[Health-care research from the German Medical Association's perspective on small-area analysis]. / Versorgungsforschung aus Sicht der Bundesärztekammer unter Berücksichtigung kleinräumiger Analysen.

As early as 2003, the German medical profession realized the necessity of not only forwarding medical research, but also analyzing the process of health care itself. Approved by a decision of the 108th German Medical Assembly in 2005, an initiative on health-care research paid by contributions of the medical profession was launched. Since then several projects have been supported with the results being published continuously. From the perspective of the German Medical Association, the success of the initiative also proves the effective approach of the scientific and medical communities' self-administration. Although the current results from health-care research can be used to support health-care politics and decision making at a macro level, a focus on small-area analysis tends to be an intrinsic attribute of health-care research, keeping a local approach toward changes so as to obtain real effects. Without local settings and without data reflecting the local situation, the"last mile" of a health-care system, which is the core subject of health-care research, will not be comprehensible.

Endothelin-1 synthesis and endothelin B receptor expression in human coronary artery smooth muscle cells and monocyte-derived macrophages is up-regulated by low density lipoproteins.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL.

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis.

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release.

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.

Separation of lipoproteins by capillary isotachophoresis combined with enzymatic derivatization of cholesterol and triglycerides.

Combining specific enzymatic derivatization of cholesterol or triglycerides with capillary isotachophoresis (CITP), human serum lipoproteins are separated into 14 lipoprotein subfractions, monitored and quantitated by direct capillary UV detection. By comparing the separation patterns of human serum with the patterns of lipoprotein particles isolated by sequential ultracentrifugation it became evident that peaks 1-5 represent lipoproteins of the high density lipoprotein (HDL) fraction, peaks 6-8 embody the very low density lipoprotein (VLDL) fraction and chylomicrons, and peaks 7-14 represent the low density lipoprotein (LDL) fraction. Peaks 7 and 8 were found in the VLDL as well as in the LDL fraction. Using triglyceride-specific staining peaks 6-8 occurred prominently; and with cholesterol-specific staining, peaks 1-5 and 7-14 were prominent. The coefficient of variation, for the sum of the peak heights of a pooled serum, was 3.94 for triglyceride-specific staining and 2.32 for cholesterol-specific staining. A linearity range between 0.23 and 2.29 mM/L was found for triglyceride-specific staining and between 0.043 and 4.33 mM/L for cholesterol-specific staining. The practicability of the method was evaluated (i) using blood of humans before and 45 min after an oral fat load. Triglyceride-specific staining revealed a prominent increase in the VLDL fraction and chylomicrones containing peaks 6 and 7, and a minor increase in the HDL fraction containing peaks 3 and 4, and (ii) in patients with manifest hypothyroidism before and after thyroxine therapy. Cholesterol-specific staining demonstrated a massive decrease in the first peak of the HDL fraction and in peaks 9 and 11 of the LDL fraction regarding the hypo versus hyperthyroid state.

[Quality assessment of medical care--a standardized scheme for the development of quality indicators]. / Qualitätsbestimmung in der medizinischen Versorgung--ein universelles Entwicklungsschema für Qualitätsindikatoren.

A reliable and valid assessment of the quality of medical interventions is an indispensable prerequisite for any initiatives targeting at quality improvement in the health system. Quality indicators are well suited tools for such tasks, e.g. in the setting of a continuous monitoring. In the German health system, previous experiences concerning the use of quality indicators are limited. Available knowledge from medical services of other nations is mainly focused on the hospital sector. Therefore, it appears to be desirable to be able to provide a highly universal and standardized way for the definition of indicators of quality, enabling measurements of performance in any kind of health sector or disease treatment. Based on the demand for continuous quality monitoring in the sector of outpatient care recognized by the Central Institute of Panel Physicians, an indicator development scheme is demonstrated.

Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells.

Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 microg/ml oxLDL. Higher oxLDL concentrations (>25 microg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 microg/ml ascorbic acid, 25, 50 and 100 microg/ml oxLDL induced apoptosis within 6-8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

Active specific immunotherapy of renal cell carcinoma: cellular and humoral immune responses.

We investigated the effect of vaccinating renal cell carcinoma (RCC) patients with irradiated autologous or allogeneic tumor cells and Newcastle disease virus (NDV) as adjuvant on cellular and humoral antitumor immunity. By Western blot analysis, we found that vaccination induced antibody formation in 33 of 34 patients against NDV proteins but not against tumor cell related antigens. NDV proteins detected had molecular weights of 53 kDa, 55-56 kDa, and 66 kDa. ADCC by patients' isolated PBMC and patients' sera against autologous or allogeneic tumor cells was not enhanced after vaccine treatment in a nonradioactive cytotoxicity assay. Target cells infected with NDV were lysed more effectively (p < 0.05) in ADCC after vaccination than noninfected targets. Natural cellular cytotoxicity of patients' isolated PBMC was not altered during vaccine treatment. Specific lysis rates against autologous and allogeneic RCC cells not exceeded 10% (effector:target ratio 50:1). Specific lysis of K-562 cells was > 20%; a slight decrease in lysis during vaccination was not significant. Numbers of lymphocyte subsets from patients' peripheral blood analyzed by FACS revealed significant expression of CD20+ (p < 0.02) and CD39+ (p < 0.03) cell numbers by vaccine therapy. Cytokine detection in patients' sera by ELISA showed significant increases (p < 0.05) for IFN-gamma and TNF-alpha but not for IFN-alpha four h post vaccination. Thus, immunomodulation with autologous or allogeneic RCC tumor cell vaccines is mainly due to cytokine induction, whereas tumor specific humoral or cellular responses are not detectable in patients' peripheral blood.

Platelet endothelial cell adhesion molecule-1 (PECAM-1): a potential prognostic marker involved in leukocyte infiltration of renal cell carcinoma.

We investigated immunohistochemically the leukocyte infiltrate [CD3, CD4, CD8, CD11a, CD11b, CD14, CD56, VLA-4 and platelet endothelial cell adhesion molecule-1 (PECAM-1)] and the endothelial expression of cell adhesion molecules (PECAM-1, VCAM-1, ICAM-1 and ICAM-2) in 23 renal cell carcinoma tumor tissues. Tumors with a moderate or high density of PECAM-1 positive endothelia showed a stronger infiltration with PECAM-1-positive leukocytes as compared to tumors with a low density of positive endothelia (p<0.0085). Additionally, overall survival of patients who presented with tumors exhibiting a moderate or high density of PECAM-1 endothelia alone or in combination with a PECAM-1-positive infiltrate was extended (median survival: 23.5 months) as compared to patients without these tumor characteristics (median survival: 6.5 months). These results suggest an involvement of PECAM-1 in the process of leukocyte migration and a potential role as a prognostic marker in renal cell carcinoma.

Lymphocyte-conditioned medium in combination with interleukin-2 effectively induces antitumour autoimmunity by adoptive transfer of short activated killer (SHAK) cells.

In this study, effective antitumour immunity was transferred by autologous short activated killer (SHAK) cells induced over four hours with lymphocyte conditioned medium (LCM) and recombinant interleukin-2 (rIL-2). Among eight patients with progressive metastatic renal cell carcinoma refractory to standard therapy, there were six objective tumour responses to SHAKs. Progression-free survival ranged from 0 to 8+ months, and overall survival ranged from 2 to 14+ months, with a median of 9+ months. Systemic toxicity of SHAKs was limited to flulike symptoms. Patient SHAKs provided a tumour-specific immunity, both cellular and humoral (expression and secretion of secondary cytokines, including IL-2, GM-CSF, INF-gamma and TNF-alpha), far superior to rIL-2 activated killer cells.

Soluble interleukin 2 receptors abrogate IL-2 induced activation of peripheral mononuclear cells.

Soluble interleukin 2 receptors (sIL-2R) exert a potential role in immunoregulation. We investigated the in vitro effects of sIL-2R on several interleukin 2 (IL-2)-dependent cellular events. Cytotoxicity of human rIL-2-stimulated PBMC against K562 and Daudi was correlated inversely to the concentration of sIL-2R in the culture medium during rIL-2 stimulation. sIL-2R concentrations higher than 4.0 pM produced a significant loss of cytotoxicity (P < 0.01). The effect of different sIL-2R concentrations added to cultured human PBMC on secondary sIL-2R production was tested by ELISA. Secondary sIL-2R production was abrogated by high initial sIL-2R dosages whereas low initial dosages were followed by a continuing production of secondary sIL-2R after five days of culture. Proliferation of the IL-2-dependent mouse cell line CTLL-2-was suppressed by sIL-2R added to the culture medium in a dose-dependent way. The neutralizing capacity of sIL-2R strongly depended on the initial number of CTLL set in per proliferation assay. In contrast, variation of rIL-2-concentration had no significant effect on reduction of proliferation by sIL-2R. Furthermore, preincubation of sIL-2R with rIL-2 did not enhance growth suppression. These last findings indicate that there is at least no functional interaction between sIL-2R and free IL-2, whereas an interaction of sIL-2R with the membrane-bound receptor for IL-2 seems possible.

Induction of cytokines and cytotoxicity against tumor cells by Newcastle disease virus.

The use of NDV as biological adjuvant in vaccines against human cancer is still actual in several clinical treatment protocols. In this study, we have investigated in vitro-effects of Newcastle disease virus (NDV) strain 73-T on isolated mononuclear blood cells and cultured tumor cells. Cellular cytotoxicity of PBMC freshly isolated from healthy donors against tumor cells was enhanced significantly (p < 0.01) after coincubation of NDV with effector cells. NDV failed to enhance cytotoxicity of effector cells when PBMC were stimulated three days with 500 IU recombinant interleukin-2 (rIL-2) per ml prior to coincubation with the virus. No significant enhancement of cellular lysis was seen when only target cells were coincubated with NDV. As shown by depletion of various lymphocyte subsets, NK cells were the predominant mediator of lysis. Enhancement of cytotoxicity correlated with the induction of interferon-alpha (IFN-alpha) in PBMC by NDV. NDV also induced high amounts of tumor necrosis factor-alpha (TNF-alpha) in PBMC. Induction of interferon-gamma (IFN-gamma) was weak. A direct cytopathic effect (CPE) of NDV on different target cells was detected by colorimetric measurement of metabolic cell activity. The human tumor cell lines A-498, A-704, Caki-1, Caki-2, and K-562 and the fibroblast line MRC-5 showed progressive cellular destruction 48 h after infection with NDV, whereas PBMC and Daudi cells remained unaffected during the observation period. The nontransformed monkey kidney cell line CV-1 and the transformed monkey kidney cell line COS-1 were both lysed by NDV with marginal difference in time course of CPE. Our results indicate a reasonable potential of pleiotropic modifications of the immune response against tumors by NDV.

Fine structure of identified neurons in the primate hippocampus: a combined Golgi/EM study in the baboon.

The hippocampi of two 1-year-old female baboons (Papio anubis) were used for a combined Golgi/electron microscope (EM) study of characteristic cell types in the hippocampus proper and fascia dentata. Results were compared with previous Golgi/EM studies of hippocampal neurons in small laboratory animals. Cell bodies of pyramidal neurons in CA1 were more loosely distributed than known from studies on the rat or guinea pig. Numerous basal and horizontal dendrites originating from the perikaryon filled in the space between neighboring cell bodies. Apical stem dendrites were varying in length, depending on the position of the parent cell body in outer or inner portions of the pyramidal layer. Dendrites were densely covered with spines which in the EM showed very complex synaptic contacts. In contrast to our observations in rats and guinea pigs, CA3 pyramidal cells in the monkey hippocampus exhibited numerous large spines or excrescences not only on apical dendrites but also on basal dendrites running through stratum oriens. These excrescences appeared to be more complex than in small rodents. They often branched, protruding deeply into presynaptic mossy fiber boutons, and formed multiple asymmetric synaptic contacts. Granule cells of the monkey fascia dentata, in contrast to those of the rodent, occasionally had basal dendrites extending into the hilar region. In the EM, granule cells either with or without basal dendrites exhibited fine structural characteristics that were very similar to those described in Golgi/EM studies of granule cells in the rat fascia dentata. Of the various types of nonpyramidal neurons the horizontal cells in stratum oriens with dendrites parallel to the alveus were analyzed. As seen in rats, these cells exhibited large amounts of rough endoplasmic reticulum, indentations of the nuclear membrane, and nuclear inclusions. Numerous terminals formed synaptic contacts on dendritic shafts. In contrast to rodents, numerous spines arose from dendrites and cell bodies of these neurons. In the EM, often single spines were found to establish synaptic contacts with several presynaptic boutons. In summary, our correlated light and EM study of four characteristic cell types, which are present in both nonprimates and primates, demonstrates a much more complex dendritic pattern and synaptic organization of these neurons in primates than in commonly studied small laboratory animals.