Assessment of oxygen-consumption by use of reverse Fick-principle and indirect calorimetry in critically ill patients

Oxygen consumption was measured simultaneously by the reverse Fick-principle (V02FICK) and by indirect calorimetry ("Metabolic Measurement Cart Horizon") (V02MMC) in 31 critically ill patients; 24 men and 7 women. Seventeen patients were breathing spontaneously, 14 patients were on mechanical ventilation. The fractional inspiratory oxygen concentration (FI02) in ventilated patients ranged from 0.21 to 0.4 (mean 0.302). Total oxygen consumption as measured by indirect calorimetry was 286.7 +/- 59.7 ml/min (mean +/- SD), and measured by reverse Fick-principle 258.9 +/- 52.2 ml/min (mean +/- SD). The coefficient of correlation between the two methods was r = 0.873. The absolute difference of oxygen consumption between reverse Fick-method and indirect calorimetry was 11.3%. Regression analysis according to Theil revealed a similar regression between spontaneously breathing and mechanically ventilated patients for the studied FI02 values below 0.4. It is concluded that indirect calorimetry is a reliable method for measuring oxygen consumption in spontaneously breathing as well as mechanically ventilated critically ill patients.     

Pharmacokinetic and pharmacodynamic studies with 4′-epi-doxorubicin in nasopharyngeal carcinoma patients

Summary The plasma pharmacokinetic profile of 4-epidoxorubicin (epirubicin) was investigated in 28 patients with nasopharyngeal carcinoma (NPC) after single i.v. rapid infusions. All patients had normal liver and renal functions. Plasma concentrations of the parent compound were specifically determined by a high-performance liquid chromatographic (HPLC) method, with UV detection at 254 nm. Plasma levels of the compound were fitted to a three-compartment open model; a triexponential decrease in plasma concentrations with a long terminal plasma halflife (44.8±21.2 h) was observed in 27 patients. The respective mean (±SD) serum concentration at 72 h and the AUC, plasma clearance, and terminal elimination rate constant in complete responders were 7.67±1.98 ng/ml, 4,002±3,080 ng· h/ml, 26.6±12.9 l/h·m², and 0.009±0.007 l/h, whereas those in nonresponders were 4.96±1.8 ng/ml, 1,88±652.8 ng·h/ml, 44.4±15 l/h·m², and 0.017±0.006 l/h, respectively; these differences were significant (P(0.05). Epirubicin produced a 52% response rate, including 6 patients with a complete response, 8 with a partial response, 11 with no change, and 2 with progressive disease. No relationship could be found between the various pharmacokinetic parameters and either leukopenia, age, or sex. These observations strongly suggest that plasma clearance may be one of the determining factors affecting the response or nonresponse of NPC patients to epirubicin, and a dose adjustment according to plasma clearance would probably increase the response rate.     

Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli

Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.     

HIV-1 Tat increases the adhesion of monocytes and T-cells to the endothelium in vitro and in vivo: implications for AIDS-associated vasculopathy

HIV-1-infected patients exhibit severe damages of the aortic endothelium, develop angioproliferative lesions such as Kaposi's sarcoma (KS), and have an increased risk of cardiovascular diseases and atherosclerosis. An increased adhesion of leukocytes to the endothelium is a common pathogenic parameter of AIDS-associated vascular diseases. Here we show that the HIV-1 Tat protein, a regulatory protein of HIV-1 released by infected cells, and TNF-alpha, a cytokine increased in sera and tissues of HIV-1-infected patients, activate synergistically the adhesion of leukocytes to endothelial cells both in vitro and in vivo. This effect is selectively mediated by HIV-1 Tat, since HIV-1 Nef, another HIV-1 regulatory protein, and the HIV-1 envelope protein gp41, had no effect. In vitro adhesion assays with PBMC and quantitative cell type analysis of adherent cells by FACS demonstrated that HIV-1 Tat selectively activates the adhesion of T-cells and monocytes but not of B-cells. Intravital microscopic studies in mice confirmed the synergistic activity of HIV-1 Tat and TNF-alpha on leukocyte adhesion to the endothelium in vivo. These data indicate that HIV-1 Tat in cooperation with TNF-alpha may contribute to the vascular damage and cardiovascular diseases observed in AIDS patients but also to the prominent extravasation of T-cells and monocytes which is a key process in the formation and progression of KS lesions.     

Expression of selectin ligands on murine effector and IL-10-producing CD4+ T cells from non-infected and infected tissues

Endothelial selectins are crucial for the recruitment of leukocytes into sites of inflammation. On T cells, ligands for selectins become induced upon differentiation into the effector/memory stage. Initial in vitro studies suggested a correlation between the Th1 phenotype and ligand expression, but whether this also holds true in vivo remained uncertain. We here analyzed selectin ligands on CD4+ T cells producing IFN-gamma, IL-4 or IL-10, prototypic cytokines of the Th1, Th2 and Tr1 subset, respectively. We analyzed mice infected with influenza virus, the bacterium Listeria, and the parasites Toxoplasma (all Th1 models) or Nippostrongylus (Th2 model). A link between the Th1 phenotype and ligand expression was not found in vivo. Surprisingly, the potentially regulatory IL-10-producing T cells displayed the highest frequency of ligand-positive cells in general. Within the inflamed tissues, the frequencies of P-selectin-binding cells increased in the dominant subset, either Th1 or Th2. Up-regulation was also found for E-selectin ligands during influenza, but not Nippostrongylus infection. In conclusion, conditions driving T cell polarization into either Th1 or Th2 in vivo do not affect the expression of selectin ligands, but acquisition of P-selectin binding and hence migration into inflamed tissues is boosted by an inflammatory milieu.     

Impaired microtubule function correctable by cyclic GMP and cholinergic agonists in the Chediak-Higashi syndrome.

The Chediak-Higashi (CH) syndrome of man and several animal species is characterized by the presence of abnormal giant granules in all granule-containing cells and by defects in chemotaxis and lysosomal degranulation during phagocytosis in polymorphonuclear leukocytes (PMNs). Since similar functional abnormalities have been reported in normal PMNs following exposure to colchicine and other agents that disrupt microtubles it was proposed that microtubule function may be impaired in the CH syndrome. The mobility of concanavalin A (con A)-receptor complexes on PMN membranes was used to test microtubule integrity. Normal PMNs showed a uniform distribution of membrane-bound con A. By contrast, con A was aggregated into surface caps on both colchicine-treated normal PMNs and untreated PMNs from mice and a patient with CH syndrome. This result is consistent with impaired microtubule function in the CH cells. The spontaneous capping response of CH PMNs was inhibited by cyclic GMP and by cholinergic agonists that can elevate cyclic GMP levels in neutrophils. This raised the possibility that the microtubule defect in CH cells may be correctable by treatments that increase cyclic GMP generation. Direct evidence for both the absence of microtubule assembly in con A-treated PMNs from the CH patient and for normal microtubule assembly in CH PMNs incubated with cyclic GMP and cholinergic agonists prior to con A treatment was obtained by electron microscopy. In addition, evidence for a direct relationship between the microtubule defect and the development of giant lysosomes in CH cells was obtained. Thus, CH fibroblasts grown in vitro developed abnormal lysosomes in the majority of cells. However, the same cells cultured in the presence of cholinergic agonists developed a majority of lysosomes that were morphologically normal at the level of the light microscope. Similarly, granule morphology appeared normal in peripheral blood leukocytes from mice treated chronically in vivo with cholinergic agonists.     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Investigations on the biology,epidemiology, pathology and control of<Emphasis Type="Italic"> Tunga penetrans</Emphasis> in Brazil: I. Natural history of tungiasis in man

Tungiasis is an important health problem in poor communities in Brazil and is associated with severe morbidity, particularly in children. The causative agent, the female flea Tunga penetrans, burrows into the skin of its host, where it develops, produces eggs and eventually dies. From the beginning of the penetration to the elimination of the carcass of the ectoparasite by skin repair mechanisms, the whole process takes 4-6 weeks. The present study is based on specimens from 86 patients, for some of whom the exact time of penetration was known. Lesions were photographed, described in detail and biopsied. Biopsies were examined histologically and by means of scanning electron microscopy (SEM). Based on clinical, SEM and histological findings, the "Fortaleza classification" was elaborated. This allows the natural history of tungiasis to be divided into five stages: (1) the penetration phase, (2) the phase of beginning hypertrophy, (3) the white halo phase, (4) the involution phase and (5) residues in the host's skin. Based on morphological and functional criteria, stages 3 and 4 are divided into further substages. The proposed Fortaleza classification can be used for clinical and epidemiological purposes. It allows a more precise diagnosis, enables the assessment of chemotherapeutic approaches and helps to evaluate control measures at the community level.     

Dominant and recessive aryl hydrocarbon hydroxylase-deficient mutants of mouse hepatoma line,Hepa-1, and assignment of recessive mutants to three complementation groups

Fifty-four benzo[a]pyrene (BP)-resistant, aryl hydrocarbon hydroxylase (AHH)-deficient mutants were found to be recessive, while five were dominant. Hybrids between the former mutants and the wild-type were killed by BP, and possessed AHH activities of at least 0.5 (relative to the wild-type). Dominant-mutant-wild-type hybrids were resistant to BP and had activities of about 0.05. Additional experiments assigned the recessive mutants to three complementation groups, designated A through C. Group-B-group-C hybrids were exceptional in possessing a mean AHH activity (0.36), less than the value (0.5) expected from gene dosage. This deficiency was probably due, in part, to instability of AHH activity in these hybrids. However, all hybrids tested retained stable DNA complements, equal to the sum of those of their parents, for 140 days in culture. Previous studies have shown that group B and group C mutations both affect the functioning of a cytosolic receptor required for AHH induction (1).     

Diurnal rhythms of leptin and ghrelin in the systemic circulation and in the gastric mucosa are related to food intake in rats

We investigated diurnal changes in leptin and ghrelin levels in the stomach and in the systemic circulation and their relation to food intake rhythms in Wistar rats housed at 22 °C with a 12-h light/dark cycle and free access to food and water. Animals were sacrificed every 3 h over a 24-h period. Leptin and ghrelin levels in serum and in the gastric mucosa were analysed by immunoassay. Leptin mRNA levels were determined in the gastric mucosa by RT-PCR and in different adipose tissue depots (epididymal, retroperitoneal and mesenteric) by Northern blot. Ghrelin mRNA levels were determined by Northern blot. Gastric and serum leptin levels displayed similar diurnal rhythms, rising during the dark phase and decreasing gradually during the light phase. Leptin expression in the different adipose tissue depots correlated positively with circulating leptin levels (P<0.05), although there were some depot-associated differences. Leptin mRNA levels in the mesenteric depot correlated positively with food intake (P<0.05). In blood, ghrelin levels rose sharply just before the onset of the dark phase and dropped suddenly just after. In the stomach, ghrelin levels were high during the fasting period of light and low during the night, and correlated inversely with food intake, gastric contents and serum leptin levels (P<0.05). Leptin and ghrelin in the stomach and in the systemic circulation thus show diurnal variations that are influenced by food intake rhythms. The results agree with a role for ghrelin as a stimulant of meal initiation.