Two isothermal challenges yield comparable physiological and subjective responses.

PURPOSE: Ventilated vests are developed to reduce thermal stress by enhancing convective and evaporative cooling from skin tissue underneath the vest. The purpose of this study is to investigate whether thermal stress is equal when a ventilated vest is worn compared to a no-vest situation with similar dry thermal resistance. METHODS: Nine healthy males walked on a treadmill (7 km h-1) for 45 min in a desert climate (34 °C, 20% relative humidity) with and without ventilated vest. Gastrointestinal temperature (Tgi), heart rate (HR), and skin temperature (Tsk) were continuously monitored. Local sweat rate (LSR) was assessed two times on six skin locations. Subjective ratings were assessed every 10 min. RESULTS: Final Tgi (37.6 ± 0.1 °C for vest and 37.6 ± 0.1 °C for no-vest), HR (133 ± 7 bpm and 133 ± 9 bpm) and mean Tsk (34.8 ± 0.7 °C and 34.9 ± 0.6 °C) were not different between conditions (p ≥ 0.163). Scapula skin temperature (Tscapula) under the vest tended to be lower (baseline to final: ΔTscapula = 0.35 ± 0.37 °C) than without vest (ΔTscapula = 0.74 ± 0.62 °C, p = 0.096). LSR at locations outside the vest did not differ with and without vest (p ≥ 0.271). Likewise, subjective responses did not differ between conditions (χ2 ≥ 0.143). CONCLUSIONS: We conclude that two systems with similar dry thermal resistance and, therefore, similar required evaporation, resulted in similar thermal stress during paced walking in a hot-dry environment. Local ventilation did not alter the sweating response on locations outside the vest.

[Moral case deliberation in complex euthanasia requests]. / Moreel beraad bij complexe euthanasieverzoeken.

Dealing with euthanasia requests can be a complex matter for a doctor. How to determine whether the due diligence criteria of the Dutch Euthanasia Act are met in cases that are not straightforward? We argue that moral case deliberation (MCD), methodically structured reflective discussions on concrete moral dilemmas, can provide support in dealing with complex euthanasia requests. In this article, we discuss a case that was talked about during a MCD (in particular the CURA method, where CURA stands for 'concentrating, postponing, reflecting and action') by a group of GPs and nurses who met in the context of a network for ambulatory palliative care.This was about an older patient with multiple chronic conditions who lost any prospects of quality of life.Among other things, it was examined whether requests could be 'well-considered' (one of the due diligence criteria) when the patients are in doubt as to when euthanasia should be carried out.The importance of recognising one's own emotions as a doctor and the quality of communication between patient and doctor were also considered.For that reason, we try to show that MCD can assist in making careful and well-considered decisions when determining a course of action in the case of complex euthanasia requests and can encourage collaborative learning processes.

Feasibility of a quantitative polymerase chain reaction assay for diagnosing pneumococcal pneumonia using oropharyngeal swabs.

Streptococcus pneumoniae is the most important pathogen causing community-acquired pneumonia (CAP). The current diagnostic microbial standard detects S. pneumoniae in less than 30% of CAP cases. A quantitative polymerase chain reaction (PCR) targeting autolysin (lytA) is able to increase the rate of detection. The aim of this study is validation of this quantitative PCR in vitro using different available strains and in vivo using clinical samples (oropharyngeal swabs). The PCR autolysin (lytA) was validated by testing the intra- and inter-run variability. Also, the in vitro specificity and sensitivity, including the lower limit of detection was determined. In addition, a pilot-study was performed using samples from patients (n = 28) with pneumococcal pneumonia and patients (n = 28) with a pneumonia without detection of S. pneumoniae with the current diagnostic microbial standard, but with detection of either a viral and or another bacterial pathogen to validate this test further. The intra- and inter-run variability were relatively low (SD's ranging from 0.08 to 0.96 cycle thresholds). The lower limit of detection turned out to be 1-10 DNA copies/reaction. In-vitro sensitivity and specificity of the tested specimens (8 strains carrying lytA and 6 strains negative for lytA) were both 100%. In patients with pneumococcal and non-pneumococcal pneumonia a cut-off value of 6.000 copies/mL would lead to a sensitivity of 57.1% and a specificity of 85.7%. We were able to develop a quantitative PCR targeting lytA with good in-vitro test characteristics.

The use of a radiotelemetric ruminal bolus to detect body temperature changes in lactating dairy cattle.

The objective of this study was to validate the efficacy of a radiotelemetric bolus (RTB) to detect changes in ruminal temperature resulting from (1) systemic illnesses that are associated with febrile responses and (2) subacute ruminal acidosis (SARA). Eight rumen-fistulated, lactating Holstein cows (586±37 kg of body weight, 106±18 d in milk) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Each period consisted of 21 d. The factors were 2 diets, a moderate forage:concentrate [MFC; 52:48; % of dry matter (DM)] or a high forage:concentrate (HFC; 65:35, % of DM) total mixed ration, and a challenge with a single intramammary injection of lipopolysaccharide (LPS; 100 µg derived from Escherichia coli 0111:B4) or no LPS (sterile saline). Thus, the 4 resulting treatments were (1) MFC with LPS challenge, (2) MFC with saline, (3) HFC with LPS challenge, and (4) HFC with saline. Cows were fed at 0800 and 1400 h daily. Cows received the intramammary injections at 0900 h of d 21. Ruminal pH and ruminal temperature were also measured on d 21 every minute via an indwelling logging system that resided in the ventral sac of the rumen and via a radiotelemetric bolus that resided in the reticulum. Vaginal temperature was also recorded every minute via temperature loggers. Prior to LPS injection, the duration of rumen pH below 5.6 (indicative of SARA) was higher in cows receiving MFC than cows receiving HFC (148±24 and 62±24 min/d, respectively). The temperature measured at the same time via RTB was higher for MFC than HFC cows (167±21 vs. 104 vs. 21 min/d above 38.8°C, respectively). The following day, cows challenged with LPS showed signs of mastitis within the injected quarters, depressed DM intake, decreased milk yield, and a peak vaginal temperature of 41.3±0.1°C 5.5h after the LPS injection. The RTB system successfully detected a fever response parallel to that measured by the vaginal loggers but temperature peak detected by RTB was, on average, 0.5°C lower than that detected by the vaginal logger. Although the RTB system was able to detect a temperature response to the diet effect before LPS challenge, it was unable to detect this effect during the LPS challenge, likely because cows receiving the LPS challenge had decreased feed consumption. In conclusion, radiotelemetry has the potential to improve the detection of SARA and fever on farm.

In vitro and in vivo measurement of cell-mediated immunity in patients with HIV-1 infection.

We studied the usefulness of the in vitro lymphoproliferation assay and the in vivo skin test in HIV-1-infected patients by using Clostridium tetani and tuberculin as testing antigens. Moreover, the relationship between data obtained from both assays was studied. In 56 HIV-infected patients not receiving antiretroviral therapy CD4+ cell counting was performed. In addition, in vitro (lymphocyte proliferation assay) and in vivo (delayed type hypersensitivity skin test) measuring of the immune status was done using C. tetani and tuberculin as testing antigens. When using C. tetani a significant correlation between the results of both tests and the CD4+ cell count was found. In contrast to earlier reports from African countries, in vivo skin testing using tuberculin did not yield clinically significant information on the degree of immunodeficiency. We explain our findings by the fact that health care policy in The Netherlands encompasses vaccination with C. tetani, which enables the application of C. tetani as testing antigen for measuring immune function both in vitro and in vivo.

Active biomonitoring of polycyclic aromatic hydrocarbons by means of mosses.

Spherical bags, packed with 20 g of peat moss (Sphagnum spp.), were exposed to ambient air at a distance of 1 km from a plant manufacturing electrodes for the production of aluminium, near Rotterdam, The Netherlands. In these bags, the concentrations of six polycyclic aromatic hydrocarbons were determined, and compared with the concentrations in moss bags that had been exposed in relatively clean areas. From the results it can be concluded that, in addition to their useful application for biomonitoring of heavy metals, mosses can be applied in active biomonitoring of polycyclic aromatic hydrocarbons in ambient air.

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).

In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro. The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively. The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation. The radical introduced, in addition, chemical DNA adducts. Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA. The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts [van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retèl J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988]. When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced. In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity. It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

The presence and measurement of secretory component in human bile and blood.

Five monoclonal antibodies which recognized three separate epitopes on the free secretory component molecule were produced using free secretory component obtained from human colostrum. Two-site immunoradiometric assays were developed to measure free secretory component and secretory IgA. Monoclonal antibody M9 was used on coated plates as the capture antibody. Monoclonal antibody M7 was used as the labelled signal antibody for the assay of free secretory component and a commercially available monoclonal anti-IgA antibody was used as the labelled signal antibody for the assay of secretory IgA. Free secretory component was found in human serum and bile. In serum, its concentration was raised in patients with high serum alkaline phosphatase due to liver disorders but not in patients with high serum alkaline phosphatase due to non-liver disorders. In bile from bile duct drains collected during the first week after liver transplantation, free secretory component was found in concentrations of up to 33 mg/l, in vast excess of that found in bile from gallstone patients (up to 0.3 mg/l). Bile from gallstone patients but not from liver transplant patients produced proteolytic degradation of free secretory component when incubated in vitro. The finding of large amounts of free secretory component, the free cleaved fragment of the polymeric IgA receptor in human bile, further supports the existence of the blood to bile transhepatocytic pathway in humans.

Milk: does it affect blood pressure? A controlled intervention study.

In a double-blind trial, the effect on blood pressure of supplementation of normal milk (1180 mg Ca2+, 1650 mg K+ and 110 mg Mg2+ d-1) vs. 'mineral-poor' milk (95 mg Ca2+, 580 mg K+ and 10 mg Mg2+ d-1) was studied. Young healthy normotensive female students consumed one of the two supplements while on a low calcium diet (less than 500 mg Ca2+ d-1) for a period of 6 weeks. In both the normal milk- and 'mineral-poor' milk-supplemented groups systolic blood pressure decreased slightly. However, this decrease was persistently greater in the milk-supplemented group. The individual mean systolic blood pressure change during normal milk treatment (-4.1%) was significantly greater (P = 0.03) than that during 'mineral-poor' milk treatment (-1.3%). An effect of normal milk supplementation on diastolic blood pressure could not be demonstrated. The results of the present study indicate a small hypotensive effect of milk consumption, which is attributable to its content of essential minerals.

Relationship between the calcium-to-protein ratio in milk and the urinary calcium excretion in healthy adults--a controlled crossover study.

Over the years, doubts have arisen concerning the use of milk as a calcium source in the prevention of osteoporosis, particularly because of potential offsetting effects of protein and phosphorus. Thus, a new milk product with a higher calcium content and lower contents of protein, phosphorus, and energy was developed. A controlled crossover study was done to determine the way in which substitution of the new milk product (860 mL) for normal milk (1000 mL) in the diet of healthy adults affected the urinary excretion of calcium and hydroxyproline. Short-term consumption of the product significantly lowered 24-h urinary calcium excretion by approximately 0.65 +/- 0.19 mmol/d (means +/- SEM, p less than 0.001). The ratio of fasting urinary hydroxyproline to creatinine did not decrease, indicating no significant reduction of bone resorption in our subjects. Elderly people in particular might benefit from the new product because it reduces their calcium loss as well as their intake of protein, phosphorus, energy, and liquid volume.

Role of the semi-quinone free radical of the anti-tumour agent etoposide (VP-16-213) in the inactivation of single- and double-stranded phi X174 DNA.

The mechanism of action of the anti-tumour agent etoposide (VP-16-213) could involve its bioactivation to metabolites which can damage DNA. Active metabolites of etoposide, generated in vitro, are the 3',4'-dihydroxy-derivative (catechol) and its oxidation product, the ortho-quinone. The conversion of the catechol into the ortho-quinone (and vice versa) proceeds via formation of a semi-quinone free radical. We investigated the role of this radical species in the inactivation of biologically active single- (ss) and double-stranded (RF) phi X174 DNA. Since the formation of semi-quinone free radicals from the ortho-quinone of etoposide is pH dependent, experiments were performed, in which the ortho-quinone was incubated at pH 4, 7.4 and greater than or equal to 9. ESR measurements showed no formation of radical species from the ortho-quinone at pH 4, but an increased rate of generation of the primary semi-quinone free radical at pH values 7.4 to 10; at still higher pH values a secondary semi-quinone free radical was produced. HPLC analyses demonstrated chemical stability of the ortho-quinone at pH 4, but an accelerated decay was observed when the pH was elevated from 7.4 to 9, with its concomitant conversion into more polar components and into the catechol of etoposide. Ss phi X174 DNA, exposed to the ortho-quinone, was inactivated at an increasing rate at pH values increasing from 4 to 7.4 and subsequently to 9. RF phi X174 DNA was only significantly inactivated in incubations with the ortho-quinone at pH 4, not at pH values 7.4 and 9. From these data it is concluded that the primary semi-quinone free radical of etoposide may to a great extent be responsible for the ortho-quinone-induced ss phi X174 DNA inactivation, but that this radical species is not lethal towards RF phi X174 DNA.

Effects of oxygen radical scavengers on the inactivation of SS phi X174 DNA by the semi-quinone free radical of the antitumor agent etoposide.

We have studied the effects of oxygen radical scavengers on the inactivation of ss phi X174 DNA by the semi-quinone free radical of the antitumor agent etoposide (VP 16-213), which was generated from the ortho-quinone of etoposide at pH greater than or equal to 7.4. A semi-quinone free radical of etoposide is thought to play a role in the inactivation of ss phi X174 DNA by its precursors 3',4'-ortho-quinone and 3',4'-ortho-dihydroxy-derivative. The possible role of oxygen radicals formed secondary to semi-quinone formation in the inactivation of DNA by the semi-quinone free radical was investigated using the hydroxyl radical scavengers t-butanol and DMSO, the spin trap DMPO, the enzymes catalase and superoxide dismutase, the iron chelator EDTA and potassium superoxide. Hydroxyl radicals seem not important in the process of inactivation of DNA by the semi-quinone free radical, since t-butanol, DMSO, catalase and EDTA had no inhibitory effect on DNA inactivation. The spin trapping agent DMPO strongly inhibited DNA inactivation and semi-quinone formation from the ortho-quinone of etoposide at pH greater than or equal to 7.4 with the concomitant formation of a DMPO-OH adduct. This adduct probably did not arise from OH. trapping but from trapping of O2-(.). DMSO increased both the semi-quinone formation from and the DNA inactivation by the ortho-quinone of etoposide at pH greater than or equal to 7.4. Potassium superoxide also stimulated phi X174 DNA inactivation by the ortho-quinone at pH less than or equal to 7. From the present study, it is also concluded that superoxide anion radicals probably play an important role in the formation of the semi-quinone free radical from the ortho-quinone of etoposide, thus indirectly influencing DNA inactivation.

The polymeric immunoglobulin A receptor is present on hepatocytes in human liver.

A monoclonal antibody raised against human colostrum secretory component produced even staining of hepatocyte plasma membranes, as well as bile duct lining cells, in all sections examined from eight normal and three abnormal human livers. Human bile samples incubated with free secretory component degraded it to varying extents, probably proteolytically; true levels of free secretory component will therefore often be higher than those reported. It seems likely that human liver resembles that of other mammals in transferring polymeric IgA through hepatocytes to the bile by means of the polymeric IgA receptor.

Reconstitution of an endosome-lysosome interaction in a cell-free system.

A cell-free model for the transfer of endocytosed material to lysosomes is described. Rat liver late endosomes, loaded in vivo with radiolabeled ligand by intravenous injection shortly before killing the animal, showed a specific interaction with lysosomes when incubated at 37 degrees C in the presence of cytosol and an ATP regenerating system. The location of the ligand, generally asialofetuin, was analyzed by isopycnic centrifugation on Nycodenz gradients. Appearance of radiolabel in the lysosomal position on such gradients was maximal after approximately 30 min at 37 degrees C and required the provision of undamaged cytosol, lysosomes, and an ATP regenerating system. It could not be accounted for by nonspecific bulk aggregation of membranes. Transfer occurred only from late endosomes; radiolabel in early endosomes was unaffected. Digestion of the asialofetuin, as shown by the appearance of TCA-soluble radioactivity, occurred on incubation at 37 degrees C and was increased by the provision of an ATP regenerating system.

Detection of carriers of the autosomal form of chronic granulomatous disease.

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT-positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

Effect of dietary calcium supplementation with lactose on bone in vitamin D-deficient rats.

The relationship between plasma calcium and bone length, chemical and histomorphometric bone parameters was studied in vitamin D-deficient rats in order to determine whether the effects of vitamin D on bone could be attributed to the effect of vitamin D on serum calcium. Plasma calcium was varied over a wide range of dietary manipulation. Four groups of vitamin D-deficient rats were given for 6 weeks: a vitamin D-deficient diet (D-, n = 6), the D--diet with calcium supplementation (D-Ca+, n = 6), the D-Ca+-diet with lactose substituted for dextrose (D-Ca+lac, n = 6) or a normal diet (D+, n = 8). After 6 weeks the mean plasma calcium concentrations were 6.1; 7.0; 9.8; and 10.4 mg/dl, respectively. In the vitamin D-deficient rats (groups D-, D-Ca+, D-Ca+lac) plasma calcium was correlated with bone length, bone ash, volumetric density of osteoid in the metaphysis of the tibia, and volumetric density of trabecular bone in the same bone section. In the D-Ca+lac group these bone parameters approximated the values of the D+ group, but were still significantly lower. It is concluded that in vitamin D-deficient rats longitudinal bone growth, bone mineral content and bone histomorphometry can be brought close to normal by supplying additional dietary calcium with lactose, without vitamin D repletion. The study does not exclude the possibility that residual amounts of vitamin D are required to obtain this effect.

The economics and evaluation of dental care and treatment.

This paper is concerned with economic evaluation in dentistry. The potential for such evaluation is great, but has not been fully realised to date. A number of issues which are common to the existing literature are discussed, and particular attention is paid to the question of measuring dental health in economic appraisal. Directions for future research are presented. The paper concludes that the future for economic evaluation in dentistry is favourable and that there is a need for greater collaboration between economic and dental researchers in this area.

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.