Development of capture assays for different modifications of human low-density lipoprotein

Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), Nepsilon(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r=0.706, P<0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.     

Rapid differentiation of the major causative agents of bacterial meningitis by use of frequency-pulsed electron capture gas-liquid chromatography: analysis of amines.

The major causative agents of bacterial meningitis (Haemophilus influenzae serogroup B, Neisseria meningitidis serogroups B and C, Klebsiella pneumoniae, Steptococcus pneumoniae, and two types of Escherichia coli) were cultured in a chemically defined medium, and selected strains were further studied in Todd-Hewitt medium. After acidic extraction of the spent media with chloroform, a basic extraction was made with chloroform to obtain amines. A third extraction was performed on re-acidified Todd-Hewitt medium with ethyl ether to obtain hydroxyacids. The extracts were derivatized with heptafluorobutyric anhydride-ethanol to form electron-capturing derivatives, and the derivatives were analyzed on a frequency-pulsed electron capture gas-liquid chromatograph (FPEC-GLC) equipped with a PEP-2 computer. The data obtained from the study showed that amines were produced by these organisms that formed characteristic patterns. Different serotypes of K. pneumoniae and the two serogroups of N. meningitidis produced different types of FPEC-GLC profiles within serotypes. E. coli produced several hydroxy acids on Todd-Hewitt medium that made it unique among the organisms studied. The methods used are practical and the techniques have potential for use in clinical laboratories and hospitals as a valuable aid for the rapid identification of the major causative agents of bacterial meningitis.     

The effect of transfected MHC class I genes on sensitivity to natural killer cells

To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line F10 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC.     

Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology

It is well established in animal models that production of the cytokine tumour necrosis factor-alpha (TNF-alpha) is essential to the proper expression of acquired specific resistance following infection with Mycobacterium tuberculosis. This gives rise to an apparent state of chronic disease which over the next 100-200 days is characterized by slowly worsening pathological changes in the lung. To determine whether continued TNF-alpha production was harmful during this phase mice were treated with a TNF-alpha inhibitor, pentoxifylline. It was observed that although this therapy did not alter the numbers of bacteria recovered from the lungs of the infected mice, tissue damage within the lung was accelerated. These data thus demonstrate that production of TNF-alpha, already known to be important during the early expression of resistance to tuberculosis, remains important and beneficial during the chronic stage of the disease.     

Ventilatory control studied with circulatory occlusion during exercise recovery

Summary Mechanisms involved in the control of pulmonary ventilation were studied in seven male subjects following 6 min of exercise on a cycle ergometer at 98w. Circulation to the legs was occluded by thigh cuffs (27 kPa) during the last 15 s of exercise and the subsequent 4 min of recovery. Respiratory gas exchange and the tidal partial pressures of O₂ and CO₂ were measured breathby-breath. The results were compared to control studies without occlusion. There was a significant increase in both systolic and diastolic blood pressures during occluded recovery. Following occlusion systolic pressure remained elevated while diastolic pressure returned to control values. Occlusion during recovery caused hyperventilation during the first 1.5 min after exercise as evidenced by significantly higher , P_ETO₂, and lower P_ETCO₂. Following the release of the cuffs P_ETCO₂, , and heart rate all increased significantly above control values, while P_ETO₂ decreased. P_ETCO₂ rose abruptly 14.5±0.9 s after the release of the cuffs. Marked increases inV _E and heart rate were seen, and occurred 30.8±1.5 s and 12.8±1.3 s, respectively, after cuff release. The 16.3±1.4 s lag between the increase in P_ETCO₂ and after occlusion suggests that the ventilatory response to a sudden load of hypercapnic blood is not mediated by a pulmonary chemoreceptor. Other receptors, probably the peripheral chemoreceptors, appear to be responsible for hypercapnic hyperventilation.     

Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat plantaris muscle

All three forms of recombinant low voltage-activated T-type Ca²⁺ channels (Ca_v3.1, Ca_v3.2 and Ca_v3.3) exhibit a small, though clearly evident, window T-type Ca²⁺ current ( I _Twindow) which is also present in native channels from different neuronal types. In thalamocortical (TC) and nucleus reticularis thalami (NRT) neurones, and possibly in neocortical cells, an I _Twindow-mediated bistability is the key cellular mechanism underlying the expression of the slow (< 1 Hz) sleep oscillation, one of the fundamental EEG rhythms of non-REM sleep. As the I _Twindow-mediated bistability may also represent one of the cellular mechanisms underlying the expression of high frequency burst firing in awake conditions, I _Twindow is of critical importance in neuronal population dynamics associated with different behavioural states.     

Serum anti-Mullerian hormone levels during controlled ovarian hyperstimulation in women with polycystic ovaries with and without hyperandrogenism

BACKGROUND: Anti-Mullerian hormone (AMH) is expressed in pre- and small-antral follicles. High serum levels are found in women with polycystic ovaries (PCO), accordant with their increased content of small follicles. To evaluate the relationship between AMH, folliculogenesis and hyperandrogenism, we compared serum AMH levels between women with PCO with and without hyperandrogenism and normal controls during controlled ovarian hyperstimulation (COH). METHODS: Nineteen women with PCO and hyperandrogenism (group A), 10 women with PCO but no hyperandrogenism (group B) and 23 ovulatory women with normal ovarian morphology (group C, controls) underwent COH with the long protocol. Serum levels of AMH, estradiol, androstenedione and follicular tracking were determined before gonadotropins treatment (day 0) and every 2-4 days up to the day of HCG administration. RESULTS: AMH levels declined gradually throughout COH in the three groups, but remained higher in groups A and B compared with the controls. Significantly higher levels were found in group A compared with group B, despite comparable numbers of small follicles. Multiple regression analysis revealed that both the number of small follicles and serum androgens were correlated to AMH. CONCLUSIONS: Women with PCO have higher serum AMH levels during COH than controls. Hyperandrogenism is associated with an additional increase in AMH. It is conceivable that hyperandrogenism may reflect more severe disruption of folliculogenesis in women with PCO or may affect AMH secretion.     

The pattern of function-related regional cerebral blood flow investigated by single photon emission tomography with 99mTc-HMPAO in patients with presenile Alzheimer's disease and Korsakoff's psychosis

Single photon emission tomography (SPET) with the lipophilic blood flow marker 99mTc-hexamethyl propyleneamine oxime (99mTc-HMPAO) has been used to determine regional uptake of radiolabel into brain regions of patients with presenile Alzheimer's disease and Korsakoff's psychosis, and age-matched controls. Using occipital cortical uptake as reference area, the pattern of relative regional cerebral blood flow (rCBF) was determined in other cortical areas and basal ganglia. In Alzheimer's disease, reduction in rCBF occurred most strikingly in posterior temporal and parietal areas. By contrast, in Korsakoff's psychosis, posterior temporal rCBF was maintained, although there was a trend to reduced tracer uptake in other cortical areas. These impairments of flow were correlated with impairments of neuropsychological function. In Alzheimer's disease, left posterior temporal and left parietal regions in particular showed rCBF to be strongly correlated with most aspects of cognitive function. In Korsakoff's psychosis, however, impaired flow in frontal regions was correlated with impaired performance on tests of memory and orientation. The findings in Alzheimer's disease show quantitative parallels with those from studies using Positron Emission Tomography (PET), and extend our understanding of the relationship between cognition and regional brain function in dementia. The findings in Korsakoff's psychosis offer the first direct evidence linking frontal lobe dysfunction with the cognitive impairment seen in the disorder.     

A pharmacological and histological examination of the microcirculation of the rat subcutaneous air-pouch: microcirculation of the rat air-pouch

The effects of histamine, 5-hydroxytryptamine (5-HT) and prostaglandin E2 (PGE2) on plasma protein extravasation in the rat subcutaneous air-pouch have been studied. Both histamine and 5-HT produced increases in plasma protein extravasation which were inhibited by specific receptor antagonists. Plasma protein extravasation induced by PGE2 was partially inhibited by either a 5-HT receptor antagonist (methysergide) or by a combination of H1 and H2 receptor antagonists (mepyramine and cimetidine). A combination of all three antagonists further reduced plasma protein extravasation. These results suggest that PGE2 increases vascular permeability indirectly via the degranulation of mast cells. This supposition was confirmed by histological evidence of extensive mast cell degranulation following the injection of PGE2 but not following histamine, 5-HT or saline injection. Using a technique of vascular labelling, following the intravenous injection of Monastral blue dye, plasma extravasation induced by histamine, 5-HT or PGE2 was observed to be restricted to post-capillary venules and was not observed in arterioles or capillaries. Electron microscopic examination of the tissue revealed the presence of monastral blue particles trapped between endothelial cells. These findings suggest that the microcirculation of the rat subcutaneous air-pouch behaves in an analogous manner to that of other tissues.     

An investigation into the relationship between salivary cortisol,stress, anxiety and depression

This study examined the relationship between indices of self-reported emotional distress and absolute versus change in cortisol levels. Fifty-four women attending a diagnostic breast clinic completed scales measuring stress, anxiety and depression and provided five saliva samples over the course of a single day for the measurement of cortisol. No significant relationships were evident between absolute cortisol levels and the distress measures. Analysis of the change in cortisol levels revealed a non-linear interaction effect between stress and anxiety and time of day. There was a non-linear relation between time of day and cortisol levels, but the extent of the non-linearity was dependent upon levels of stress and anxiety, not depression. A relationship was apparent between indices of distress and change in cortisol levels, but not absolute levels of the hormone.