A protocol for the in situ polymerase chain reaction (IS-PCR) detection of viral nucleic acid in the heart tissue of four-to-five-week-old CD1 mice infected with coxsackievirus B3 (CBV3) Nancy strain is described. To compare the effects of formalin concentration on the IS-PCR process, two different concentrations (10 and 37%) were employed. Using 37% formalin, 25 PCR cycles were sufficient and a permeabilization step could be omitted. However, postfixation of tissues with 4% paraformaldehyde and 100% ethanol after the deparaffinization, reverse transcriptase and amplification steps was required in order to minimize artefacts. When the tissues were fixed in 10% formalin, postfixation with 4% paraformaldehyde was not required, but a permeabilization step had to be employed and 40 cycles of PCR amplification were needed. To detect the PCR product in the 10% formalin-fixed samples, incubation with 0.3 U/ml of an anti-digoxigenin antibody conjugated to alkaline phosphatase was performed for 90 min. When 37% formalin-fixed samples were used, the concentration of the antibody conjugate had to be increased to 3 U/ml and the exposure time was decreased to 30 min. Enterovirus (EV) nucleic acid was detected in the cytoplasm of myocytes. Thus, IS-PCR was successful in localizing EV nucleic acid in the cytoplasm of myocytes in mice infected with a cardiotropic strain of CBV3. Using this technique, 10% formalin-fixed tissues gave better results than 37% formalin-fixed tissues.

Animals↗

PubMedDate not supplied

Influence of large intakes of trace elements on recovery after major burns.

Because Cu, Se, and Zn are involved in immune and antioxidative defense mechanisms and tissue repair, deficiencies might aggravate complications classically observed with burns. After measuring massive cutaneous trace element losses in 10 burn patients, our aim in this study was to determine whether large intravenous intakes of Cu, Zn, and Se can modify serum trace element levels and recovery after major burns. Ten patients, aged 34 +/- 6 yr (mean +/- SD), admitted to the burns center of a Swiss university hospital with thermal burns on 41 +/- 9% of their body surface were studied prospectively, with trace element balance studies from day 1 (D1) to D7 postinjury. Urine and blood samples were also collected on D10, D15, D20, and D25. The patients were divided into two groups of five and received either standard (group 1, control) or greatly increased (group 2, treatment: 4.5 mg Cu, 190 micrograms Se, and 40 mg Zn/day) trace element intakes. Energy and protein intake and wound treatment were similar in both groups. The treatment group was characterized by improved Cu, Se, and Zn status (increase in serum levels and various protein indicators), a much larger leukocyte increase between D4 and D14 (mainly neutrophils), and shorter hospital stay (45 days) compared with the untreated group (57 days). Grafting requirements were more extensive in group 1. Although severity of injury and wound treatment were similar in the groups, the duration of hospitalization was lower in the treated group. Further studies are required to determine whether this is related to trace element supplementation.

Adult↗