Martin Albrecht

Exploring Human Testicular Peritubular Cells: Identification of Secretory Products and Regulation by Tumor Necrosis Factor-α

Testicular peritubular cells are myofibroblastic cells, which represent the major cellular components of the wall of the seminiferous tubules. In men their phenotypic characteristics, including possible secretory activity and regulation, are not well known, in neither normal nor pathologically altered testes. Especially in testes of men with impaired spermatogenesis, the cytoarchitecture of the tubular wall is frequently remodeled and presents fibrotic thickening, increased innervation, and infiltration by macrophages and mast cells. The latter are two sources of TNF-alpha. The purpose of our study was to explore human testicular peritubular cells and mechanisms of their regulation. To this end we primarily studied cultured human testicular peritubular cells (HTPCs), isolated from adult human testes. Having established that HTPCs express TNF-alpha receptors 1 and 2 and respond to recombinant human TNF-alpha by a rapid phosphorylation of ERK1/2, we used complementary approaches, including gene array/RT-PCR studies, Western blotting/immunocytochemistry, and ELISA techniques to study phenotypic characteristics of HTPCs and actions of TNFalpha. We found that HTPCs express the nerve growth factor gene and TNF-alpha-stimulated mRNA levels and secretion of nerve growth factor in a dose- and time-dependent manner. Similarly, monocyte chemoattractant protein-1 was identified as a product of HTPCs, which was regulated by TNF-alpha in a concentration- and time-dependent manner. TNF-alpha furthermore strongly enhanced expression and/or synthesis of other inflammatory molecules, namely IL-6 and cyclooxygenase-2. Active cyclooxygenase-2 is indicated by increased prostaglandin D2 levels. In addition, intercellular adhesion molecule-1, which was not detected at protein level in the absence of TNF-alpha, was induced upon TNF-alpha stimulation. In conclusion, these results provide novel insights into the nature of human peritubular cells, which are able to secrete potent signaling molecules and are regulated by TNF-alpha. These results also hint to an as-yet-unknown role of peritubular cells in normal human testis and involvement in the pathomechanisms associated with impaired spermatogenesis in men.

Hypothermia and Postconditioning after Cardiopulmonary Resuscitation Reduce Cardiac Dysfunction by Modulating Inflammation, Apoptosis and Remodeling

Background Mild therapeutic hypothermia following cardiac arrest is neuroprotective, but its effect on myocardial dysfunction that is a critical issue following resuscitation is not clear. This study sought to examine whether hypothermia and the combination of hypothermia and pharmacological postconditioning are cardioprotective in a model of cardiopulmonary resuscitation following acute myocardial ischemia. Methodology/Principal Findings Thirty pigs (28–34 kg) were subjected to cardiac arrest following left anterior descending coronary artery ischemia. After 7 minutes of ventricular fibrillation and 2 minutes of basic life support, advanced cardiac life support was started according to the current AHA guidelines. After successful return of spontaneous circulation (n = 21), coronary perfusion was reestablished after 60 minutes of occlusion, and animals were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group n = 7) for 24 hours. The effects on cardiac damage especially on inflammation, apoptosis, and remodeling were studied using cellular and molecular approaches. Five animals were sham operated. Animals treated with hypothermia had lower troponin T levels (p<0.01), reduced infarct size (34±7 versus 57±12%; p<0.05) and improved left ventricular function compared to normothermia (p<0.05). Hypothermia was associated with a reduction in: (i) immune cell infiltration, (ii) apoptosis, (iii) IL-1β and IL-6 mRNA up-regulation, and (iv) IL-1β protein expression (p<0.05). Moreover, decreased matrix metalloproteinase-9 activity was detected in the ischemic myocardium after treatment with mild hypothermia. Sevoflurane conferred additional protective effects although statistic significance was not reached. Conclusions/Significance Hypothermia reduced myocardial damage and dysfunction after cardiopulmonary resuscitation possible via a reduced rate of apoptosis and pro-inflammatory cytokine expression.

Mild hypothermia alone or in combination with anesthetic post-conditioning reduces expression of inflammatory cytokines in the cerebral cortex of pigs after cardiopulmonary resuscitation

IntroductionHypothermia improves survival and neurological recovery after cardiac arrest. Pro-inflammatory cytokines have been implicated in focal cerebral ischemia/reperfusion injury. It is unknown whether cardiac arrest also triggers the release of cerebral inflammatory molecules, and whether therapeutic hypothermia alters this inflammatory response. This study sought to examine whether hypothermia or the combination of hypothermia with anesthetic post-conditioning with sevoflurane affect cerebral inflammatory response after cardiopulmonary resuscitation.MethodsThirty pigs (28 to 34 kg) were subjected to cardiac arrest following temporary coronary artery occlusion. After seven minutes of ventricular fibrillation and two minutes of basic life support, advanced cardiac life support was started according to the current American Heart Association guidelines. Return of spontaneous circulation was achieved in 21 animals who were randomized to either normothermia at 38°C, hypothermia at 33°C or hypothermia at 33°C combined with sevoflurane (each group: n = 7) for 24 hours. The effects of hypothermia and the combination of hypothermia with sevoflurane on cerebral inflammatory response after cardiopulmonary resuscitation were studied using tissue samples from the cerebral cortex of pigs euthanized after 24 hours and employing quantitative RT-PCR and ELISA techniques.ResultsGlobal cerebral ischemia following resuscitation resulted in significant upregulation of cerebral tissue inflammatory cytokine mRNA expression (mean ± SD; interleukin (IL)-1β 8.7 ± 4.0, IL-6 4.3 ± 2.6, IL-10 2.5 ± 1.6, tumor necrosis factor (TNF)α 2.8 ± 1.8, intercellular adhesion molecule-1 (ICAM-1) 4.0 ± 1.9-fold compared with sham control) and IL-1β protein concentration (1.9 ± 0.6-fold compared with sham control). Hypothermia was associated with a significant (P < 0.05 versus normothermia) reduction in cerebral inflammatory cytokine mRNA expression (IL-1β 1.7 ± 1.0, IL-6 2.2 ± 1.1, IL-10 0.8 ± 0.4, TNFα 1.1 ± 0.6, ICAM-1 1.9 ± 0.7-fold compared with sham control). These results were also confirmed for IL-1β on protein level. Experimental settings employing hypothermia in combination with sevoflurane showed that the volatile anesthetic did not confer additional anti-inflammatory effects compared with hypothermia alone.ConclusionsMild therapeutic hypothermia resulted in decreased expression of typical cerebral inflammatory mediators after cardiopulmonary resuscitation. This may confer, at least in part, neuroprotection following global cerebral ischemia and resuscitation.

Increased Exposure to Estrogens Disturbs Maturation, Steroidogenesis, and Cholesterol Homeostasis via Estrogen Receptor α in Adult Mouse Leydig Cells

Deteriorated male reproductive health has been connected to overexposure to estrogens or to imbalanced androgen-estrogen ratio. Transgenic male mice expressing human aromatase (AROM(+) mice) serve as an apt model for the study of the consequences of an altered androgen-estrogen ratio. Our previous studies with AROM(+) mice showed that low androgen levels together with high estrogen levels result in cryptorchidism and infertility. In the present study, the AROM(+) mice were shown to have severe abnormalities in the structure and function of Leydig cells before the appearance of spermatogenic failure. Decreased expression of adult-type Leydig cell markers (Ptgds, Vcam1, Insl3, Klk21, -24 and -27, Star, Cyp17a1, and Hsd17b3) indicated an immature developmental stage of the Leydig cells, which appears to be the first estrogen-dependent alteration. Genes involved in steroidogenesis (Star, Cyp17a1, and Hsd17b3) were suppressed despite normal LH levels. The low expression level of kallikreins 21, 24, and 27 potentially further inhibited Leydig cell function via remodeling extracellular matrix composition. In connection with disrupted steroidogenesis, Leydig cells showed enlarged mitochondria, a reduced amount of smooth endoplasmic reticulum, and an accumulation of cholesterol and precursors for cholesterol synthesis. The results of studies with AROM(+) mice crossed with estrogen receptor alpha or beta (ERalpha and ERbeta, respectively) knockout mice lead to the conclusion that the structural and functional disorders caused by estrogen exposure were mediated via ERalpha, whereas ERbeta was not involved.

Postoperative neurocognitive dysfunction in patients undergoing cardiac surgery after remote ischemic preconditioning: a double-blind randomized controlled pilot study.

Background Remote ischemic preconditioning (RIPC) has been shown to enhance the tolerance of remote organs to cope with a subsequent ischemic event. We hypothesized that RIPC reduces postoperative neurocognitive dysfunction (POCD) in patients undergoing complex cardiac surgery. Methods We conducted a prospective, randomized, double-blind, controlled trial including 180 adult patients undergoing elective cardiac surgery with cardiopulmonary bypass. Patients were randomized either to RIPC or to control group. Primary endpoint was postoperative neurocognitive dysfunction 5–7 days after surgery assessed by a comprehensive test battery. Cognitive change was assumed if the preoperative to postoperative difference in 2 or more tasks assessing different cognitive domains exceeded more than one SD (1 SD criterion) or if the combined Z score was 1.96 or greater (Z score criterion). Results According to 1 SD criterion, 52% of control and 46% of RIPC patients had cognitive deterioration 5–7 days after surgery (p = 0.753). The summarized Z score showed a trend to more cognitive decline in the control group (2.16±5.30) compared to the RIPC group (1.14±4.02; p = 0.228). Three months after surgery, incidence and severity of neurocognitive dysfunction did not differ between control and RIPC. RIPC tended to decrease postoperative troponin T release at both 12 hours [0.60 (0.19–1.94) µg/L vs. 0.48 (0.07–1.84) µg/L] and 24 hours after surgery [0.36 (0.14–1.89) µg/L vs. 0.26 (0.07–0.90) µg/L]. Conclusions We failed to demonstrate efficacy of a RIPC protocol with respect to incidence and severity of POCD and secondary outcome variables in patients undergoing a wide range of cardiac surgery. Therefore, definitive large-scale multicenter trials are needed. Trial Registration ClinicalTrials.gov NCT00877305

Cytoprotective effects of the volatile anesthetic sevoflurane are highly dependent on timing and duration of sevoflurane conditioning: findings from a human, in-vitro hypoxia model.

Using animal models, volatile anesthetics have been recognized for their neuroprotective effects. Nevertheless, there is still disagreement about the optimal duration and timing of conditioning with the volatile anesthetic sevoflurane in the human system. In the study presented, we employed a human neuronal cell culture model to investigate the effects of hypoxia and to evaluate potential cytoprotective properties of different sevoflurane conditioning strategies. Sevoflurane was applied to human IMR-32 cells in which hypoxic conditions were induced for 2h using our recently described two-enzyme model (Zitta et al., Eur. J. Pharmacol., 2010). Cellular effects of hypoxia and sevoflurane conditioning were evaluated by lactate dehydrogenase (LDH) measurements, brightfield microscopy, ELISAs, cytometric bead arrays, Westernblotting and RT-PCR. Hypoxia increased the release of LDH into the culture medium after 24h (normoxia: 0.15+/-0.02 a.u; hypoxia: 0.69+/-0.08 a.u, P<0.001) and expression of hypoxia associated genes HIF-1alpha, VEGF, catalase. Cytoprotective effects were observed in cultures that received sevoflurane for 30 min before hypoxia (preconditioning: 0.41+/-0.07 a.u., P<0.01) and for 30 min during the hypoxic period (intraconditioning: 0.20+/-0.01 a.u., P<0.001). Application of sevoflurane after the hypoxic insult did not lead to cytoprotection (postconditioning: 0.73+/-0.12a.u., P>0.05). Conditioning with sevoflurane for a total of 3h before, during and after hypoxia, however, resulted in an enhanced release of LDH (periconditioning: 0.97+/-0.10a.u., P<0.01) and additional cell damage. Hypoxia and sevoflurane intraconditioning were associated with changes in erk1/2 phosphorylation (T202/Y204) and HIF-1alpha protein levels, whereas phosphorylation of akt (S473) was not significantly altered. Our results suggest short pre- and intraconditioning with sevoflurane as most potent strategies to reduce hypoxia induced neuronal cell damage.

Serum from patients undergoing remote ischemic preconditioning protects cultured human intestinal cells from hypoxia-induced damage: involvement of matrixmetalloproteinase-2 and -9.

Remote ischemic preconditioning (RIPC) can be induced by transient occlusion of blood flow to a limb with a blood pressure cuff and exerts multiorgan protection from ischemia/reperfusion injury. Ischemia/reperfusion injury in the intestinal tract leads to intestinal barrier dysfunction and can result in multiple organ failure. Here we used an intestinal cell line (CaCo-2) to evaluate the effects of RIPC-conditioned patient sera on hypoxia-induced cell damage in vitro and to identify serum factors that mediate RIPC effects. Patient sera (n = 10) derived before RIPC (T0), directly after RIPC (T1) and 1 h after RIPC (T2) were added to the culture medium at the onset of hypoxia until 48 h after hypoxia. Reverse transcription-polymerase chain reaction, lactate dehydrogenase (LDH) assays, caspase-3/7 assays, silver staining, gelatin zymography and Western blotting were performed. Hypoxia led to morphological signs of cell damage and increased the release of LDH in cultures containing sera T0 (P < 0.01) and T1 (P < 0.05), but not sera T2, which reduced the hypoxia-mediated LDH release compared with sera T0 (P < 0.05). Gelatin zymography revealed a significant reduction of activities of the matrixmetalloproteinase (MMP)-2 and MMP-9 in the protective sera T2 compared with the nonprotective sera T0 (MMP-2: P < 0.01; MMP-9: P < 0.05). Addition of human recombinant MMP-2 and MMP-9 to MMP-deficient culture media increased the sensitivity of CaCo-2 cells to hypoxia-induced cell damage (P < 0.05), but did not result in a reduced phosphorylation of prosurvival kinases p42/44 and protein kinase B (Akt) or increased activity of caspase-3/7. Our results suggest MMP-2 and MMP-9 as currently unknown humoral factors that may be involved in RIPC-mediated cytoprotection in the intestine.

Hypoxia-induced cell damage is reduced by mild hypothermia and postconditioning with catalase in-vitro: Application of an enzyme based oxygen deficiency system

Mild hypothermia and pharmacological postconditioning are widespread therapeutical treatment options that positively influence the clinical outcome after tissue hypoxia. In the study presented, a two-enzyme based in-vitro oxygen deficiency model in combination with cultured HT-1080 fibrosarcoma cells was employed to mimic the in-vivo situation of hypoxia and to evaluate the influence of mild hypothermia and postconditioning with catalase on hypoxia-mediated cell damage. Using the in-vitro oxygen deficiency model, partial pressure of oxygen was rapidly reduced to levels below 5mmHg in the culture media and cells responded with an increased expression of hypoxia inducible factor-1 on protein level. Hypoxia resulted in significant cell rounding and retraction of cytoplasmic cell extensions. Evaluation of cytotoxicity revealed a 3.5-fold increase in lactate dehydrogenase levels which was accompanied by 40-fold elevated levels of hydrogen peroxide. The hypoxia-induced increase of lactate dehydrogenase was 2.5-fold reduced in the hypothermia group, although morphological correlates of cytotoxicity were still visible. Hypothermia did not significantly influence hydrogen peroxide concentrations in the culture media. Pharmacological postconditioning with catalase however dose-dependently decreased hypoxia-induced lactate dehydrogenase release. This cytoprotective effect was accompanied by a dose-dependent, up to 50-fold reduction of hydrogen peroxide concentrations and retention of normal cell morphology. We suggest that the described in-vitro oxygen deficiency model is a convenient and simple culture system for the investigation of cellular and subcellular events associated with oxygen deficiency. Moreover, our in-vitro results imply that catalase postconditioning may be a promising approach to attenuate hypoxia-induced and hydrogen peroxide-mediated cell and tissue damage.

Divergent effects of the major mast cell products histamine, tryptase and TNF-alpha on human fibroblast behaviour

Abstract.Fibroblast proliferation is a key process in tissue remodeling and mast cells (MCs) are thought to play a crucial role. Having established that the three major MC products, tryptase, histamine and TNF-alpha (TNF) are normally present in human skin MCs, which are in close proximity to dermal fibroblasts, we studied their individual effects on cell cycle-controlled human dermal fibroblasts (HFFF2). These cells express receptors (H1, PAR2, TNFR1/2) for the major MC mediators, but only tryptase or a PAR2 agonist peptide stimulated proliferation and gene expression. TNF was antimitotic, and histamine, while elevating intracellular Ca2+ levels at high concentrations, did not affect proliferation. We conclude that MC products but also composition and numbers of respective receptors on fibroblasts are crucially responsible for fibroproliferative events.

Ionizing radiation induces degranulation of human mast cells and release of tryptase.

Purpose: Skin fibrosis is a hallmark of ionizing radiation-induced tissue injury and we hypothesized that mast cells via their products (especially tryptase) are involved in this event. We therefore investigated whether: (i) irradiation with 5 Gray (Gy) is able to induce the release of the typical mast cell mediator tryptase from human mast cells (HMC-1) in vitro, (ii) this effect can be influenced by application of clinically relevant mast cell blockers, and (iii) irradiation leads to mast cell degranulation in ex vivo skin culture models. Materials and methods: The human mast cell line (HMC)-1, as well as ex vivo skin tissue served as experimental models. Fluorescence activated cell sorting (FACS), Enzyme linked immunosorbent assays (ELISA), mast cell degranulation assays and immunohistochemistry were applied. Results: Ionizing radiation induces a time-dependent, statistically significant increase in the release of tryptase by HMC-1 cultured in vitro. Mast cell degranulation and secretion of tryptase was partially, but not significantly, inhibited by pre-incubation with the histamine-1 receptor (H1) blocker cetirizine. Mast cell degranulation was also clearly evident after irradiation using an ex vivo skin culture model of mastocytoma tissue. Conclusions: We propose that ionizing radiation leads to a degranulation of dermal mast cells, an event which is accompanied by the release of tryptase.