Cerebrospinal fluid-filtration reduces TNF alpha in bacterial meningitis-CSF

A 37 year old male was admitted with the diagnosis of bacterial meningitis. Pneumococci were seen in the Gram stain of the cerebrospinal fluid. The clinical condition did not suggest severely raised intracranial pressure, there were no localizing signs and symptoms. CSF was turpid, with 20.100/3/mm³, mainly polymorphonuclear cells. Tumor necrosis factor alpha in CSp was greatly increased with 813 pg/ml. Parallel to the application of intravenous Penicillin G a CSF filtration was carried out. Within 214 h 225 ml CSF were filtrated through a Pall-filter, using a bidirectional pump. Cell count dropped to 720/3 cells/mm³, TNF-alpha to 39 pg/ml. The clinical course was uneventful, on day 12 the patient could be discharged without sequelae. CSF filtration may be a highly effective method to reduce from the CSF pathogenetically important cytokines, such as TNF-alpha, being responsible for intrathecal/meningeal inflammatory processes and triggered by cell-wall components of bacteria, e.g. pneumococci.     

Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses.     

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells

The vitamin D₃ derived hormone 1,25 (OH)₂ vitamin D₃ (1,25 D₃) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1,25 D₃ reduced proliferation as measured by ³H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D₃ 65% inhibition was achieved at 48 n M . The MC 1288 stereoisomer of 1,25 D₃ was more potent and had the same activity at 48 n M .
The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4.
The antiproliferative effect of liposomal 1,25 D₃ was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33.
When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)₂ vitamin D₃ may offer a novel approach to treatment of myelomonocytic leukaemia.     

Pharmacokinetics of the acetylcholinesterase oxime reactivator, HI-6, in rhesus monkeys (Macaca mulatta): effect of atropine, diazepam, and methoxyflurane anesthesia

The pharmacokinetics of the bispyridinium oxime HI-6 (CAS reg. no. 34433-31-3; 1-(((4-aminocarbonyl)pyridinio)methoxy)methyl)-2-[hydroxy i mino)methyl)- pyridinium dichloride) was investigated in rhesus monkeys (Macaca mulatta). The effects of methoxyflurane anesthesia, administration of atropine with and without diazepam were determined on the serum half-life (t1/2), clearance rate (CL), and the volume of distribution (Vd) following intramuscular (IM) administration of HI-6 (30 mg kg-1). The control t1/2, CL and Vd of HI-offere 27 min, 8.6 ml min-1 kg-1 and 0.34 l kg-1, respectively. These parameters were unaffected by the co-administration of either atropine (0.5 mg kg-1, IM) or atropine and diazepam (0.5 mg kg-1, IM + 0.2 mg kg-1 IV, respectively). Methoxyflurane anesthesia resulted in a significant increase in the HI-6 t1/2 to 61 min concomitant with a decrease in the CL to 4.1 ml min-1 kg-1 with no change in the Vd. The increase in the t1/2 of HI-6 in methoxyflurane anesthetized monkeys is probably the result of a decrease in the clearance rate and, thus, excretion of HI-6 by the kidneys.     

Are hypodense eosinophils in children activated or immature?

In patients with allergic asthma and rhinitis high numbers of hypodense eosinophils (HE) have been demonstrated. In a previous study we reported that asthmatic and healthy children had more HE than their adult counterparts. We assumed that this might, in part, he due to the presence of immature eosinophils in children. To distinguish between immature and activated eosinophils, determination of eosinophil cationic protein (ECP) might be interesting as it is known that high serum levels of ECP are associated with increased activation of eosinophiis. In this study we determined (he levels of ECP in scrum in asthmatic and healthy children and adults trying to distinguish activated from immature eosinophils. We found that ECP levels were not increased in children (healthy and asthmatic) compared to adults (healthy and asthmatic). This supports the hypothesis that increased numbers of HE in childhood are, at least in part, immature eosinophils. Nevertheless, we could confirm that inflammation was present in children because soluble interleukin-2-receptor (slL-2R), a marker of lymphocyte activation, was higher in asthmatic children as compared to healthy children. IL-6, a marker of macrophage/monocyte activation, was not different in the different patient groups. We conclude that although signs of inflammation are present in childhood asthma, the increased numbers of HE in children are in part due to the presence of immature eosinophils.     

The in vivo microdialysis recovery of dopamine is altered independently of basal level by 6-hydroxydopamine lesions to the nucleus accumbens

The present study was designed to test the hypothesis that the active neurotransmitter processes of release and uptake affect the in vivo microdialysis recovery of dopamine (DA) in the nucleus accumbens (N ACC) of the rat. The in vivo recovery for DA was established for rats which had received either unilateral infusions of the neurotoxin 6-hydroxydopamine (6-OHDA, 8 μg) or vehicle (0.2 μg ascorbate). In the quantitative dialysis method used (point of no net flux method), DA is added to the perfusate at concentrations above and below the expected extracellular concentration (0, 5, 10 and 20 nM) and DA is measured in the dialysate from the brain to generate a series of points. A linear fit is performed, the slope of which is the in vivo recovery of the dialysis probe. The in vivo recovery of the 6-OHDA group was 30 ± 3% which was significantly lower (P < 0.002) than the in vivo recovery of the control group which was 60 ± 3% (mean ± SEM; n = 6/group). The zero intercept of this regression is the point of no net flux, which is the extracellular concentration of DA independent of the probe sampling characteristics. The extracellular DA concentration for the 6-OHDA group was 7.8 ± 1.1nM, which was not significantly different than the control group which was 6.9 ± 0.7nM. The tissue DOPAC/DA ratios of the 6-OHDA lesioned hemispheres were significantly higher than the contralateral hemispheres of the same animals (0.62 ± 0.1vs.0.27 ± 0.1; P < 0.02) while the DOPAC/DA ratios in the control group were not significantly different (0.24 ± 0.1vs.0.27 ± 0.1). The fractional DA efflux from the terminals in the 6-OHDA group was significantly higher than the fractional DA efflux of the control group (0.52 ± 0.08vs.0.03 ± 0.003; P < 0.0001), indicating that the remaining terminals have increased turnover of DA. Despite the increased turnover, however, the number of remaining release and uptake sites are not sufficient to maintain the high in vivo recovery observed in the control group.     

Repair of DNA lesion O 6-methylguanine in hepatocellular carcinogenesis

Evidence from both experimental carcinogenesis and studies in human cirrhotic liver suggest that defective repair of the promutagenic DNA base lesion, O ⁶-methylguanine, is a factor in the multistep process of hepatocellular carcinogenesis. Ubiquitous environmental alkylating agents such as N-nitroso compounds can produce O ⁶-methylguanine in cellular DNA. Unrepaired, O ⁶-methylguanine can lead to the formation of G ? A transition mutations, a known mechanism of human oncogene activation and tumour suppressor gene inactivation. Combined treatment of rodents with an agent producing O ⁶-methylguanine in DNA, and an agent promoting cell proliferation, leads to development of hepatic nodules and hepatocellular carcinoma (HCC), cell division, hence DNA replication, being required for the propagation of tumorigenic mutation(s) in hepatocyte DNA. The paramount importance of O ⁶-methylguanine in hepatocellular carcinogenesis is indicated by the observation that transgenic mice engineered to have increased hepatic levels of repair enzyme O ⁶-methylguanine-DNA methyltransferase (MGMT) are significantly less prone to hepatocellular carcinogenesis following alkylating agent treatment. Cirrhosis is a universal risk factor for development of human HCC, and a condition that is characterized by increased hepatocyte proliferation as a result of tissue regeneration. Levels of the human repairing enzyme for O ⁶-methylguanine were found to be significantly lower in cirrhotic liver than in normal tissue. In accord with findings from animal models, this suggested a mechanism in which persistence of O ⁶-methylguanine due to defective DNA repair by MGMT, together with increased hepatocyte proliferation, might lead to specific gene mutation(s) and hepatocellular carcinogenesis. Screening for the presence and persistence of O ⁶-methylguanine in human DNA presently involves formidable technical difficulty. Indications are that such limitations might be overcome by the use of an ultrasensitive method such as immuno-polymerase chain reaction (PCR). This approach should allow parallel measurement of DNA adduct and repair enzyme in routine liver biopsy samples. It might also enable investigation of O ⁶-methylguanine in human genes specifically associated with hepatocellular carcinogenesis. Given the wide variation in human MGMT levels observed between individuals, tissues, and cells, this technology should be adapted to permit the ultrasensitive localisation and measurement of adducts and repairing enzyme in liver biopsy tissue sections. Ability to ultrasensitively measure O ⁶-methylguanine, and its repair enzyme, should prove valuable in the risk assessment of cirrhotic patients for developing hepatocellular carcinoma. Received for publication on July 6, 1998; accepted on Aug. 12, 1998     

Are cytokines possible mediators of cancer cachexia?

The possible role of cytokines in the development of cancer cachexia was reviewed from the literature. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, interferon (IFN)-gamma and leukemia inhibitory factor (LIF) can elicit many but not all host changes seen in cancer cachexia, including loss of appetite, loss of body weight, and the induction of acute-phase protein synthesis. However, these cytokines are not always demonstrated in the circulation of the cancer patients. The inability to detect circulating cytokines may be due to their low rate of production, their short half-life and rapid clearance from plasma, or their mode of action (autocrine or paracrine). Different cytokines are induced to stimulate the same response. This is very different from hormonal regulation, where a hormone acts on a cell directly through a specific receptor without depending on other mediators. Specific antibodies including anti-IFN-gamma, anti-TNF and anti-IL-6 antibodies, as well as the cyclooxygenase inhibitor indomethacin, have been used to reverse cancer cachexia. Overlapping physiologic activities make it unlikely that a single substance is the sole cause of cancer cachexia. It is hoped that further investigation on other cytokines and their possible relationships with hormones will help to clarify the mechanisms of cancer cachexia in the near future.This work was supported by a grant from the Japan-Sweden Foundation in 1991.     

Differing time courses between Δlactate and mitochondrial respiration during coronary occlusion and after reperfusion in canine hearts

Summary The present study was designed to clarify whether or not a difference between arterial and venous lactate (lactate) levels is useful for evaluation of mitochondrial function in ischemia-reperfused myocardium. In the first experiment, 12 dogs were divided into 2 groups: 10-min occlusion of the left anterior descending coronary artery (LAD) followed by 10-min reperfusion, or 30-min occlusion followed by 40-min reperfusion, were performed. The lactate levels in the femoral artery and the great cardiac vein were measured enzymatically. Lactate was reversed immediately after occlusion. Ten min and 20 min were required for the recovery of lactate in the 10-min-occlusion with 10-min-reperfusion, and 30-min-occlusion with 40-min-reperfusion groups, respectively. In the second experiment, 36 dogs were divided into 6 groups: 10-min occlusion of LAD; 10-min occlusion with 10-min reperfusion; 30-min occlusion; and 30-min occlusion with 10-, 20-, or 40-min reperfusion were performed. Mitochondria from normal and occluded or reperfused areas were prepared, and the respiratory function of the mitochondria was measured polarographically. No significant decreases in the mitochondrial function were observed in the 10-min-occlusion, and 10-min-occlusion with 10-min-reperfusion groups. On the other hand, respiratory function of mitochondria was impaired by 30-min occlusion and was not improved by 10- or 20-min reperfusion. Significant recovery in the mitochondrial function was observed after 40-min reperfusion. That is, differing recovery time courses between lactate and the mitochondrial function were observed.     

Monoamine oxidase-B inhibition: a comparison of in vivo and ex vivo measures of reversible effects

Summary A behavioural test involving potentiation of the effects of an acute injection of -phenylethylamine (10 mg kg^–1 i.p.) was used to assess the time-course of type-B MAO inhibition after administration of (–)deprenyl (5 mg kg^–1 i.p.) and of MD 240928 (20 mg kg^–1 i.p.) respectively. Potentiation of the effects of -phenylethylamine was observed 1 h after injection of (–)deprenyl or MD 240928. This effect was still evident 120 h after administration of (–)deprenyl but not 24 h after administration of MD 240928. Comparisons of ex vivo estimates of MAO activity yielded a corresponding time-course for the recovery of this enzyme. The extent of MAO inhibition required for potentiation of the effects of -phenylethylamine was inferred from a comparison of the behavioural test results and the ex vivo MAO activity observed after (–)deprenyl administration. These comparisons indicate a significant underestimation of MD 240928-induced MAO inhibition using ex vivo measures. This underestimation is interpreted as evidence fordilution effects in the ex vivo assay of MAO inhibition. The potentiation of effects of -phenylethylamine under the present conditions is proposed as a useful and simple test for effects of reversible type-B MAO inhibitors.