Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain.

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

Hemodynamic, renal, and endocrine effects of 4-h infusions of human atrial natriuretic peptide in normal volunteers.

A synthetic human atrial natriuretic peptide of 26 aminoacids [human (3-28)ANP or hANP] was infused into normal male volunteers. Six subjects were infused for 4 h at 1-wk intervals with either hANP at the rate of 0.5 or 1.0 microgram/min or its vehicle in a single-blind randomized order. Human (3-28)ANP at the dose of 0.5 microgram/min raised immunoreactive plasma ANP levels from 104 +/- 17 to 221 +/- 24 pg/ml (mean +/- SEM), but it induced no significant change in blood pressure, heart rate, effective renal plasma flow, glomerular filtration rate, or renal electrolyte excretion. At the rate of 1.0 microgram/min, human (3-28)ANP increased immunoreactive plasma ANP levels from 89 +/- 12 to 454 +/- 30 pg/ml. It reduced effective renal plasma flow from 523 +/- 40 to 453 +/- 38 ml/min (P less than 0.05 vs. vehicle), but left glomerular filtration rate unchanged. Natriuresis rose from 207 +/- 52 to 501 +/- 69 mumol/min (P less than 0.05 vs. vehicle) and urinary magnesium excretion from 3.6 +/- 0.5 to 5.6 +/- 0.5 mumol/min (P less than 0.01 vs. vehicle). The excretion rate of the other electrolytes, blood pressure, and heart rate were not significantly modified. At both doses, human (3-28)ANP tended to suppress the activity of the renin-angiotensin-aldosterone system. In 3 additional volunteers, the skin blood flow response to human (3-28)ANP, infused for 4 h at the rate of 1.0 microgram/min, was studied by means of a laser-doppler flowmeter. The skin blood flow rose during the first 2 h of peptide administration, then fell progressively to values below baseline. After the infusion was discontinued, it remained depressed for more than 2 h. Thus, in normal volunteers, human (3-28)ANP at the dose of 1.0 microgram/min produced results similar to those obtained previously with rat (3-28)ANP. It enhanced natriuresis without changing the glomerular filtration rate while effective renal plasma flow fell. It also induced a transient vasodilation of the skin vascular bed.

Increased frequency of regulatory T cells and selection of highly potent CD62L+ cells during treatment of human lung transplant recipients with rapamycin.

The currently available immunosuppressive agents applied in human transplantation medicine are highly potent in the protection from acute allograft rejection. However, long-term allograft survival is still poor as these drugs fail to sufficiently prevent chronic allograft rejection. Naturally occurring regulatory T cells have been postulated as the key players to establish long-lasting transplantation tolerance. Thus, the development of immunosuppressive regimens which shift the pathological balance of cytopathic versus regulatory T cells of human allograft recipients towards a protective T-cell composition is a promising approach to overcome limitations of current transplantation medicine. Thirty-three patients that received rapamycin (RPM) or calcineurin inhibitor treatment following lung transplantation were included and their T-cell compartments analysed. Twelve healthy volunteers without history of lung disease served as controls. In this article, we show that treatment of human lung transplant recipients with RPM is associated with an increased frequency of regulatory T cells, as compared with treatment with calcineurin inhibitors or to healthy controls. Moreover, regulatory T cells during treatment with RPM were CD62Lhigh, a phenotype that displayed an enhanced immunosuppressive capacity ex vivo. Our data support the use of RPM in human lung transplant recipients and undertaking of further prospective studies evaluating its impact on allograft and patient survival.

Monitoring of innate and adaptive immune responses in the context of human immunotherapy

Summary1 SummaryCancer patients have a better clinical outcome when their tumours display marked infiltration by memory Τ cells. Moreover, the overrepresentation of Th1 gene signatures in primary tumours correlates with favourable prognosis. Thus, vaccination to induce Τ cells capable of infiltrating and eradicating the tumour seems a promising strategy for the treatment of cancer. Here, I monitored CD4 Τ cell responses in melanoma patients vaccinated with the long synthetic peptides Melan- A16-35(A27L) and NY-ESO-179.108. Most of the patients developed strong and diverse peptide antigen specific CD4 Τ cell responses. Analysis of the fine specificity of CD4 Τ cell antigen recognition led to the identification of two new epitopes. The peptide Melan-A16_35(A27L) was delivered by virus-like particles (VLPs) derived from bacteriophage Οβ, which themselves displayed strong immunogenicity. I show evidence for induction of Οβ- and Melan-A specific CD4 Τ cell responses that developed a Th1 functional profile after repeated vaccination cycles. They also specifically released the chemokines CCL-3 and CCL-4, which play important roles in attracting CD8 Τ cells to the APC surface for priming and formation of Τ cell memory. We further found induction of robust humoral IgG responses upon VLP vaccination, and the lgG1-lgG4 isotype composition depended on the adjuvant used. Since heavy chain class switching largely dépends on the presence of CD4 Τ cell help, this result suggests that the adjuvant can influence the differentiation of elicited CD4 Τ cells, thereby contributing to the quality and function of both Β cells and CD8 Τ cells. The nature of the inflammatory processes in the tumour microenvironment can modulate CD8 Τ cell function. A collaboration was established for the investigation regulation of inflammasome activation in human primary monocytes. We identified IL- 4 and TGF-β as strong inhibitors of IL-1 β secretion, Indicating some level of regulation from effector Th2 and Treg responses. We further found a potent inhibition of inflammasome activation by type I interferon, and demonstrated in vivo inhibition of IL-1 β responses in monocytes from active multiple sclerosis patients under IFN-β therapy. This finding further offers a possible explanation for its success, which mechanism of action is still largely unclear. Interestingly, type I interferon is also being used as adjuvant treatment for tumour free metastatic cutaneous melanoma patients. While its clinical benefit has remained controversial, recent data suggest that the subset of patients with ulcerated primary melanoma lesions can benefit from this therapy. Future investigations will shed light on the implication of the inflammasome in this context, and may offer new strategies for improved adjuvant treatments of melanoma.2 RésuméLes patients atteints de cancer ont une meilleure chance de survie si leurs tumeurs s'avèrent être largement infiltrées par des cellules Τ mémoires. De plus, la surreprésentation d'une signature génique Th1 est en corrélation avec un pronostic favorable. Ainsi, la vaccination visant à induire des cellules Τ capables d'infiltrer et de détruire la tumeur parait être une stratégie prometteuse pour le traitement du cancer. Dans ce travail, j'ai procédé au monitoring de la réponse des cellules Τ CD4 dans des patients atteints de mélanome vaccinés avec les longs peptides synthétiques Melan-A16_35(A27L) et NY-ESO-179_108. Ces peptides représentent des antigènes tumoraux reconnus par des lymphocytes T. La majorité des patients a développé une réponse forte et diversifiée des cellules Τ CD4 spécifiques contre les peptides. L'analyse de la spécificité fine de la reconnaissance antigénique des cellules Τ CD4 nous a conduits à l'identification de deux nouveaux épitopes. Le peptide Melan-Aie. 35(A27L) a été délivré par des particules de type viral (VLPs) dérivés de bactériophages Qβ, qui ont eux-mêmes démontré une forte immunogénicité. Mon travail montre les preuves d'une induction de réponses spécifiques des cellules Τ CD4 contre les Qβ et Melan-A développant un profil fonctionnel Th1 après plusieurs cycles de vaccination. Elles secrètent aussi spécifiquement les chimiokines CCL-3 et CCL-4, qui jouent un rôle important dans l'attraction des cellules Τ CD8 à la surface des cellules présentatrices d'antigènes et contribuent ainsi à induire et former la mémoire cellulaire Τ CD8. Nous avons également remarqué une induction de fortes réponses humorales IgG après vaccination avec les VLPs, et que la composition des isotypes lgG1-lgG4 dépendait de l'adjuvant utilisé. Etant donné qu'une commutation de classe de la chaîne lourde dépend largement ùie l'aide des cellules Τ CD4, ce résultat suggère que l'adjuvant puisse influencer la différeritiation de cellules Τ CD4 en différent types, contribuant ainsi à la qualité et à la fonction des cellules Β et des cellules Τ CD8.La nature des processus d'inflammation dans le microenvironnement tumoral peut moduler la fonction des cellules Τ CD8. Une collaboration a été établie pour investiguer la régulation de l'activation de l'inflammasome dans des monocytes primaires humains. Nous avons identifié l'IL-4 et le TGF-β comme étant de puissants inhibiteurs de la sécrétion de IL-Ιβ, indiquant une certaine régulation de la réponse inflammatoire induite par les cellules Th2 et Τ régulatrices. Nous avons également trouvé une forte inhibition de l'activation de l'inflammasome par l'interféron type I, et nous avons démontré une inhibition in vivo de la réponse IL-1 β dans des monocytes de patients atteints d'une sclérose en plaque active sous traitement IFN-β. Ce résultat nous offre une possible explication du succès de cette thérapie, dont le mécanisme reste à ce jour encore largement obscur. Il est intéressant de noter que l'interféron de type I est également utilisé pour le traitement de patients atteints de mélanome cutané métastasique sans tumeurs. Bien que le bénéfice clinique de ce traitement reste controversé, des études récentes montrent qu'une partie des patients atteints de mélanome primaire ulcéré peut tirer bénéfice de cette thérapie. De futures investigations pourront mieux nous renseigner sur l'implication de l'inflammasome dans ce contexte et offrir de nouvelles stratégies pour améliorer les traitements adjuvants du mélanome.

Mechanisms regulating the proliferative potential of human CD8+ T lymphocytes overexpressing telomerase.

In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.

Evolutionary impact assessment: accounting for evolutionary consequences of fishing in an ecosystem approach to fisheries management

Managing fisheries resources to maintain healthy ecosystems is one of the main goals of the ecosystem approach to fisheries (EAF). While a number of international treaties call for the implementation of EAF, there are still gaps in the underlying methodology. One aspect that has received substantial scientific attention recently is fisheries-induced evolution (FIE). Increasing evidence indicates that intensive fishing has the potential to exert strong directional selection on life-history traits, behaviour, physiology, and morphology of exploited fish. Of particular concern is that reversing evolutionary responses to fishing can be much more difficult than reversing demographic or phenotypically plastic responses. Furthermore, like climate change, multiple agents cause FIE, with effects accumulating over time. Consequently, FIE may alter the utility derived from fish stocks, which in turn can modify the monetary value living aquatic resources provide to society. Quantifying and predicting the evolutionary effects of fishing is therefore important for both ecological and economic reasons. An important reason this is not happening is the lack of an appropriate assessment framework. We therefore describe the evolutionary impact assessment (EvoIA) as a structured approach for assessing the evolutionary consequences of fishing and evaluating the predicted evolutionary outcomes of alternative management options. EvoIA can contribute to EAF by clarifying how evolution may alter stock properties and ecological relations, support the precautionary approach to fisheries management by addressing a previously overlooked source of uncertainty and risk, and thus contribute to sustainable fisheries.

Hypotensive effect of human factor XII active fragment in conscious normotensive rats: role of bradykinin.

The active fragment derived from factor XII (factor XIIf) was purified from human plasma and administered intravenously to normotensive conscious rats. Factor XIIf-mediated hypotension was dose-dependent and augmented by pretreatment with captopril, an inhibitor of the bradykinin-processing enzyme kininase II. These results therefore suggest that factor XIIf-mediated hypotension is due to the formation of bradykinin.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8⁺ T cells.

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures

Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types. Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols. However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally. Defining optimal culture parameters for ex vivo expansion has been a major challenge. We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions. Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved. Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion. Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture. With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.

Identification and modelling of human breast cancer subtypes

Résumé Le cancer du sein est le cancer le plus commun chez les femmes et est responsable de presque 30% de tous les nouveaux cas de cancer en Europe. On estime le nombre de décès liés au cancer du sein en Europe est à plus de 130.000 par an. Ces chiffres expliquent l'impact social considérable de cette maladie. Les objectifs de cette thèse étaient: (1) d'identifier les prédispositions et les mécanismes biologiques responsables de l'établissement des sous-types spécifiques de cancer du sein; (2) les valider dans un modèle ín vivo "humain-dans-souris"; et (3) de développer des traitements spécifiques à chaque sous-type de cancer du sein identifiés. Le premier objectif a été atteint par l'intermédiaire de l'analyse des données d'expression de gènes des tumeurs, produite dans notre laboratoire. Les données obtenues par puces à ADN ont été produites à partir de 49 biopsies des tumeurs du sein provenant des patientes participant dans l'essai clinique EORTC 10994/BIG00-01. Les données étaient très riches en information et m'ont permis de valider des données précédentes des autres études d'expression des gènes dans des tumeurs du sein. De plus, cette analyse m'a permis d'identifier un nouveau sous-type biologique de cancer du sein. Dans la première partie de la thèse, je décris I identification des tumeurs apocrines du sein par l'analyse des puces à ADN et les implications potentielles de cette découverte pour les applications cliniques. Le deuxième objectif a été atteint par l'établissement d'un modèle de cancer du sein humain, basé sur des cellules épithéliales mammaires humaines primaires (HMECs) dérivées de réductions mammaires. J'ai choisi d'adapter un système de culture des cellules en suspension basé sur des mammosphères précédemment décrit et pat décidé d'exprimer des gènes en utilisant des lentivirus. Dans la deuxième partie de ma thèse je décris l'établissement d'un système de culture cellulaire qui permet la transformation quantitative des HMECs. Par la suite, j'ai établi un modèle de xénogreffe dans les souris immunodéficientes NOD/SCID, qui permet de modéliser la maladie humaine chez la souris. Dans la troisième partie de ma thèse je décris et je discute les résultats que j'ai obtenus en établissant un modèle estrogène-dépendant de cancer du sein par transformation quantitative des HMECs avec des gènes définis, identifiés par analyse de données d'expression des gènes dans le cancer du sein. Les cellules transformées dans notre modèle étaient estrogène-dépendantes pour la croissance, diploïdes et génétiquement normales même après la culture cellulaire in vitro prolongée. Les cellules formaient des tumeurs dans notre modèle de xénogreffe et constituaient des métastases péritonéales disséminées et du foie. Afin d'atteindre le troisième objectif de ma thèse, j'ai défini et examiné des stratégies de traitement qui permettent réduire les tumeurs et les métastases. J'ai produit un modèle de cancer du sein génétiquement défini et positif pour le récepteur de l'estrogène qui permet de modéliser le cancer du sein estrogène-dépendant humain chez la souris. Ce modèle permet l'étude des mécanismes impliqués dans la formation des tumeurs et des métastases. Abstract Breast cancer is the most common cancer in women and accounts for nearly 30% of all new cancer cases in Europe. The number of deaths from breast cancer in Europe is estimated to be over 130,000 each year, implying the social impact of the disease. The goals of this thesis were first, to identify biological features and mechanisms --responsible for the establishment of specific breast cancer subtypes, second to validate them in a human-in-mouse in vivo model and third to develop specific treatments for identified breast cancer subtypes. The first objective was achieved via the analysis of tumour gene expression data produced in our lab. The microarray data were generated from 49 breast tumour biopsies that were collected from patients enrolled in the clinical trial EORTC 10994/BIG00-01. The data set was very rich in information and allowed me to validate data of previous breast cancer gene expression studies and to identify biological features of a novel breast cancer subtype. In the first part of the thesis I focus on the identification of molecular apacrine breast tumours by microarray analysis and the potential imptìcation of this finding for the clinics. The second objective was attained by the production of a human breast cancer model system based on primary human mammary epithelial cells {HMECs) derived from reduction mammoplasties. I have chosen to adopt a previously described suspension culture system based on mammospheres and expressed selected target genes using lentiviral expression constructs. In the second part of my thesis I mainly focus on the establishment of a cell culture system allowing for quantitative transformation of HMECs. I then established a xenograft model in immunodeficient NOD/SCID mice, allowing to model human disease in a mouse. In the third part of my thesis I describe and discuss the results that I obtained while establishing an oestrogen-dependent model of breast cancer by quantitative transformation of HMECs with defined genes identified after breast cancer gene expression data analysis. The transformed cells in our model are oestrogen-dependent for growth; remain diploid and genetically normal even after prolonged cell culture in vitro. The cells farm tumours and form disseminated peritoneal and liver metastases in our xenograft model. Along the lines of the third objective of my thesis I defined and tested treatment schemes allowing reducing tumours and metastases. I have generated a genetically defined model of oestrogen receptor alpha positive human breast cancer that allows to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasis.

Induction of MC-1 immunoreactivity in axons after injection of the Fc fragment of human immunoglobulins in macaque monkeys.

Although previous studies have suggested an increased activation of humoral immunity in neurodegenerative diseases, it remains unclear whether this phenomenon is secondary to lesion formation or contributes directly to their development. Using stereotaxic injections in macaque monkey cerebral cortex, we studied the effects of human immunoglobulins on the neuronal cytoskeleton. Under these conditions, several MC-1-immunoreactive axons were observed in the vicinity of injection site. No MC-1 or TG-3 staining was detected in neuronal soma. Ultrastructurally, several axons in the same area displayed curly formations and accumulation of twisted tubules but not paired helical filaments. These data suggest that Fc fragment induce conformational changes of tau and subtle structural alterations in axons in this model. Immunocytochemical analyses in human autopsy materials revealed the presence of human Fc fragments as well as Fc receptors only in large pyramidal neurons known to be vulnerable in brain aging and Alzheimer's disease, further supporting a possible role of immunoglobulins in neurodegeneration.

Lack of antitumour activity of human recombinant tumour necrosis factor-alpha, alone or in combination with melphalan in a nude mouse human melanoma xenograft system.

The most promising developments in the field of isolated limb perfusion have centred around the use of the recombinant cytokine tumour necrosis factor-alpha (rTNF-alpha) in combination with melphalan. While the results of clinical trials are impressive, the exact antitumour mechanisms of rTNF-alpha and its role in combination with melphalan remain unclear. Our aim was to study the antitumour activity of human rTNF-alpha with or without the combination of melphalan in a nude mouse human melanoma xenograft system. In a first attempt to define the maximal tolerated single dose of rTNF-alpha in this setting, 15 animals were exposed to increasing doses of rTNF-alpha (60-2500 microg/kg intraperitoneally). All but one animal survived and tumour growth was not influenced by these single dose applications of rTNF-alpha even at the very high doses. Anti-tumour activity of repeated application of melphalan (three times 9 mg/kg in group 2 and three times 6 mg/kg in group 3), of rTNF-alpha alone (nine doses of 50 microg/kg in group 4), and of rTNF-alpha in combination with melphalan (nine doses of 50 microg/kg rTNF-alpha and three times 6 mg/kg melphalan in group 5) was further compared with non-treated animals (group 1). Tumour growth was significantly inhibited in all animals treated with melphalan (group 2, 3 and 5), but was not decreased in animals treated with rTNF-alpha alone (group 4). Mean final tumour volumes and mean tumour weight were not different in group 2 (789 +/- 836 mm3, 0.38 +/- 0.20 g), group 3 (1173 +/- 591 mm3, 0.55 +/- 0.29 g) and group 5 (230 +/- 632 mm3, 0.37 +/- 0.29 g), but significant lower than group 1 (3156 +/- 1512 mm3, 2.35 +/- 0.90 g) and group 4 (3228 +/- 1990 mm3, 2.00 +/- 1.16 g). There were no significant differences between high and low dose melphalan treatment and between melphalan treatment in combination with rTNF-alpha. Histological examination did not show differences between treated and non-treated animals besides slightly inhibited mitotic activities of tumour cells in melphalan-treated animals. While tumour growth of human xenotransplanted melanoma in nude mice could be inhibited by melphalan, we failed to demonstrate any antitumour effect of rTNF-alpha. The combination of melphalan and rTNF-alpha did not enhance the antiproliferative effect of melphalan alone. Human xenotransplanted tumours on nude mice might not be the ideal experimental setting for studies of potential direct antineoplastic activity of rTNF-alpha, and these results support the concept that TNF-alpha exerts its antitumour activity indirectly, possibly by impairing the tumour vasculature and by activating the immune system.

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.