Cell-cell adherence as a selection method for the generation of anti-melanoma monoclonal antibodies.

The aim of the present study was to obtain monoclonal antibodies (mAbs) recognising human melanoma-associated antigens after immunisation of BALB/c mice with a 70-150 kDa membrane fraction from melanoma tumour tissues. Screening of specific antibody- producing hybridomas was performed using a novel cell-cell adherence method with the melanoma cell line M-14. Three mAbs of IgG1 isotype were selected: Mel-1, Mel-2 and Mel-3 which recognised the immunogen by ELISA and stained several melanoma cell lines positive in immunofluorescence. The molecular weight of the antigen was studied by different methods; a 170-kDa band was identified following immunoblotting of tumour lysate and a 72-kDa band was observed following immunoaffinity purification. Cell-cell adherence appears to be a reliable procedure for the generation of mAbs against native cellular antigens.

Thioredoxin, thioredoxin reductase and tumour necrosis factor-alpha expression in melanoma cells: correlation to resistance against cytotoxic attack.

Although malignant melanomas are often associated with cytotoxic lymphocyte infiltration, these cells are largely ineffective in inducing tumour cell kill, indicating that the melanoma cells have protective mechanisms. These mechanisms are not fully understood, but cytokines and redox-active antioxidant proteins such as catalase, superoxide dismutase, thioredoxin (Trx) and Trx reductase (TrxR) present in the tumour cells constitute part of this protection. In this study firstly we investigated the constitutive intracellular expression of Trx, TrxR, the cytokines interleukin (IL)-1alpha, IL1beta, IL2, IL4, IL6, IL8, IL10, tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) in normal melanocytes and ten primary and metastatic malignant melanoma cell lines. Secondly, we analysed whether redox stimulation by Trx alone or in combination with the phorbol ester PMA affected the expression and release of TNFalpha. Thirdly, we explored the possible correlation between Trx/TrxR expression and resistance to exogenous TNFalpha. All the cultured cells showed intracellular overexpression of Trx and TrxR, which was not always the case for melanoma cells in vivo (tissue sections). The predominant intracellular cytokines found were TNFalpha, IL1alpha and IL1beta. In spite of its presence in the Golgi apparatus, none of the cell lines secreted TNFalpha constitutively, and only one melanoma, FM3, released detectable amounts after stimulation. In contrast, U-937 monocyte control cells released high amounts of TNFalpha on identical stimulation. All the melanoma cell lines were relatively resistant against exogenous TNFalpha, and there was a significant correlation (P < 0.01) between intracellular Trx/TrxR expression and TNFalpha resistance (IC50). In conclusion, Trx and TrxR, as well as TNFalpha, IL1alpha and IL1beta, were highly expressed in cultured normal skin melanocytes and malignant melanoma cell lines. In contrast to U-937 monocytic cells, TNFalpha showed a secretory block in these cells, suggesting a cytoprotective and possible autocrine role for TNFalpha. The intracellular expression of Trx and TrxR together with endogenous TNFalpha was correlated with the resistance to TNFalpha-induced cytotoxicity.

Phenotypical study of human lymphokine-activated killer (LAK) cells.

In the present study, T-cell enriched lymphocyte populations were stimulated with recombinant interleukin-2 (IL-2) to obtain lymphokine-activated killer (LAK) cells, and their cytotoxicity was tested against different target cells. The expression of phenotypical markers with a panel of monoclonal antibodies (Moabs) using indirect immunofluorescence techniques and cytofluorometric analysis was studied. The results showed slight variations in T-cell antigen expression and an increase in the expression of immature NK and proliferation associated antigens.

Toxic effects of interleukin-2-activated lymphocytes on vascular endothelial cells.

IL-2-activated lymphocytes (LAK cells) show increased adherence to, and killing of, human vascular endothelial cells compared to resting lymphocytes. In the present work, we have found that supernatants from LAK cell cultures also are toxic to human umbilical vein endothelial cells (HUVEC) when tested for 48 h in a neutral red uptake assay. Recombinant TNF-alpha and IFN-gamma at high concentrations are also toxic under the same test conditions, and TNF-alpha was directly detected in LAK cell supernatants. An inconsistent inhibition of toxicity was found with anti-TNF-alpha whereas anti IFN-gamma antibodies had a partial inhibitory effect. The susceptibility of HUVEC to cellular killing by LAK cells could be up- and down-regulated with insulin-like growth factor I and IFN-gamma, respectively. It is concluded that damage to vascular endothelium during high dose IL-2 treatments may be partially related to an excessive production of lymphokines such as IFN-gamma and TNF-alpha. IFN-gamma may, in addition, be protective for HUVEC during cellular interactions with LAK cells.

Thioredoxin expression and localization in human cell lines: detection of full-length and truncated species.

Thioredoxin (Trx) is an intracellular multifunctional 12-kDa protein with a reduction/oxidation (redox) active disulfide constitutively expressed by most cells of the human body. Trx can also be released by cells such as lymphocytes upon activation or oxidative stress exposure and exert a cocytokine and cytoprotective activity. In addition, a truncated 10-kDa form of Trx has been reported. In order to better understand the function of full-length and truncated Trx, we have produced, for the first time, specific monoclonal antibodies, which can discriminate between the two forms. Using these novel antibodies, designated alpha Trx1 to alpha Trx4, a panel of cell lines derived from human B and T lymphocytes, monocytes, granulocytes, and melanomas was analyzed by immunochemical techniques. The cellular distribution differed between the two forms. All lines contained full-length Trx, also located to a minor extent on the cell surface. One exception was the melanoma cell line FM28.4, which did not show any Trx expression. Truncated Trx was present in most cells in minimal amounts only, whereas the monocytic cell lines THP-1 and U-937 expressed high amounts on the cell surface, as shown by flow cytometric analysis of living cells and confocal laser-scanning microscopy. The biological importance and function of the short versus long forms of Trx as detected by the antibodies are discussed.

Production of active anti-CD6 mouse/human chimeric antibodies in the milk of transgenic mice.

The expression of chimeric genes in the mammary gland of transgenic farm animals has become an alternative for the large-scale production of recombinant proteins and for the modification of milk composition. In this paper, we show that a mouse/human chimeric antibody against the human CD6 leukocyte antigen can be assembled and correctly folded by the mammary gland, and secreted to milk, where it maintains its specificity. The base sequences encoding for the heavy and light chain variable regions of the anti-CD6 mouse monoclonal antibody IOR-T1 were cloned by the polymerase chain reaction from hybridoma cDNA, coupled to human heavy and light chain constant region genes, and inserted in a vector containing the 5' regulatory region of the rabbit whey acidic protein gene. Transgenic mice were produced by conventional pronuclei microinjection techniques. Integration and transgene copy number were determined by Southern blot. Assembled human immunoglobulin was detected in milk using a sandwich ELISA. Expression levels of chimeric antibodies in milk were determined to be around 400 micrograms/ml by Western blot, using CHO-derived chimeric IOR-T1 antibodies as reference. The chimeric antibodies produced in milk recognized human peripheral blood T lymphocytes by indirect immunofluorescence, with the classical patch-like pattern of IOR-T1.

Estudio comparartivo del proceso de purificación de anticuerpos monoclonales mediante cromatografía de adsorción en hidroxiapatita / Comparative study of the purification of monoclonal antibodies using hydroxiapatite absortion chromatography

En el presente trabajo se compara la purificación de anticuerpos monoclonales de ratón de diferentes clases y subclases (IgG1, IgG2 e IgM) por cromatografía de adsorción en hidroxiapatita (BioRad) y en hidroxiapatita-Ultrogel (LKB). Se analizaron los factores capacidad de adsorción, rendimiento, tiempo de corrida (velocidad de flujo), reciclaje y capacidad de enlazamiento específicos de los anticuerpos obtenidos inmediatamente después de la purificación y posterior a su almacenamiento. Se concluye que la cromatografía de adsorción en hidroxiapatita, y en específico, luego de que esta sustancia acoplada a un soporte adecuado (HA Ultrogel), constituye un método fácil, sencillo, rápido y eficiente para la purificación de anticuerpos monoclonales