Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

AIM: To investigate the proteolytic contribution of tumor-associated macrophages (TAM) in tumor invasion, we analyzed whether TAM at the invasive front of small HCC in Abcb4^-/--mice show an enhanced expression of MMP-9.METHODS: Liver cryosections of the hepatocellular carcinoma (HCC) invasive front from 12 mo old Abcb4^-/--mice were stained for collagen type I and MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies. Afterwards, the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies. Finally, photographs of the invasive tumor front were digitally overlaid and analyzed.RESULTS: After complete bleaching of the primary dye, specific fluorescence staining of a third antigen, here F4/80, was successfully performed on the same histological section. With this method, we were able to identify conglomerates of matrix metalloproteinase (MMP-9) expressing macrophages within the tumor capsule of HCC.CONCLUSION: MMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC. The described staining protocol provides a simple yet powerful extension of conventional immuno-histochemistry, facilitating visualization of at least three different antigens plus nuclei in one single histological section.     

A free fatty acid tolerance test identifies patients with coronary artery disease among individuals with a low conventional coronary risk profile

This study investigated whether the presence of coronary artery disease (CAD) in patients with a low conventional coronary risk profile is associated with perturbations of free fatty acid (FFA) metabolism. All patients studied were non-smokers, normoglycemic, normotensive, nonobese, and had triglycerides, low-density lipoprotein and high-density lipoprotein (HDL) cholesterol in the reference ranges. An FFA tolerance test was designed, consisting of a heparin injection 4 h after an oral fat load which induced a marked increase in plasma FFA concentrations. Measurements were made before the fat load, after 4 h (immediately before heparin injection), and after 4.5, 8, and 10 h. The test was carried out in 28 male CAD patients and in 25 male controls free of CAD as verified by coronary angiography. In the fasting state the two groups showed no differences in conventional risk factors with the exception of HDL cholesterol (patients 0.97 ± 0.04 mmol/l, controls 1.13 ± 0.05 mmol/l, P = 0.013). During the test the best discriminator found was FFA at 8 h (P = 0.0009) and, very pronounced, at 10 h (P = 0.000). We conclude that perturbed FFA metabolism in an FFA tolerance test can indicate the presence of CAD in men with a low conventional coronary risk profile, possibly as an early indicator of the metabolic syndrome. Received: May 1, 2001 / Accepted: October 13, 2001     

In heavy drinkers fatty acid ethyl esters in the serum are increased for 44 hr after ethanol consumption

BACKGROUND: Fatty acid ethyl esters (FAEEs) have been proposed as a marker of ethanol consumption because they can be detected for up to 24 hr after a moderate intake of ethanol, even though blood ethanol remains increased for only 8 hr. Therefore, this study investigated whether FAEEs can be found during a time period exceeding 24 hr in a group of patients who were hospitalized for ethanol detoxification. A second aim was to study the distribution of FAEEs between lipoproteins during that time. METHODS: Serum samples of 12 patients with acute ethanol intoxication were assayed for FAEEs. Blood samples were drawn 8.2, 20.2, 32.2, and 44.2 hr after hospitalization. FAEEs were quantified by gas chromatography-mass spectrometry. RESULTS: Ethanol was no longer detectable after 20.2 hr from hospitalization, whereas FAEEs were still found after 32.2 and 44.2 hr. These late FAEEs were significantly higher than the FAEEs in 15 different healthy men who had abstained from ethanol for 4.5 days (p < 0.001 and p = 0.001). FAEEs were associated mainly with lipid-free serum but tended to accumulate in very-low-density lipoprotein in patients with moderate hypertriglyceridemia. CONCLUSIONS: In heavy drinkers, the FAEEs were increased after ethanol consumption for at least 44 hr. It remains to be studied whether they originate from a single ethanol intake or, in addition, from a slow release out of body storage compartments.     

Dosage finding and outcome of venlafaxine treatment in psychiatric outpatients and inpatients: results of a drug utilization observation study

BACKGROUND: Venlafaxine is an antidepressive drug with the special characteristic of inhibiting both synaptic serotonin and norepinephrine reuptake. This double action is dosage dependent, with the relatively weaker inhibition of norepinephrine becoming clinically relevant only at higher dosages. This allows treatment to be tailored towards the needs of individual patients through differential dosing. It is unknown, however, how physicians use this unique feature in prescribing venlafaxine in routine treatment. METHOD: Data from a drug utilization observation (DUO) study, including 6706 patients, are used to investigate which patient and setting variables predict dosage of venlafaxine as prescribed by psychiatrists in inpatient and outpatient settings. Treatment outcome and adverse drug reactions (ADR) were analyzed for different dosage groups. RESULTS: Treatment setting is the most important factor in predicting high (> 75 mg/day) or low (up to 75 mg/day) dosage of venlafaxine, with inpatients receiving higher dosages. Severity of illness and a history of previous treatment with major antidepressives are also related to higher dosages. Although the total rate of ADR did not increase with increased dosage, the profile of drug reactions changed. Response to therapy was better in cases of non-chronic, major depression with no treatment history of antidepressives. Additionally, increased dosage increased the likelihood of response in outpatients. In both settings, very high dosages predicted better response to venlafaxine among severely ill patients. CONCLUSION: Venlafaxine at a dosage of 75 mg/day is sufficient for the majority of cases. In extremely ill patients, higher dosages are associated with additional benefits. Therefore, a stepwise dosage regimen is suggested, with an increase of dosage to upper limits in cases of non-response before discontinuation of treatment with venlafaxine.     

Antihypertensive treatment and homocysteine concentrations

Thiazides and angiotensin-converting enzyme (ACE) inhibitors are first-choice drugs for lowering elevated blood pressure and hence risk of cardiovascular disease. Homocysteine (tHcy) is another and independent cardiovascular risk factor and has been reported to be elevated in patients on antihypertensive therapy. As these studies reported only associations, a preliminary, randomized, prospective treatment study was performed in 40 hypertensive patients. We investigated the major determinants of tHcy concentrations after treatment with hydrochlorothiazide (HCT) or captopril: vitamins B6, B12, folic acid, and creatinine and cystatin C as parameters of renal function. A total of 21 Patients were treated with HCT and 19 with captopril, for, respectively, 31 and 29 days. HCT, but not captopril, raised tHcy by 16% (P =.003) and also creatinine and cystatin C (P =.025 and P =.004, respectively). This tHcy increase may offset the desired cardioprotection conferred by lowering the blood pressure.     

Evidence for associations between common polymorphisms of estrogen receptor beta gene with homocysteine and nitric oxide.

BACKGROUND: Homocysteine and asymmetric dimethylarginine (ADMA) affect nitric oxide (NO) concentration, thereby contributing to cardiovascular disease (CVD). Both amino acids can be reduced in vivo by estrogen. Variation in the estrogen receptor (ER) may influence homocysteine and ADMA, yet no information is available on associations with single nucleotide polymorphisms in the estrogen receptor genes ERalpha (PvuII and XbaI) and ERbeta (1730G-->A and cx + 56 G-->A). OBJECTIVE: To find relationships between common polymorphisms associated with cardiovascular disease and cardiovascular risk factors homocysteine and ADMA. METHODS: In a cross-sectional study with healthy postmenopausal women (n = 89), homocysteine, ADMA, nitric oxide metabolites (NOx), plasma folate and ERalpha and beta polymorphisms ERalpha PvuII, ERalpha XbaI; ERbeta 1730G-->A (AluI), ERbeta cx + 56 G-->A (Tsp509I) were analyzed. RESULTS: Women who are homozygotic for ERbetacx + 56 G-->A A/A exhibited higher homocysteine (p = 0.012) and NOx (p = 0.056) levels than wildtype or heterozygotes. NOx concentration was also significantly affected by ERbeta 1730 G -->A polymorphism (p = 0.025). The ERbeta (p < 0.001) and ERalpha (p < 0.001) polymorphisms were in linkage disequilibrium. CONCLUSIONS: Women who are homozygotic for ERbetacx + 56 G-->A A/A may be at increased risk for cardiovascular disease due to higher homocysteine levels.     

Adenosine in the noninvasive diagnosis of dual AV nodal conduction: use as a follow-up parameter after slow pathway ablation in AVNRT

OBJECTIVE: The aim of this study was to evaluate if administration of adenosine during sinus rhythm to patients with PSVT of unknown mechanism is capable to detect dual AV nodal conduction and furthermore to evaluate this diagnostic parameter as a controlling test after slow pathway ablation in AVNRT. METHODS AND RESULTS: Before electrophysiological study 35 consecutive patients with PSVT were given adenosine during sinus rhythm. After radiofrequency ablation the adenosine test was repeated in a subset of 19 patients. The electrophysiological study revealed 19 patients (54%) with typical AVNRT (study group), 10 (29%) with atrioventricular reentry tachycardia (AVRT), 4 (11%) with ectopic atrial tachycardia (EAT) and 2 patients (6%) with inducible atrial flutter (AF) (control group). We observed a sudden increment of the PQ interval of more than 50 msec between two consecutive beats in 15 of 19 patients (79%) in the study group (75+/-35 msec) and in 2 patients (1 with EAT, AF) of the control group (19+/-12 msec) (p<0.001). After slow pathway radiofrequency ablation the sudden increment of PQ interval persisted in 4 of 12 patients (33%) of the study group. Three of these 4 patients had a relapse of AVNRT during a follow-up of 3 months. CONCLUSION: The administration of adenosine during sinus rhythm is an excellent noninvasive diagnostic test for identifying dual AV nodal conduction and additionally for verifying radiofrequency ablation results in patients with AVNRT.     

Plasma pyridoxal-5-phosphate and future risk of myocardial infarction in the European Prospective Investigation into Cancer and Nutrition Potsdam cohort

BACKGROUND: Retrospective studies indicate that low concentrations of plasma pyridoxal-5-phosphate (PLP) are associated with cardiovascular events; however, few prospective studies of this issue have been conducted. OBJECTIVE: We therefore investigated whether PLP concentrations are independently associated with myocardial infarction (MI) in the European Investigation into Cancer and Nutrition (EPIC) Potsdam Study. DESIGN: After exclusion of prevalent MI or stroke, incident cases of MI were identified among 26 761 participants (aged 35-65 y at baseline). The current analysis is based on a nested case-cohort study consisting of a control group of 810 subjects without MI or stroke at baseline and a case group of 148 subjects who had an MI during a mean follow-up period of 6.0 +/- 1.5 y. Cox proportional hazard models were used to evaluate the association between plasma PLP and risk of MI. RESULTS: In the age- and sex-adjusted analysis, subjects in the highest quintile of PLP had a significantly reduced risk of MI (hazard ratio: 0.50; 95% CI: 0.29, 0.83). Adjustment for either low-grade inflammation or smoking diminished this association. When both low-grade inflammation and smoking were adjusted for, the association was abolished. In addition, adjustment for established risk factors also abolished the association between PLP and risk of MI. CONCLUSION: These findings from a prospective German cohort study suggest that PLP is not independently associated with risk of MI.     

Analysis of the transcobalamin II 776C>G (259P>R) single nucleotide polymorphism by denaturing HPLC in healthy elderly: associations with cobalamin, homocysteine and holo-transcobalamin II.

A relatively new method for the detection of single nucleotide polymorphisms is the use of denaturing high-performance liquid chromatography (DHPLC). DHPLC was used to analyse the transcobalamin II 776C>G polymorphism in DNA from 159 healthy elderly. Furthermore, cobalamin, folate, homocysteine and holo-transcobalamin II (holo-TC II) were measured. The allele frequency of the G-allele was 17% with n = 55 harbouring the CC genotype, n = 77 being heterozygous and n = 27 showing the GG genotype. Holo-TC II concentrations were significantly decreased in patients harbouring the GG genotype. There was no effect on cobalamin, methylmalonyl-CoA, folate or homocysteine concentrations. A new G>A variant at nucleotide position 810 in the TC II gene was detected by an altered peak pattern in the DHPLC and further elucidated by direct sequencing. The TC II G810A variant is a silent mutation without replacement of the corresponding amino acid (alanine) at position 270 in the TC II protein and was only found as a heterozygous genotype in a single patient. The new variant would have been undetected by other methods used for single nucleotide polymorphism detection, e.g., restriction fragment length polymorphism analysis. The results suggest that the common TC II 776C>G polymorphism has no major influence on vitamin B12 metabolism.     

Supplementation with vitamin B12 decreases homocysteine and methylmalonic acid but also serum folate in patients with end-stage renal disease.

Hyperhomocysteinemia is frequently found in patients with end-stage renal disease (ESRD). Plasma total homocysteine (tHcy) concentrations may be reduced by supplementation with folic acid or combinations of folic acid, vitamin B12, and vitamin B6. Supplementation studies with vitamin B12 alone in patients with ESRD have not yet been published. In this study, we investigated the effects of intravenous injection of cyanocobalamin (1 mg/wk for 4 weeks) in ESRD patients (N = 14) with low serum cobalamin concentrations (<180 pmol/L). All patients had elevated levels of plasma tHcy, methylmalonic acid (MMA), and cystathionine before supplementation. After supplementation, plasma tHcy and MMA decreased 35% and 48%, respectively; however, cystathionine levels were unchanged. The extent of the plasma tHcy reduction tended to be influenced by the C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR). Serum cobalamin increased significantly upon supplementation, whereas serum folate levels were substantially reduced by 47%. In contrast, red blood cell (RBC) folate was unchanged. This study shows that vitamin B12 supplementation effectively decreases both MMA and plasma tHcy in ESRD patients with low B12 levels. Furthermore, it illustrates the close interrelation between vitamin B12 and folate metabolism.