Serum values of tumour necrosis factor-alpha and of soluble tumour necrosis factor-R55 in melanoma patients.

Tumour necrosis factor-alpha (TNFalpha) is a cytokine with a variety of biological activities, including an effect on tumour growth. The soluble TNF receptor TNF-R55 (sTNF-R55) inhibits TNFalpha functioning. Serum values of TNFalpha and TNF-R55 have been observed in patients with different cancers. To determine the serum values of TNFalpha and its soluble receptors and to investigate the prognostic value of sTNF-R55, we studied the sera of 68 patients with metastatic melanoma, 109 patients with no recurrent disease after surgical removal of melanoma, and 69 healthy controls. At least four different monoclonal antibodies against human TNFalpha and human TNF-R55, respectively, were prepared to obtain sensitive and reliable sandwich enzyme-linked immunosorbent assays. We found that in patients with metastatic melanoma the serum values of sTNF-R55 were significantly higher (2.41 ng/ml; range 0.02 23.0 ng/ml; P < 0.05) than in the melanoma patients without recurrence (0.54 ng/ml; range 0.02-6.25 ng/ml) and healthy controls (0.5 ng/ml; range 0.02 5.0 ng/ml). The sTNF-R55 concentrations increased as the disease progressed. Patients with metastatic melanoma also had significantly higher concentrations of TNFalpha (3.34 ng/ml; range 0.03-30.0 ng/ml; P<0.05) than patients without recurrence (1.24 ng/ml; range 0.02 23.0 ng/ml). Patients with metastatic melanoma, a high sTNF-R55 concentration, a low TNFalpha concentration and a low TNF:sTNF-R55 ratio had the worst prognosis. Low values of sTNF-R55 (<0.6 ng/ml) were associated with favourable response to chemotherapy (P = 0.007). According to our findings, patients with metastatic melanoma have higher values of sTNF-R55 than the controls and melanoma patients without recurrence. sTNF-R55 values higher than 0.6 ng/ml and a TNF:sTNF-R55 ratio lower than 1.5 are unfavourable prognostic factors for response to chemotherapy.

Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.

BACKGROUND: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. MATERIALS AND METHODS: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). RESULTS: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. CONCLUSION: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vaccine.

Genetic risk factors associated with Creutzfeld-Jakob disease in Slovenians and a rapid typing for PRNP codon 129 single nucleotide polymorphism.

PRNP has been the most informative marker for the predisposition to variant Creutzfeld-Jakob disease (vCJD). All victims of the vCJD carried methionine (M) at the position 129 of the PrP. Prions could travel through the immune system to get from the gut to the brain, and human leucocyte antigens (HLAs) could be involved in this carriage, with HLA-DQ7 being less efficient. Contradictory reports have raised the question of the influence of sampling in population studies. We developed a fast and reliable real-time polymerase chain reaction for codon 129 single nucleotide polymorphism (SNP) using TaqMan technology, which overcomes the main drawbacks of other methods and analysed Slovenian population (n = 97). The comparison with other populations served for the estimation of the genetic risk for the development of vCJD in Slovenians. The frequencies at the codon 129 SNP in the Slovenian population were 43.3% M, 45.4% M/V 11.3% V. Considerable differences between the DQ7 frequencies in diverse samples from the same population can be seen, especially when compared to Slovenian population. This could be because of the diverse criteria for including subjects into the study and the sampling of geographically distinct subpopulations. Analysing the adequacy of HLA-DQ7 as a possible predictive factor for developing Creutzfeld-Jakob disease (CJD) by case - control studies could be improved with exact and equal sampling of groups of patients and controls. CJD genetic risk factors in the Slovenians were not found significantly different than those in British.

Weak D and partial D in Slovenian population through serology and genotyping.

Weak D red cell phenotype (formerly D(II)) exhibits weaker serological reaction with anti-D antibodies. Weak D occurs in 0.2% to 1% of whites and is caused by qualitatively altered RhD proteins called partial D or normal, only weakly expressed RhD proteins that are called weak D. Partial D genes are hybrid alleles between RHD an RHCE genes. 23 partial RHD alleles are described. Weak D phenotypes with reduced expression are likely to possess the normal RHD gene, but the latest findings indicate that weak D alleles carry at least one point mutation. The aim of the present work was to answer an important question how to approach partial and weak D identification in diagnostic use and if it is possible to distinguish between partial D and weak D using commercially available anti-D reagents for routine use. We also wanted to evaluate D-screen kit for partial D identification. We compared phenotypes identified by serological testing and genotypes identified by RHD Multiplex PCR and D(VII) specific ASPA PCR. Our results showed that it is not possible to distinguish between partial and weak D using commercially available anti-D reagents for routine use. D-screen proved to be useful for D(VI) and D(VII) identification, whereas for partial D(DFR) identification we must look for another set of monoclonal antibodies or simply use genotyping methods. In 44 samples with not interpretable serological results out of 80 we found all RHD specific exons present and we classified the samples as weak D. Fourteen types of weak D with at least one point mutation were recently proposed. Designing of allele specific PCRs for identification of proposed types of weak D is in progress.

Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method.

Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.

Murine monoclonal antibodies directed against human recombinant Macrophage Migration Inhibitory Factor.

Macrophage Migration Inhibitory Factor (MIF) is a crucial component of the immune system acting together with glucocorticosteroids to regulate immunity and inflammation. Understanding of its many putative functions and action mechanisms is still ambiguous. Due to the newest findings that a local MIF expression is up regulated in allograft rejection and in glomerulonephritis, an interest in MIF research is increasing and is focused on possibilities of anti-MIF treatment. In the present work new murine monoclonal antibodies (MAbs) directed against human recombinant MIF (hrMIF) are described. hrMIF protein used for the immunisation was tested for its biological activity and has evident macrophage migration inhibitory activity. The selected MAbs were purified and further characterised. They recognised MIF in a Western blot experiment after a native IEF. Anti-MIF MAb designated as M1 inhibited MIF activity in the test, which was performed in the 48 well Boyden chamber system. It is presumed that M1 MAb could be used as a potential therapeutic agent.

Soluble tumor necrosis factor receptor I (sTNFRI) as a prognostic factor in melanoma patients in Slovene population.

Tumor necrosis factor alpha (TNF-alpha) and its receptors (TNFRI and TNFRII) which exist in soluble form as a product of cleavage of the extracellular domain of membrane integrated receptors, still rise debate about their importance. It was reported that TNF-alpha has numerous actions in diseases such as inflammation, autoimmunity, infectious diseases, septic shock and many types of cancer [1, 2]. Several authors have reported the significance of sTNFRI level in serum of cancer patients [3, 4]. This study was performed in collaboration with the Institute of Oncology of Slovenia. At least two different mouse monoclonal antibodies (MAbs) against human sTNFRI have been prepared to obtain a sensitive and reliable sandwich ELISA. It was compared with commercially available R&D and Endogen ELISAs for the determination of sTNFRI. Groups of patients with different stages of melanoma and epithelial ovarian carcinoma were tested and their clinical records were reexamined. Levels of sTNFRI were measured and compared with the normal serum levels of sTNFRI.