Ralf Lichtinghagen

Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain.

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunit expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit‐specific sorting and segregation. This was investigated by studying in detail the expression of six different α‐subunits of voltage‐gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five α‐subunits (KV1.1, KV1.2, KV1.3, KV1.4, KV1.6) of the Shaker‐related subfamily of rat Kv channels, which encode delayed‐rectifier type and rapidly inactivating A‐type potassium channels. Their distribution was compared to that of an A‐type potassium channel (KV3.4), belonging to the Shaw‐related subfamily of rat Kv channels. Our results show that these Kv channel α‐subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of Kv channel α‐subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different Kv channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of Kv1 α‐subunits are co‐localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of Kv channel α‐subunits may contribute to Kv channel diversity and thereby to presynaptic and postsynaptic membrane excitability.

Effects of a cinnamon extract on plasma glucose, HbA1c, and serum lipids in diabetes mellitus type 2

Background  According to previous studies, cinnamon may have a positive effect on the glycaemic control and the lipid profile in patients with diabetes mellitus type 2. The aim of this trial was to determine whether an aqueous cinnamon purified extract improves glycated haemoglobin A1c (HbA1c), fasting plasma glucose, total cholesterol, low‐density lipoprotein (LDL), high‐density lipoprotein (HDL) and triacylglycerol concentrations in patients with type 2 diabetes.

Diagnostic potential of circulating TIMP-1 and MMP-2 as markers of liver fibrosis in patients with chronic hepatitis C.

BACKGROUND Circulating levels of tissue inhibitor of metalloproteinase (TIMP)-1 and matrix metalloproteinase (MMP)-2 are investigated as parameters for the diagnosis of fibrosis in chronic liver disease. We evaluated their diagnostic potential in comparison to hepatic histology, serum hyaluronate and standard liver function tests. METHODS Commercially available ELISA assays were used to study circulating values of TIMP-1 and MMP-2 (Bindazyme, Biotrak, Quantikine) in patients with chronic hepatitis C (CAH; n=59), hepatitis C virus-induced cirrhosis (n=19) and 30 healthy controls. Hepatic histology was evaluated using the Hepatitis-Activity-Index according to Ishak et al. [J. Hepatol., 22 (1995) 696-699], quantifying separately inflammatory activity and fibrosis. RESULTS Normal ranges for TIMP-1 and MMP-2 values differed for the different assays. Nevertheless, the various assays showed similar diagnostic ability and linear correlation. MMP-2 values were similar in controls and in CAH patients with and without fibrosis, but increased significantly in cirrhosis. TIMP-1 values showed a steady increase from normal to CAH without fibrosis, hepatitis with fibrosis, and cirrhosis. The diagnostic potential of serum MMP-2 to detect fibrosis was low with a sensitivity of 7% in the two assays used and an overall diagnostic efficiency of 56% and 58%. The potential of circulating MMP-2 to detect cirrhosis was higher with sensitivities of 74% and 83% and specificities of 96% and 100%, resulting in a diagnostic efficiency of 92% in the different assays. Plasma TIMP-1 values detect fibrosis with a sensitivity of 52% and 67% and a specificity of 68% and 88% resulting in overall efficiency rates of 68% and 71%, respectively. TIMP-1 values detect cirrhosis with 100% sensitivity but only 56% and 75% specificity. The diagnostic potential of circulating TIMP-1 was similar to that of hyaluronate and better than that of enzymes or albumin values. CONCLUSION Plasma values of TIMP-1 and MMP-2 are able to detect cirrhosis with high sensitivity. TIMP-1 values also detect fibrosis with comparable efficiency. Regular determinations of both TIMP-1 and MMP-2 in CAH patients may be used as indicators of increasing fibrosis and the development of cirrhosis.

Different mRNA and protein expression of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 in benign and malignant prostate tissue

OBJECTIVE The aim of this study was to assess the behavior of the matrix metalloproteinases (MMPs) 2 and 9 and the tissue inhibitor of metalloproteinases 1 (TIMP-1) in human prostate cancer. METHODS mRNA and protein expression patterns of MMP-2, MMP-9, and TIMP-1 were studied in cancerous and noncancerous parts of 17 prostates removed by radical prostatectomy. Competitive RT-PCR, gelatin-substrate zymography, and ELISA techniques were used for quantification. RESULTS On the mRNA level, MMP-2 expression was decreased and MMP-9, TIMP-1, the ratios of MMP-2 and MMP-9 to TIMP-1 were unchanged in cancerous tissue compared to the normal counterparts. On the protein level, expression of MMP-9 was significantly higher and TIMP-1 expression was significantly lower, MMP-2 was unchanged and the ratios of MMP-2 and MMP-9 to TIMP-1 were increased in tumor tissue. CONCLUSIONS The higher concentration of MMP-9 as well as the increased ratios of MMP-2 and MMP-9 to TIMP-1 in malignant tissue prove the proteolytic dysbalance in prostate cancer, which does not seem to be associated with the stage and grade of the tumor. Comparison of mRNA and protein expression of MMP-2, MMP-9 and TIMP-1, respectively, did not show any significant relationships illustrating the necessity to study these components at both molecular levels.

Antibodies specific for distinct Kv subunits unveil a heterooligomeric basis for subtypes of .alpha.-dendrotoxin-sensitive potassium channels in bovine brain

The authentic subunit compositions of neuronal K+ channels purified from bovine brain were analyzed using a monoclonal antibody (mAb 5), reactive exclusively with the Kv1.2 subunit of the latter and polyclonal antibodies specific for fusion proteins containing C-terminal regions of four mammalian Kv proteins. Western blotting of the K+ channels isolated from several brain regions, employing the selective blocker alpha-dendrotoxin (alpha-DTX), revealed the presence in each of four different Kvs. Variable amounts of Kv1.1 and 1.4 subunits were observed in the K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex, and brain stem; on the other hand, contents of Kv1.6 and 1.2 subunits appeared uniform throughout. Each Kv-specific antibody precipitated a different proportion (anti-Kv1.2 > 1.1 >> 1.6 > 1.4) of the channels detectable with radioiodinated alpha-DTX in every brain region, consistent with a widespread distribution of these oligomeric subtypes. Such heterooligomeric combinations were further documented by the lack of additivity upon their precipitation with a mixture of antibodies to Kv1.1 and Kv1.2; moreover, cross-blotting of the multimers precipitated by mAb 5 showed that they contain all four Kv proteins. Collectively, these findings demonstrate that subtypes of alpha-DTX-susceptible K+ channels are prevalent throughout mammalian brain which are composed of different Kv proteins assembled in complexes, shown previously to also contain auxiliary beta-subunits [Parcej, D. N., Scott, V. E. S., & Dolly, J.O. (1992) Biochemistry 31, 11084-11088].

Matrix metalloproteinase (MMP)-2, MMP-7, and tissue inhibitor of metalloproteinase-1 are closely related to the fibroproliferative process in the liver during chronic hepatitis C

BACKGROUND/AIMS To study whether expression of matrix metalloproteinases and their inhibitors correlate with ongoing fibrogenesis, we measured hepatic mRNA levels of matrix metalloproteinase (MMP) -2, MMP-7, and MMP-9 as well as tissue inhibitor of metalloproteinase (TIMP) -1, TIMP-2, and TIMP-3 and compared it to histology, procollagen IV alpha-1 chain mRNA levels, and biochemical parameters in patients with chronic active hepatitis C (CAH). METHODS Quantitative reverse transcription-polymerase chain reaction/enzyme-linked immunossorbent assay using in vitro transcribed competitor and standard RNA were performed from ten normal livers (N), 29 CAH liver biopsies and seven samples with hepatitis C virus (HCV)-induced end-stage cirrhosis (Ci). RESULTS From N to Ci both TIMP and MMP RNA expression increased. However, none of the RNA levels differed significantly between CAH patients with and without fibrosis. Non-parametric correlation analysis and receiver operating characteristics curves show that MMP-2, MMP-7, and TIMP-1 provide the best discrimination between cirrhosis and pre-cirrhotic stages. They also correlate with histologic and biochemical inflammatory activity and with procollagen IV mRNA. CONCLUSION Hepatic fibroproliferation is associated with alterations of hepatic TIMP and MMP expression. The relation of hepatic TIMP and MMP mRNA levels to disease stage and inflammatory activity underlines their potential as diagnostic markers in chronic liver disease.

Serum neutrophil gelatinase-associated lipocalin at inception of renal replacement therapy predicts survival in critically ill patients with acute kidney injury

IntroductionNeutrophil gelatinase-associated lipocalin (NGAL) is a promising novel biomarker that correlates with the severity and outcome of acute kidney injury (AKI). However, its prognostic utility during the late course of AKI, especially in patients that require renal replacement therapy (RRT) remains unknown. The aim of this study was to evaluate the predictive value of serum NGAL in patients with established AKI at inception of RRT in the intensive care unit (ICU).MethodsSerum NGAL (ELISA methodology) was measured in 109 critically ill patients with AKI at inception of RRT in 7 ICUs of a tertiary care university hospital. The primary outcome studied was 28-day mortality. Secondary outcome measures were ICU length of stay, ventilator-free days, and renal recovery at day 28.ResultsThere was a significant difference in serum NGAL between healthy subjects (median [interquartile range] 39.0 [37.5-42.75] ng/mL), critically ill patients with systemic inflammatory response syndrome (SIRS) (297 [184-490] ng/mL), and critically ill patients with sepsis (708 [365-1301] ng/mL; P < 0.0001), respectively. Multiple linear regression showed that NGAL levels were independently related to the severity of AKI and the extent of systemic inflammation. NGAL levels were higher in non-survivors (430 [303-942] ng/mL) compared to survivors (298 [159-506] ng/mL; P = 0.004). Consistently, Cox proportional hazards regression analysis identified NGAL as a strong independent predictor for 28-day survival (hazard ratio 1.6 (95% confidence interval [CI] 1.15 - 2.23), P = 0.005).ConclusionsThis is the first prospective evaluation of serum NGAL as an outcome-specific biomarker in critically ill patients at initiation of RRT. The results from this study indicate that serum NGAL is as an independent predictor of 28-day mortality in ICU patients with dialysis-dependent AKI.

Blood specimen collection methods influence the concentration and the diagnostic validity of matrix metalloproteinase 9 in blood.

BACKGROUND Matrix metalloproteinases (MMP) in blood are promising new diagnostic tools. It was shown that the blood sampling process resulted in different blood concentrations of MMPs. To clarify whether the sampling process also influences the diagnostic validity of MMPs, MMP-9 measurements were performed in plasma and serum samples of patients with prostate carcinoma and renal cell cancer. METHODS MMP-9 ELISAs were performed in samples of heparin plasma and serum collected in blood tubes with and without clot accelerator. Measurements were undertaken in 78 healthy persons, 33 patients with prostate carcinoma and 33 patients with renal cell carcinoma. RESULTS MMP-9 showed higher concentrations in serum samples than in heparin plasma and was about threefold higher in serum samples collected in tubes with clot activator than in native serum samples. Both patient groups had lower MMP-9 concentrations in serum, whereas in plasma, patients with renal cell carcinoma had higher, but patients with prostate cancer unchanged MMP-9 concentrations. 13 of 33 patients with renal cell carcinoma had increased MMP-9 plasma values but no patient had increased serum concentrations. CONCLUSIONS To optimise the diagnostic validity of the MMP-9 in blood, measurements should be performed in heparin plasma but not in serum.

Expression of matrix metalloproteinase-9 and its inhibitors in mononuclear blood cells of patients with multiple sclerosis

Inflammatory mononuclear cells invading the nervous system in demyelinating diseases are supposed to be a major source of matrix metalloproteinases which are involved in damaging the blood-brain barrier and facilitating cellular migration through the extracellular matrix. Several studies revealed a crucial role of 92 kDa gelatinase (matrix metalloproteinase-9, MMP-9) in these processes. In this study, we determined MMP-9, TIMP-1 and TIMP-2 (tissue inhibitor of metalloproteinase) mRNA in peripheral blood mononuclear cells of multiple sclerosis (MS) patients by competitive reverse transcription PCR and plasma protein levels by ELISA. In active MS patients, both with relapsing-remitting and chronic progressive disease MMP-9 mRNA and plasma protein levels were significantly increased compared to healthy controls. No significant changes in mRNA expression were found for TIMP-1 and TIMP-2. However, unbound TIMP-2 in plasma was significantly higher in MS patients compared to healthy controls. MMP-9 and TIMP-1 expression was significantly correlated in MS with a significantly higher MMP-9/TIMP-1 ratio in active MS than in healthy controls. These results are in support of an important pathogenic role of MMP-9 activity in MS.

Quantitation of DNA Extracted after Micropreparation of Cells from Frozen and Formalin-Fixed Tissue Sections

Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafields and Mayers alum or Weigerts iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.