Efficacy of Intraoperative Cooling Methods

Background: Patients may require perioperative cooling for a variety of reasons including treatment of a malignant hyperthermia crisis and induction of therapeutic hypothermia for neurosurgery. The authors compared heat transfer and core cooling rates with five cooling methods.

Methods: Six healthy volunteers were anesthetized with desflurane and nitrous oxide. The cooling methods were 1) circulating water (5 [degree sign] Celsius, full-length mattress and cover), 2) forced air (10 [degree sign] Celsius, full-length cover), 3) gastric lavage (500 ml iced water every 10 min), 4) bladder lavage (300 ml iced Ringer's solution every 10 min), and 5) ice-water immersion. Each method was applied for 40 min or until the volunteers' core temperatures approached 34 [degree sign] Celsius. The volunteers were rewarmed to normothermia between treatments. Core cooling rates were evaluated using linear regression.

Results: The first volunteer developed abdominal cramping and diarrhea after gastric lavage. Consequently, the technique was not again attempted. Bladder lavage increased heat loss 10 [nearly =] 10 W and decreased core temperature 0.8 +/- 0.3 [degree sign] Celsius/h (r2 = 0.99 +/- 0.002; means +/- SD). Forced-air and circulating-water cooling comparably increased heat flux, [nearly =] 170 W. Consequently, core cooling rates were similar during the two treatments at 1.7 +/- 0.5 [degree sign] Celsius/h (r2 = 0.99 +/- 0.001) and 1.6 +/- 1.1 [degree sign] Celsius/h (r2 = 0.98 +/- 0.02), respectively. Immersion in an ice water slurry increased heat loss [nearly =] 600-800 W and decreased core temperature 9.7 +/- 4.4 [degree sign] Celsius/h (r sup 2 = 0.98 +/- 0.01). Immersion cooling was associated with an afterdrop of [nearly =] 2 [degree sign] Celsius.     

Lack of Nonshivering Thermogenesis in Infants Anesthetized with Fentanyl and Propofol

Background: Sweating, vasoconstriction, and shivering have been observed during general anesthesia. Among these, vasoconstriction is especially important because-once triggered-it minimizes further hypothermia. Surprisingly, the core-temperature plateau associated with vasoconstriction appears to preserve core temperature better in infants and children than adults. This observation suggests that vasoconstriction in anesthetized infants may be accompanied by hypermetabolism. Consistent with this theory, unanesthetized infants rely on nonshivering thermogenesis to double heat production when vasoconstriction alone is insufficient. Accordingly, the authors tested the hypothesis that intraoperative core hypothermia triggers nonshivering thermogenesis in infants.

Methods: With Ethics Committee approval and written parental consent, the authors studied six infants undergoing abdominal surgery. All were aged 1 day to 9 months and weighed 2.4-9 kg. Anesthesia was maintained with propofol and fentanyl. The infants were mechanically ventilated and allowed to cool passively until core (distal esophageal) temperatures reached 34-34.5 degrees Celsius. Oxygen consumption-the authors' index of metabolic rate- was recorded throughout cooling. Because nonshivering thermogenesis triples circulating norepinephrine concentrations, arterial blood was analyzed for plasma catecholamines at [nearly equal] 0.5 degrees Celsius intervals. Thermoregulatory vasoconstriction was evaluated using forearm - fingertip, skin-surface gradients, with gradients exceeding 4 degrees Celsius, indicating intense vasoconstriction. The patients were subsequently rapidly rewarmed to 37 degrees Celsius. Regression analysis was used to correlate changes in oxygen consumption and plasma catecholamine concentrations with core temperature.

Results: All patients were vasoconstricted by the time core temperature reached 36 degrees Celsius. Further reduction in core temperature to 34-34.5 degrees Celsius did not increase oxygen consumption. Instead, oxygen consumption decreased linearly. Hypothermia also failed to increase plasma catecholamine concentrations.     

Indomethacin-Dipalmitoylphosphatidylcholine Interaction. A Calorimetric Study of Drug Release from Poly(Lactide-co-glycolide) Microspheres into Multilamellar Vesicles

A comparative study of indomethacin controlled release from poly(lactide-co-glycolide) (50:50, molecular weight 3000) (PLGA) microspheres loaded with two different amounts of drug (10.9 ± 1%, and 34.1 ± 1% w/w) and pure free indomethacin, considering the effects exerted by the drug on the thermotropic behavior of dipalmitoylphosphatidylcholine multilamellar vesicles, was carried out by differential scanning calorimetry (DSC). The release was monitored by comparing the effect exerted by the free indomethacin on lipid thermotropic behavior with that of the drug released by the microspheres and relating these effects to a lipid aqueous dispersion containing the molar ratio of drug able to cause it. By DSC measurements, the pure free indomethacin was found to be able to have a fluidifying effect on the model membrane, causing a shift toward lower values of the transitional temperature (T_m), characteristic of phospholipid liposomes, without variations in the enthalpic changes (ΔH). This shift was found to be modulated by the drug molar fraction with respect to the lipid concentration in the aqueous dispersion. Successively, calorimetric measurements were performed on suspensions of blank liposomes added to weighed amounts of unloaded and indometha-cin-loaded microspheres as well as free powdered indomethacin, and the T_m shifts of the lipid bilayer caused by the drug released from the polymeric system, as well as by the free drug, were compared with that caused by free drug increasing molar fractions dispersed directly on the membrane, employed as a calibration curve to obtain the fraction of drug released. This drug release model could be employed to determine the different kinetics involved in the drug transfer from the microspheres to a membrane. This in vitro study suggests that the kinetic process involved in drug release is influenced by the amount of drug loaded in the microspheres. This calorimetric study shows that the PLGA microspheres are a good delivery system able to sustain drug release. Moreover, the DSC technique applied to the drug interaction with biomembranes constitutes a good tool for determining the drug release representing an innovative alternative in vitro model.     

Differential apoptotic response of J774 macrophages to alumina and ultra-high-molecular-weight polyethylene particles.

We recently identified apoptosis in in vitro wear particle-stimulated macrophages. The recent explosion of interest in apoptosis lies in the fact that it is under positive and negative regulation through evolutionary conserved biochemical pathways. It may also be possible to modulate macrophage apoptosis in the treatment of periprosthetic osteolysis. The purpose of this study was to compare the macrophage response to identically sized particles of alumina ceramic (Al2O3) and ultra-high-molecular-weight polyethylene (UHMWPE) in terms of TNF-alpha release and induction of apoptosis. J774 mouse macrophages were incubated for 0-24 h in the presence of Al2O3 and UHMWPE particles. TNF-alpha release was measured by ELISA; Poly(ADP-ribose)polymerase (PARP) and caspase-3 expression was analyzed by Western blot; DNA fragmentation (DNA laddering) was visualized on agarose gel containing ethidium bromide. Al2O3 particles induced TNF-alpha release after 4 h incubation with concentrations reaching 483 and 800 pg/ml after 24 h with 125 and 250 particles/macrophage, respectively (control = 161 pg/ml) (P < 0.05 vs. control). The same concentrations of UHMWPE particles induced a much larger and significant TNF-alpha release after only 1 h incubation, increasing up to 6250 pg/ml after 24 h (P < 0.05 vs. control). Western blot analysis demonstrated that the active caspase-3 fragment (17 kDa) and the proteolytic PARP fragment (85 kDa) were expressed after 2 h incubation with 125 and 250 Al2O3 particles/macrophage. The active caspase-3 and the PARP fragment had lower expression and appeared after a longer incubation time (8 h) with 125 and 250 UHMWPE particles/macrophage. Finally, DNA fragmentation (DNA laddering) was observed after 16 h with 125 and 250 particles of Al2O3 per macrophage whereas no laddering was induced by UHMWPE particles even after 24 h incubation. This study shows that although both Al2O3 and UHMWPE particles induce TNF-alpha release, this stimulation was much greater (8-10 times higher) with UHMWPE than Al2O3 (P < 0.05 vs. control). As well, the induction of apoptosis, as measured by activation of caspase-3, PARP cleavage and DNA laddering, is different for these two particles, being faster and more important with Al2O3 than UHMWPE. We hypothesize that the ability of Al2O3 to induce macrophage apoptosis may explain the lower TNF-alpha release observed with these particles and explain the differences seen in osteolysis patterns of ceramic-ceramic (CC) vs. metal-polyethylene (Mpe) articulations. In conclusion, apoptosis may be a major internal mechanism to decrease macrophage activity and may be a desired therapeutic endpoint. The identification of an apoptosis-related pathway in the macrophage response to ceramic particles provides crucial data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis.     

Evaluation of a Diagnostic Algorithm Using Immunoglobulin M Enzyme-Linked Immunosorbent Assay To Differentiate Human West Nile Virus and St. Louis Encephalitis Virus Infections during the 2002 West Nile Virus Epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was ≥1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.     

Fusion and Matrix Protein Gene Sequence Analysis of Paramyxoviruses of Type 1(PMV-1) Isolated from Pigeons in Slovenia

Paramyxoviruses of type 1 (PMV-l) isolated from pigeons were genetically analyzed. A part of the fusion and the matrix protein genes were amplified and sequenced, Typical amino acid sequences associated with virulence were determined at the fusion protein cleavage site in all PMV-1 isolates. All Slovene pigeon PMV-1 strains share high amino acid sequence similarity with other pigeon strains. In the phylogenetic tree, they are clustered together with pigeon PMV-1 isolates with moderate pathogenicity. Phylogenetic analysis obtained from the fusion and the matrix protein gene alignments showed the same branching order. Viruses circulating among pigeons were found to form quite unique lineage of virulent NDV strains.     

Induction of protein oxidation by cobalt and chromium ions in human U937 macrophages

Metal particles and ions from hip prostheses have the potential to induce the production of reactive oxygen species (ROS), making them prime suspects for disturbing the cellular balance of oxidants/antioxidants (redox state of the cell). To better understand the cellular effect of metal ions from metal-on-metal prostheses, the aim of this study was to examine the effect of cobalt (Co2+) and chromium (Cr3+) ions on protein oxidation in human U937 macrophages. Protein oxidation was measured by Western blot using antibodies directed against dinitrophenylhydrazine (DNP)-derivatized protein carbonyls, the most commonly measured products of protein oxidation in biological samples. Three DNP-derived proteins were detected. The first has a molecular weight of 16 kDa and is expressed at a very low level. The second has a molecular weight of 48 kDa and its level is not regulated by metal ions. The third is a 69 kDa protein and its level is regulated by Co2+ and Cr3+ ions. Therefore, the last band served as a marker of protein oxidation in the present study. Results showed that Co2+ and Cr3+ ions induced a time- and dose-dependent protein oxidation reaching 6.5 and 2.9 times the control after 72 h, respectively, which were inhibited by the antioxidant glutathione monoethyl-ester. Finally, results showed that the oxidized proteins are mainly found in the cytoplasmic fraction of the cells and are absent from the nucleus. In conclusion, our results suggest that metal ions from metal-on-metal prostheses have the potential to modify the redox state of cells both locally (periprosthetic environment) or systematically (circulating cells). The long term effect of these ions on protein oxidation in vivo remains to be investigated.     

Effect of cobalt and chromium ions on bcl-2, bax, caspase-3, and caspase-8 expression in human U937 macrophages

The bcl-2 and caspase families of proteins play a central role in the modulation of apoptosis. The purpose of this study was to analyze the effect of Co(2+) and Cr(3+) ions on the expression of bcl-2, bax, caspase-3 and caspase-8 to better understand the mechanisms leading to ion-induced apoptosis in macrophages. U937 human macrophages were exposed to Co(2+) and Cr(3+) ions. The expression of proteins was measured by Western blot while caspase activities were measured by colorimetric assay. Results show that Co(2+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 52% inhibitory effect after 24 h. Co(2+) stimulated bax expression with a significant stimulation (p<0.05) after 8 h and a maximal 1.75-fold increase after 16 h. Co(2+) also stimulated the expression of the active fragment of caspase-3 as well as caspase-3 activity maximal increase after 24 h. Co(2+) ions had no effect on caspase-8 expression or activity.Cr(3+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 43% inhibitory effect after 24 h. Cr(3+) stimulated bax expression with significant stimulation (p<0.01) after 8h and a maximal 2.25-fold increase after 24 h. Cr(3+) ions also stimulated the expression of the active fragments of caspase-3 and -8, as well as the activities of both proteases. The effect of Cr(3+) ions on the expression of both caspase active fragments was maximal after 16 h incubation. In conclusion, our results suggest that the modulation of the expression of proteins from the bcl-2 and the caspase families of proteins are implicated in the induction of macrophage apoptosis by Co(2+) and Cr(3+) ions.     

Coamplification of 12p11 and 12q13 approximately q22 in multiple ring chromosomes in a spindle cell sarcoma resolved by novel multicolor fluorescence in situ hybridization analysis

An 80-year-old male presented with a lobulated mass in the lower abdominal wall. A diagnosis of an intermediate grade myofibroblastic spindle cell sarcoma was made. Cytogenetic analysis demonstrated a complex karyotype with a der(6), a small marker and five, different in size, ring chromosomes. Fluorescence in situ hybridization (FISH), multiplex FISH, and multicolor banding analysis was used to further delineate this complex karyotype. The der(6) was shown to be a der(18)t(6;18;9;12;18), the marker chromosome was identified as del(17), and the ring chromosomes as r(9) and r(12;18)x4. Amplification of 18 and coamplification of 12p and 12q was detected in the ring and marker chromosomes. No intercellular heterogeneity was observed although a few micronuclei containing chromosome 18 and anaphase bridges, containing chromosome 12 material, the result of bridge-fusion-bridge (BFB) cycles, were observed. Our findings combined with results from others indicate that amplification of chromosomes 12 and 18 as well as BFB phenomena characterize this type of sarcoma.