Wulf Hildebrandt

Role of Mononuclear Cells and Inflammatory Cytokines in Pancreatic Cancer-Related Cachexia

Background and Purpose: The mechanism behind aggressive development of cachexia in patients suffering from pancreatic cancer is not well understood. In this study, we investigated which factors are associated with the cachectic status of the patients and evaluated cachexia-promoting capacity of cancer and inflammatory cells. Experimental Design: DNA microarray analysis and quantitative reverse transcription-PCR were used to screen for cachexia-associated factors in pancreatic specimens obtained from noncachectic and cachetic patients diagnosed with pancreatic ductal adenocarcinoma. The expression pattern of the most prominently altered cachexia-associated factor, interleukin-6 (IL-6), was further analyzed in patients sera by ELISA, in pancreatic specimens by immunohistochemistry, and in a coculture system by quantitative reverse transcription-PCR using pancreatic cancer cell lines T3M4 (IL-6 positive) and Panc-1 (IL-6 negative) and peripheral blood mononuclear cells (PBMC) obtained from donors and noncachectic and cachectic patients. Results: Among numerous analyzed factors, IL-6 was significantly overexpressed in pancreatic specimens and elevated in serum of cachectic patients. The coculture system revealed that pancreatic cancer T3M4 cells but not Panc-1 cells were able to stimulate IL-6 exclusively in cachectic PBMC (by 14-fold) and this triggering was reduced by half in the presence of IL-6-neutralizing antibodies. Conclusion: IL-6 represents a prominent cachexia-associated factor in pancreatic cancer. IL-6 overexpression in cachectic patients is related to the ability of certain tumors to sensitize PBMC and induce cytokine expression in cachectic PBMC.

Morphology, metabolism, microcirculation, and strength of skeletal muscles in cancer-related cachexia

Purpose. Cancer-related cachexia is an obscure syndrome leading to muscle wasting, reduced physical fitness and quality of life. The aim of this study was to assess morphology, metabolism, and microcirculation in skeletal muscles of patients with cancer-related cachexia and to compare these data with matched healthy volunteers. Methods. In 19 patients with cancer-induced cachexia and 19 age-, gender-, and body-height-matched healthy volunteers body composition and aerobic capacity (VO2max) were analyzed. Skeletal muscle fiber size and capillarization were evaluated in biopsies of the vastus lateralis muscle. The cross-sectional area (CSA) of the quadriceps femoris muscle was measured by magnetic resonance imaging as well as its isokinetic and isometric force. The energy and lipid metabolism of the vastus lateralis muscle was quantified by 31P and 1H spectroscopy and parameters of its microcirculation by contrast-enhanced ultrasonography (CEUS). Results. Morphologic parameters were about 30% lower in cachexia than in volunteers (body mass index: 20±3 vs. 27±4 kg m−2, CSA: 45±13 vs. 67±14 cm2, total fiber size: 2854±1112 vs. 4181±1461 µm2). VO2max was reduced in cachexia (23±9 vs. 32±7 ml min−1 kg−1, p=0.03), whereas histologically determined capillary density and microcirculation in vivo were not different. Both concentrations of muscular energy metabolites, pH, and trimethyl-ammonium-containing compounds were comparable in both groups. Absolute strength of quadriceps muscle was reduced in cachexia (isometric: 107±40 vs. 160±40 Nm, isokinetic: 101±46 vs. 167±50 Nm; p=0.03), but identical when normalized on CSA (isometric: 2.4±0.5 vs. 2.4±0.4 Nm cm−2, isokinetic: 2.2±0.4 vs. 2.5±0.5 Nm cm−2). Conclusions. Cancer-related cachexia is associated with a loss of muscle volume but not of functionality, which can be a rationale for muscle training.

Assessment of Skeletal Muscle Perfusion Using Contrast-Enhanced Ultrasonography

The purpose of this study was to develop a clinically applicable examination method to assess perfusion of the skeletal muscle using contrast‐enhanced ultrasonography (CEUS) analyzing replenishment kinetics of microbubbles.

Plasma cystine concentration and redox state in aging and physical exercise.

Because redox-regulated signalling pathways are often modulated by the thiol/disulfide redox state (REDST), changes in plasma REDST may possibly account for pathological processes. We, therefore, investigated the mechanisms that account for changes in the plasma REDST as derived in first approximation from the cystine and acid soluble thiol (mainly cysteine) concentrations. Elderly subjects (studies A) and younger subjects after intensive physical exercise (IPE) (study B) i.e. subjects in conditions typically associated with decreased insulin responsiveness, showed, on the average, an increase in the plasma total free amino acid (TAA) concentration to approximately 3000 microM, including an increase in cystine but no increase in the thiol concentration if compared with controls. The REDST was decreased accordingly. A study on the postabsorptive amino acid exchange rates across the lower extremities (study C) indicated that a TAA level > or =2800 microM supports a balanced net protein synthesis even under conditions of weak insulin stimulation, suggesting that high TAA levels may prevent the release of cysteine into the blood in the postabsorptive state. Collectively, these studies indicate that the age-related oxidative shift in plasma REDST may result from the decrease in amino acid clearance capacity and may be aggravated by excessive physical exercise.

Effect of thiol antioxidant on body fat and insulin reactivity.

Insulin signaling is enhanced by moderate concentrations of reactive oxygen species (ROS) and suppressed by persistent exposure to ROS. Diabetic patients show abnormally high ROS levels and a decrease in insulin reactivity which is ameliorated by antioxidants, such as N-acetylcysteine (NAC). A similar effect of NAC has not been reported for non-diabetic subjects. We now show that the insulin receptor (IR) kinase is inhibited in cell culture by physiologic concentrations of cysteine. In two double-blind trials involving a total of 140 non-diabetic subjects we found furthermore that NAC increased the HOMA-R index (derived from the fasting insulin and glucose concentrations) in smokers and obese patients, but not in nonobese non-smokers. In obese patients NAC also caused a decrease in glucose tolerance and body fat mass. Simultaneous treatment with creatine, a metabolite utilized by skeletal muscle and brain for the interconversion of ADP and ATP, reversed the NAC-mediated increase in HOMA-R index and the decrease in glucose tolerance without preventing the decrease in body fat. As the obese and hyperlipidemic patients had lower plasma thiol concentrations than the normolipidemic subjects, our results suggest that low thiol levels facilitate the development of obesity. Supplementation of thiols plus creatine may reduce body fat without compromising glucose tolerance.

Cerebrospinal Fluid Concentrations of Functionally Important Amino Acids and Metabolic Compounds in Patients with Mild Cognitive Impairment and Alzheimer’s Disease

Cerebrospinal fluid (CSF) biomarkers play an important role in the differential diagnosis of neurodegenerative diseases such as Alzheimer’s disease (AD) and its postulated precursor stage mild cognitive impairment (MCI). While CSF tau protein, phospho-tau protein and β-amyloid have become part of the diagnostic process in clinical routine, the importance of several other biomarkers remains quite unclear. Among these, amino acids and metabolic compounds have been studied in clinical conditions mostly other than AD and, to our knowledge, never in MCI. In patients with AD (n = 14) and MCI (n = 13) we now determined CSF levels of 36 different amino acids and metabolic compounds by high-performance liquid chromatography. We found that 8 out of 36 amino acids (urea, threonine, glutamate, citrulline, α-aminobutyric acid, ornithine, ammonia and arginine) were significantly elevated in the CSF of patients with AD compared to those with MCI. As most of these amino acids and metabolic compounds are functionally important for brain-specific metabolic processes, neurotransmitter pathways or compensatory mechanisms, our findings might reflect these changes occurring within the brain of patients with MCI and those who developed manifest AD.

Novel Systemic Markers for Patients with Alzheimer Disease? – A Pilot Study

Almost 2% of the population of western industrialized countries are affected by Alzheimers disease (AD). Nevertheless the pathogenetic process leading to this neurodegenerative disease is widely unknown. Thus, we focus on novel pathophysiological aspects of AD. We hypothesize that AD patients reveal increased levels of peripheral blood mononuclear cells (PBMCs) expressing proinflammatory (COX-2, TNF-alpha, CD40), proapoptotic (PARP-1), adhesion-relevant (CD38) or AD associated (C99, BACE1, Presenilin-1) proteins as well as elevated proinflammatory biochemical plasma parameters. Therefore, PBMCs of AD patients and age-matched control subjects were studied by two color fluorescence-activated cell sorter (FACS) analysis. Furthermore, concentration of plasma oxidized low-density lipoprotein (oxLDL) and TNF-alpha were measured by enzyme-linked immunosorbent assay (ELISA). We found a significantly increased percentage of TNF-alpha, COX-2, PARP-1, CD38, C99 or presenilin-1 positive PBMCs in AD patients compared with healthy subjects. FACS analyses revealed that the percentage of C99 or presenilin-1 positive PBMCs, which also express TNF-alpha, COX-2, PARP-1 or CD38 is also increased in AD patients. Additionally, AD patients had significantly increased plasma oxLDL and TNF-alpha levels. Furthermore, we found positive correlations between plasma oxLDL or TNF-alpha concentrations and the percentage of TNFalpha+, COX-2+ or PARP-1+, as well as PS-1+, C99+ or BACE+ PBMCs. Our findings suggest that immunocytological investigations, based on immunophenotyping of AD relevant proteins combined with measurement of proinflammatory, proapoptotic and adhesion-relevant proteins in PBMCs may provide more insight into the pathophysiology of AD.

The early immunological response to acute ischemic stroke: Differential gene expression in subpopulations of mononuclear cells

UNLABELLED Peripheral blood mononuclear cells (PBMCs), i.e. lymphocytes, monocytes and macrophages are key players in the development of innate and adaptive immune responses. However, little is known about their properties in patients with acute stroke. EXPERIMENTAL PROCEDURES We presently characterized the early time course of PBMC subpopulations in 19 patients with acute ischemic stroke and symptom onset below 6 h compared to 19 age-matched healthy subjects. Immediately after acute ischemic stroke, as well as 1 and 3 days thereafter, PBMC subpopulations (cluster of differentiation [CD]3+, CD14+, CD19+, CD68+) were isolated by magnetic bead system and the expression of proinflammatory (CD40, tumor necrosis factor-alpha [TNFalpha]), proapoptotic (caspase-3 [CPP32], poly(ADP-ribose) polymerase [PARP]) and adhesion relevant (CD38) genes was measured by quantitative polymerase chain reaction (PCR). Furthermore, besides routine parameters, plasma levels of oxidized low-density lipoproteins (oxLDL) were studied. RESULTS In comparison to healthy subjects, patients revealed (i) twofold elevated plasma oxLDL concentrations, (ii) decreased (15%) blood cholesterol levels, and (iii) a 40% decrease in total number of lymphocytes. Furthermore, the majority of PBMC subpopulations revealed an increased expression of proinflammatory, proapoptotic or adhesion-relevant genes. Significant positive correlations were observed between expression of most of these genes in PBMCs and individual plasma oxLDL concentrations. CONCLUSION Elevated expression of proinflammatory, proapoptotic and adhesion genes in subsets of PBMCs after ischemic stroke may contribute to an immunodepressive syndrome, possibly due to increased plasma oxLDL levels.

Concentric resistance training increases muscle strength without affecting microcirculation

PURPOSE While the evidence is conclusive regarding the positive effects of endurance training, there is still some controversy regarding the effects of resistance training on muscular capillarity. Thus, the purpose was to assess whether resistance strength training influences resting skeletal muscle microcirculation in vivo. MATERIALS AND METHODS Thirty-nine middle-aged subjects (15 female, 24 male; mean age, 54+/-9 years) were trained twice a week on an isokinetic system (altogether 16 sessions lasting 50 min, intensity 75% of maximum isokinetic and isometric force of knee flexors and extensors). To evaluate success of training, cross-sectional area (CSA) of the quadriceps femoris muscle and its isokinetic and isometric force were quantified. Muscular capillarization was measured in biopsies of the vastus lateralis muscle. In vivo, muscular energy and lipid metabolites were quantified by magnetic resonance spectroscopy and parameters of muscular microcirculation, such as local blood volume, blood flow and velocity, by contrast-enhanced ultrasound analyzing replenishment kinetics. RESULTS The significant (P<0.001) increase in CSA (60+/-16 before vs. 64+/-15 cm(2) after training) and in absolute muscle strength (isometric, 146+/-44 vs. 174+/-50 Nm; isokinetic, 151+/-53 vs. 174+/-62 Nm) demonstrated successful training. Neither capillary density ex vivo (351+/-75 vs. 326+/-62) nor ultrasonographic parameters of resting muscle perfusion were significantly different (blood flow, 1.2+/-1.2 vs. 1.1+/-1.1 ml/min/100g; blood flow velocity, 0.49+/-0.44 vs. 0.52+/-0.74 mms(-1)). Also, the intensities of high-energy phosphates phosphocreatine and beta-adenosintriphosphate were not different after training within the skeletal muscle at rest (beta-ATP/phosphocreatine, 0.29+/-0.06 vs. 0.28+/-0.04). CONCLUSION The significant increase in muscle size and strength in response to concentric isokinetic and isometric resistance training occurs without an increase in the in vivo microcirculation of the skeletal muscles at rest.

Increased gene expression of scavenger receptors and proinflammatory markers in peripheral blood mononuclear cells of hyperlipidemic males

Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaques are suggested to be pathophysiologically relevant to lipid-induced arteriosclerosis. In this study, gene expressions of scavenger receptors (CD36, CD68), LPS receptor (CD14), proinflammatory (tumor necrosis factor alpha [TNFα], CD40, interleukin-1 beta [IL-1β]) and oxidative stress-related (manganese superoxide dismutase [MnSOD]) markers were analyzed in PBMCs of clinically asymptomatic males with classical proatherogenic risk factors such as smoking and/or hyperlipidemia. PBMCs were isolated from venous blood of normolipidemic non-smokers (n = 10) and smokers (n = 8), and hyperlipidemic non-smokers (n = 9) and smokers (n = 8). RNA from PBMCs was used for PCR analyses. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA. The gene expressions of CD36, CD68, CD40, TNFα, and MnSOD were significantly higher in PBMCs of hyperlipidemics than in normolipidemics, irrespective of whether they were smoking or not. The individual expression of these genes showed significant positive correlations with each other but also with serum cholesterol or plasma oxLDL concentrations. The higher expressions of scavenger receptors, proinflammatory and oxidative stress-related genes of PBMCs are suggested to result mainly from hyperlipidemia and the accompanied increase of oxLDL concentrations.