and serum levels of granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) were analyzed in patients with continuous ambulatory peritoneal dialysis (CAPD). Samples from the peritoneal effluent and from serum were obtained during the first months of dialysis and during peritonitis from the first three dialysate bags drained on the day of admittance and from nightbags on days three and ten. Serum samples were drawn on days one and ten. On the first day of infection G-CSF was detected in twelve out of fifteen samples in the dialysate and reached its peak median level, 443 pg/ml, in the first drained bag and thereafter decreased significantly. Also in serum a peak, 190 pg/ml, was observed on the first day. LIF was found in six of ten analyzed dialysate samples, with a peak median level of 77 pg/ml on day one, while only four of ten patients had detectable GM-CSF. Peripheral blood mononuclear cells from non-infected CAPD patients were stimulated with lipopolysacharide and G-CSF levels in the supernatants increased significantly (P < 0.05) after 6 h stimulation. We conclude that G-CSF is produced locally in the dialysate during the acute stage of peritonitis and to a lesser extent also systemically. These findings are in line with G-CSF production after LPS stimulation of peripheral blood mononuclear cells.     

Monocyte and iscom enhancement of cell-mediated response to cytomegalovirus

Cell-mediated and humoral responses to cytomegalovirus were studied in a monkey model. Repeated low doses of virus antigen gave poor reactivities in both respects. High antigen doses gave a good humoral IgG response. When autologous monocytes were incubated with the CMV antigen as the immunizing injection, the specific cellular response to CMV antigen increased. The monocytes themselves did not contribute to the in vitro specific proliferation response. When iscoms were the carrier particles for CMV antigens, cellular response was even more strongly enhanced. In immunization schedules where specific cellular responses are important, we suggest that autologous monocytes or iscoms may be employed as antigen carriers.     

Inhibitory Effect of Glucocorticosteroids on Anti-IgE-Induced Histamine Release from Human Basophilic Leukocytes: Evidence for a Dual Mechanism of Action

Anti-IgE-induced histamine release from human leukocytes is inhibited when the cells before challenge are cultured overnight in the presence of glucocorticoids (GCSs). The present report suggests that the GCSs might exert their effect by at least a dual mechanism of action. Histamine release was induced by a suboptimum concentration of anti-IgE. When the release recorded in the presence of the steroid is plotted against the release recorded in its absence, the data points of several experiments fit a regression line characterized by two parameters: its slope and its intercept with the abscissa. Structure-activity examination with selected GCSs indicates that the orders of potency for affecting these two parameters are not identical. Furthermore, pulse experiments suggest that the cells require different times of contact with the steroid to express inhibition according to the two parameters. The removal of adherent cells or platelets did not markedly affect the degree of leukocyte histamine release or its inhibition by a given GCS, suggesting that the steroid interacts directly with the basophil. Finally, steroid-induced inhibition was not affected by the putative phospholipase A2-inhibitor p-bromophenacylbromide (BPB) or the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA).     

Evaluation of a synthetic-peptide enzyme-linked immunosorbent assay for immunoglobulin M to human parvovirus B19.

A synthetic peptide corresponding to a part of the virus protein 1-virus protein 2 overlapping region of human parvovirus B19 was used in an indirect enzyme-linked immunosorbent assay. Antibodies of the immunoglobulin (Ig) M class were measured in serum samples from patients with erythema infectiosum and controls. In comparison with an IgM assay using native B19 viral antigen, the peptide antigen assay was 92% sensitive and 87% specific. B19 IgM reactivities were seen in a limited number of children with other viral diseases. Specific IgM reactivities to short synthetic viral peptides have previously been reported only with Epstein-Barr virus. Since other sources of viral antigen are limited, the peptide antigen assay may be a useful alternative for the diagnosis of B19-associated disease in human beings.     

Infusion of ornithine-alpha-ketoglutarate in healthy subjects: effects on protein metabolism

Infusion of ornithine-alpha-ketoglutarate (Ornicetil) has been suggested to improve nitrogen balance in trauma patients. Whether this anticatabolic effect is localised to the liver or to skeletal muscle is as yet unknown. Consequently, the splanchnic and leg exchange of amino acids, urea and ammonia were measured in seven healthy non-obese subjects in the basal state and during infusion of ornithine-alpha-ketoglutarate at a rate of 28 mg/min for 150 min. The results demonstrate a six-fold rise in arterial ornithine and an increased uptake by both splanchnic and leg tissues during infusion. The splanchnic uptake of threonine and lysine also increased, while no other alterations were seen in leg amino acid exchange. The arterial urea concentration decreased slightly (-6%, P<0.01) during the infusion in spite of an unchanged urea production from the liver. The ammonia concentration fell by 20% (P<0.05), while glycerol and ketone body concentrations did not change significantly. It is concluded that intravenous infusion of ornithine-alpha-ketoglutarate in healthy subjects does not significantly influence hepatic or skeletal muscle protein metabolism.