Fernando Bittinger

Response to transarterial chemoembolization as a biological selection criterion for liver transplantation in hepatocellular carcinoma

Criteria to select patients with hepatocellular carcinoma (HCC) for liver transplantation (LT) are based on tumor size and number of nodules rather than on tumor biology. The present study was undertaken to assess the role of transarterial chemoembolization (TACE) in selecting patients with tumors suitable for LT. Ninety‐six consecutive patients with HCC were treated by repeatedly performed TACE, 62 of them exceeding the Milan criteria. Patients meeting the Milan criteria were immediately listed, and patients beyond the listing criteria were listed upon downstaging of the tumor following successful TACE. Fifty patients were finally transplanted. Of these 50 patients, 34 exceeded the Milan criteria. In these 96 patients, overall 5‐year survival was 51.9%. However, it was 80.9% for patients undergoing LT and 0% for patients without transplantation (P < 0.0001). Tumor recurrence was primarily influenced by the control of the disease through continued TACE during the waiting time. Freedom from recurrence after 5 years was 94.5% in patients (n = 39) with progress‐free TACE during the waiting time. Tumor recurrence was significantly higher in patients (n = 11) who after initial response to TACE progressed again before LT (freedom from recurrence 35.4%; P = 0.0017). Progress‐free course of TACE during the waiting time (P = 0.006; risk ratio, 8.95), and a limited number of tumor nodules as assessed in the surgical specimen (P = 0.025; risk ratio, 0.116) proved to be significant predictors for freedom from recurrence in the multivariate analysis. Milan criteria were without impact on recurrence. Our data suggest that sustained response to TACE is a better selection criterion for LT than the initial assessment of tumor size or number. Liver Transpl 12:1260‐1267, 2006.

The non-neuronal cholinergic system in humans: Expression, function and pathophysiology

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter. In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.

Staging small cell lung cancer: Veterans Administration Lung Study Group versus International Association for the Study of Lung Cancer—what limits limited disease?

Small cell lung cancer (SCLC) is usually classified into a two-stage system, limited (LD) and extensive disease (ED). However, the criteria for these two categories remain controversial. The widely used Veterans Administration Lung Study Group (VALG) definition of LD includes patients with primary tumor and nodal involvement limited to one hemithorax. In contrast, the International Association for the Study of Lung Cancer (IASLC) recommends that LD should additionally include all patients without distant metastasis. As a consequence, since treatment modalities for LD and ED could be different, individual clinical outcome of SCLC patients may be influenced by the staging system chosen. Among 109 consecutive SCLC patients treated in our clinic between 1989 and 1999 (mean age 68+/-9.1 years, 81% male) 23 patients (21%) could be either classified as LD or ED (LD-ED), depending on the staging system used. The prognosis of this overlapping group (LD-ED: median survival 291 days) was not statistically different from patients with limited disease defined by VALG criteria (LD-VALG: 385 days, log-rank test P = 0.42). On the other hand the survival difference between LD-ED patients and the ED-IASLC population was relevant (ED-IASLC: 208 days, P = 0.05), indicating that LD-ED patients should rather be included in the LD category. This is further supported by the results of a multivariate Cox regression analysis with all clinically relevant data. Only stage as defined by IASLC criteria was an independent prognostic factor in the likelihood-ratio-forward (hazard ratio = 1.94, CI = 1.26-2.99; P = 0.005) and backward model (hazard ratio = 1.76, CI: 1.12-2.76; P = 0.012), confirming the higher discriminatory power of the IASLC definition. In conclusion, the IASLC staging criteria for SCLC patients have a higher prognostic impact and are therefore preferable in clinical practice and future therapeutic trials.

Microcirculatory dysfunction in sepsis: a pathogenetic basis for therapy?

Sepsis is a frequent complication of multiple organ dysfunction syndrome and remains a major problem of intensive care medicine. It is also a common factor in the final cause of death in hospital populations. Clinical observations, assisted by invasive monitoring techniques as well as pathological–anatomical studies, clearly indicate that microcirculatory dysfunction lies at the centre of sepsis pathogenesis. Numerous animal models, from rodents to primates, many of which employ bacteria or their toxins, especially endotoxins, have helped to shed light on the pathomechanisms leading to this dysregulation in the peripheral circulation. Among these are activation of humoral and cellular inflammatory mediator systems, with special emphasis on neutrophil–endothelial interactions, affecting endothelial barrier function and vasoregulation and ultimately leading to severely perturbed oxygen transport and utilization. In vitro studies have provided more insight into the molecular mechanisms involved in this microcirculatory dysfunction, although much more attention must be directed towards microvascular endothelial cells and the role of heterogeneity of response in various vascular beds. These experimental data must in turn be validated by comparing with the human in situ situation, both clinical and morphological. This review aims at a critical appraisal of the clinical and experimental evidence for sepsis‐induced dysregulation of the microcirculation and how knowledge of the underlying cellular and molecular pathology could be used to make therapy more rational and effective. To date, therapeutic approaches, such as anti‐cytokine and anti‐oxidant regimens, which have been highly successful in experimental models, have failed to demonstrate clinical efficacy. Newer approaches, such as targeting the coagulation system, nitric oxide synthesis or intracellular signal transduction, are also discussed. The necessity to focus on the role of anti‐inflammatory mediators, as well as the pathogenetic significance of important molecular groups, such as the heat shock proteins, which until now have been given scant attention, will be stressed. Copyright

Generation of human pulmonary microvascular endothelial cell lines

The limited lifespan of human microvascular endothelial cells in cell culture represents a major obstacle for the study of microvascular pathobiology. To date, no endothelial cell line is available that demonstrates all of the fundamental characteristics of microvascular endothelial cells. We have generated endothelial cell lines from human pulmonary microvascular endothelial cells (HPMEC) isolated from adult donors. HPMEC were cotransfected with a plasmid encoding the catalytic component of telomerase (hTERT) and a plasmid encoding the simian virus 40 (SV40) large T antigen. Cells transfected with either plasmid alone had an extended lifespan, but the cultures eventually entered crisis after several months of proliferation. Only those cells that were transfected with both plasmids acquired the capacity to grow in vitro without demonstrating major crisis, and these cells have been in culture for 24 months. HPMEC isolated from two different donors were used, generating two populations of immortalized cells, HPMEC-ST1 and HPMEC-ST2. Single cell–derived clones of the immortalized cells HPMEC-ST1 exhibited growth characteristics that were similar to those of the parental HPMEC. One selected clone, HPMEC-ST1.6R, displayed all major constitutively expressed and inducible endothelial phenotypic markers, including platelet endothelial cell adhesion molecule (PECAM-1, CD31), von Willebrand factor (vWF), and the adhesion molecules, intercellular adhesion molecule (ICAM-1), vascular adhesion molecule (VCAM-1), and E-selectin. In addition, an angiogenic response was demonstrated by sprout formation on a biological extracellular matrix (Matrigel). The HPMEC-ST1.6R cells did not form tumors in nude mice. The microvascular endothelial cell line, HPMEC-ST1.6R, will be a valuable tool for the study of microvascular endothelial physiology and pathology including gene expression, angiogenesis, and tumorigenesis.

Release of non-neuronal acetylcholine from the isolated human placenta is mediated by organic cation transporters.

The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non‐neuronal cholinergic system. Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration‐dependent manner with an IC50 value of 5 μM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 μM. Procaine (100 μM), a sodium channel blocker, and vesamicol (10 μM), an inhibitor of the vesicular acetylcholine transporter, were ineffective. Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration‐dependent manner with an IC50 value of 2 μM. Substrates of OCT subtype 1, 2 and 3 (amiloride, cimetidine, guanidine, noradrenaline, verapamil) inhibited acetylcholine release, whereas carnitine, a substrate of subtype OCTN2, exerted no effect. Long term exposure (48 and 72 h) of villus strips to anti‐sense oligonucleotides (5 μM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti‐sense oliogonucleotides were ineffective. It is concluded that the release of non‐neuronal acetylcholine from the human placenta is mediated via organic cation transporters of the OCT1 and OCT3 subtype.

Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock

In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn2+ and Cd2+ are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni2+ and Co2+ only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn(2+) and Cd(2+) are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni(2+) and Co(2+) only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.

Biomaterial-induced sarcoma: A novel model to study preneoplastic change.

In the study of carcinogenesis most interest has focused on carcinomas, as they represent the majority of human cancers. The recognition of the adenoma-carcinoma sequence both in humans and in animal experimental models has given the field of basic oncology the opportunity to elucidate individual mechanisms in the multistep development of carcinoma. The relative scarcity of human sarcomas coupled with the lack of adequate animal models has hampered understanding of the molecular genetic steps involved. We present an experimental model in the rat in which a high incidence of malignant mesenchymal tumors arise around a subcutaneously implanted biomaterial. Nine commercially available biomaterials were implanted in a total of 490 rats of the Fischer strain for 2 years. On average, macroscopic tumors were found in 25.8% of implantation sites over a period from 26 to 110 weeks after implantation. The most frequent tumors were malignant fibrous histiocytomas and pleomorphic sarcomas, although fibrosarcomas, leiomyosarcomas, and angiosarcomas readily developed, the latter especially around polyurethane implants. Of particular interest are the results of a detailed histological study of the capsules around the implanted biomaterials without tumors. Here a spectrum of change from focal proliferative lesions through preneoplastic proliferation to incipient sarcoma could be observed. A parallel immunohistochemical study of peri-implant capsules showed that proliferating cell nuclear antigen was of particular help in identifying these atypical proliferative lesions. To our knowledge this is the first description of a sarcoma model in which preneoplastic lesions can be readily identified and also reproducibly induced. This model provides the molecular biologist with defined stages in the development of mesenchymal malignancy, with which the multistage tumorigenesis hypothesis can be tested, analogous to the well-known adenoma-carcinoma sequence.

Tissue response and biomaterial integration: the efficacy of in vitro methods

Implantation involves tissue trauma, which evokes an inflammatory response, coupled to a wound healing reaction, involving angiogenesis, fibroblast activation and matrix remodelling. Until now the type and extent of such reactions to give optimal integration of various biomaterials are practically unknown. Three principal fields of research can yield useful data to understand these phenomena better: studies on explanted biomaterials, animal models and relevant in vitro techniques. This paper will present examples of the latter field and the application of endothelial cell (EC) culture systems to study the effects of important tissue (e.g. pro-inflammatory cytokines, chemokines) and material (e.g. metal ions, particulate debris) factors on the regulation of the inflammatory and angiogenic response. A central feature is the use of microvascular endothelial cells (MEC), which can be used in both 2-and 3-dimensional (3-D) assays. We have also used genetic manipulation to develop a permanent MEC line from the human lung (HPMEC-ST1), which is being tested for its suitability to study cell-biomaterial interactions. In addition, suitable in vitro techniques are being developed in order to investigate drug delivery systems (DDS). Of particular interest is the targeting of the central nervous system, our approach being to establish a human model of the blood-brain barrier (BBB). A mainstay of our scientific philosophy is that such in vitro methods can make an important contribution to understanding biological reactions at the tissue-biomaterial interface and thus further a causal approach to tissue engineering (TE) and drug delivery applications.

Basaloid squamous cell carcinoma of the esophagus

Basaloid squamous cell carcinoma (BSCC) is a recently recognized, poorly differentiated variant of squamous cell carcinoma (SCC), which is located predominantly in the upper aerodigestive tract.