HLA homozygous stem cell lines derived from human parthenogenetic blastocysts.

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S. population, and is shared by different racial groups. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. G-banded karyotyping demonstrates a normal 46,XX karyotype in lines hpSC-Hhom-1 and hpSC-Hhom-4, and chromosomal anomalies in lines hpSC-Hhom-2 and hpSC-Hhom-3, both derived from the same donor. HLA genotyping of all four hpSC-Hhom lines demonstrates that they are HLA homozygous. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. Single-nucleotide polymorphism (SNP) data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by SNP analysis. The protocol used for deriving these HLA homozygous stem cell lines minimizes the use of animal-derived components, which makes them more appealing for potential clinical application.

Patient-specific stem cell lines derived from human parthenogenetic blastocysts.

Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.

Separation of the pore-forming and cytotoxic activities from natural killer cell cytotoxic factor.

The influence of Ca2+ on the cytotoxic activity of natural killer cell cytotoxic factor (NKCF) was analyzed. When the natural killer susceptible cell line K562 was exposed to NKCF in the presence of 5 mM Ca2+, two peaks of cell damage were found. The first peak was observed after 30-40 min of incubation as a result of pore formation on the surface of target cells. The second was a peak of cytolytic activity which appeared after 24 h of incubation. Upon dilution of the NKCF preparation, only the first peak was observed. Therefore, NKCF produced by large granular lymphocytes in response to K562 consists of different proteins and represents pore-forming and cytolytic activities.

[Effect of oxoplatin and platidiam on human melanoma strains and functional activity of blood lymphocytes in athymic mice]. / Deistvie oksoplatiny i platidiama na shtammy melanomy cheloveka i funktsional'nuiu aktivnost' limfotsitov krovi u bectimusnykh myshei.

Oxoplatinum (USSR) caused a more pronounced anticancer effect than platidiam (CSSR) when treating nude mice with the transplantable human melanoma. Moderate lymphopenia was found with therapeutic doses of oxoplatinum and platidiam, but there was no direct inhibition of the energy metabolism of lymphocytes in the peripheral blood. In the cancer cells and in the lymphocytes infiltrating the tumour oxoplatinum caused a significant decrease in the alpha-GPDG activity realizing the binding of the glycolysis and oxidation which might evidence for a definite selective action of oxoplatinum on the human melanoma.

[Therapeutic effect of blastolysin in athymic mice with transplanted strains of human tumors]. / Terapevticheskii éffekt blastolizina u bestimusnykh myshei s perevivnymi shtammami opukholei cheloveka.

9 transplanted strains were used for the evaluation of antitumour effect of blastolysin. The short-term inhibition of the tumour growth was observed on the following models: breast cancer, stomach cancer, kidney cancer and chorionepithelioma.

[Effects of cortiphen on human tumor strains transplanted into athymic mice and rats]. / Deistvie kortifena na shtammy opukholei cheloveka, transplantiruemye bestimusnym mysham and krysam.

The antitumour activity of cortiphen synthesized at the All-Union Cancer Research Centre of the USSR Academy of Medical Sciences was studied on human tumour strains transplanted into nude mice and rats. Cortiphen was found to possess an expressed activity to kidney cancer, cancer of corpus uteri, chorionepithelioma, fibrosarcoma. Two strains of colon adenocarcinoma out of three have displayed an expressed sensitivity to cortiphen, while melanoma and Jewing sarcoma strains proved to be weakly sensitive to the preparation.

[Testosterone stimulation of the growth of a human osteogenic sarcoma strain transplanted into athymic rats]. / Stimuliatsiia testosteronom rosta shtamma osteogennoi sarkomy cheloveka, transplantirovannogo bestimusnym krysam.

Intraperitoneal testosterone administration stimulated the growth of subcutaneously transplanted cell line of human osteogenic sarcoma in nude rats but did not provoke tumour metastasizing to other organs and tissues. The specific connection of androgens with receptor proteins in the cytosol fraction was not found in the studied osteogenic sarcomas, whereas cytoplasmic estrogen receptors were revealed in all tumours. It is supposed that the mechanism of the stimulating effect of androgens on the tumour growth is mediated through other biologic systems being not related to androgen receptors.

[Immunohistochemical and radiologic study of the accumulation of antibodies to the beta-1-MA antigen in human tumors transplanted into athymic mice]. / Immunogistokhimicheskoe i radiologicheskoe izuchenie nakopleniia antitel k antigenu beta-1-MA v opukholiakh cheloveka, transplantirovannykh bestimusnym mysham.

Human tumours and tissues of nude mice were studied immunohistochemically using rabbit antibody to the beta-1-MA. Specific reaction was observed in colonic carcinomas, beta-1-MA was not found in other tumours. The specific reaction was found in intestinal epithelium of nude mice, and was absent in other tissues. 131I-labelled antibody to the beta-1-MA was intravenously administered to tumour-bearing nude mice. Specifically connected antibodies were revealed by immunohistochemical method in colonic carcinomas only. Radioimmunological investigation has shown accumulation of 131I-labelled antibodies to the beta-1-MA in colonic carcinomas.

[Enhanced transcription of genes of the intracisternal A particles and B2 elements in mouse tumors]. / Povyshennyi uroven' transkriptsii genov intratsistral'nykh A-chastits i B2-élementov v opukholiakh myshei.

A comparison of the expression of two mobile genetic elements A1 and B2 was studied in normal and tumor tissues. The A1 element is a chromosomal homolog of IAP genes, and B2 is a short ubiquitous repetitive sequences of the mouse genome. These sequences were earlier cloned in our laboratory and in this study were used as probes in hybridization experiments with RNA isolated from different mouse tumor and normal tissues. Both elements were efficiently transcribed in tumor cells. The level of expression of A1 sequences in tumors was 100-200 times higher than in normal tissues. The amount of B2 small cytoplasmic RNA significantly varied in different normal tissues. The content of this RNA was much higher in tumors. Closed circular DNA molecules containing IAP sequences were found in Ehrlich carcinoma cells. These DNA molecules are considered as intermediate forms of the mobile elements. The role of these mobile elements in the regulation of RNA expression and tumor progression is discussed.

[Study of transcription of the DNA sequence of a mts271 clone in various tumor and normal mouse cells]. / Issledovanie transcriptsii posledovatelnostei DNK klona mts271 v razlichnykh opukholevykh i normal'nykh kletkakh myshi.

Transcription of the mts271 gene was studied in 27 mouse cell tumor lines. Transcripts of the gene have been found in 6 out of 7 different metastatic lines. No transcription of the gene has been observed in non-metastatic tumor lines. In normal tissues, specific gene DNA was only found in hemopoietic cells--in bone marrow, spleen, thymus and lymphocytes. Transcription of the gene mts271 is not dependent on the level of DNA methylation.

[Transforming activity of the DNA from human hepatoma cells. The transcription of integrated viral sequences and their cloning]. / Transformiruiushchaia aktivnost' DNK kletok gepatomy cheloveka. Transkriptsiia integrirovannykh virusnykh posledovatel'nostei i ikh klonirovanie.

A monolayer culture of mouse fibroblasts, line NIH/3T3, was transformed by DNA from human hepatoma cell line PLC/PRF/5. The resulting lines of transformed cells differ in their growth characteristics and are capable of producing tumors in nude mice. DNA of these cell lines was found to contain integrated sequences of human DNA and hepatitis B virus DNA. Analysis of transcription showed the amount of virus-specific RNA in the original cell line PLC/PRF/5 to be 20 times as large as in one of the transformed lines. Fragments of DNA of the transformed cells containing virus sequences were cloned, and 400,000 clones were obtained. A clone was identified the insert of which contains a portion of viral genome.

[Ascitic form the human cancer transplantable to thymus-deprived mice]. / Astsitnaia forma raka molochnoi zhelezy cheloveka, transplantiruemaia na bestimusnykh myshakh.

An ascitic form of tumor was obtained in thymus-free BALB/c mice from a solid strain of human mammary carcinoma. It passed 32 serial transplantations. The paper describes the kinetics of the tumor growth. Cytological and biochemical examinations of the ascitic form of mammary carcinoma demonstrated its appurtenance to human malignant tumors of the mammary gland.

[The evaluation of the mutagenic activity of pertussis vaccinal preparations]. / Otsenka mutagennoi aktivnosti kokliushnykh vaktsinal'nykh preparatov.

Two vaccine preparations obtained from Bordetella pertussis, whole-cell vaccine constituting one of the components of adsorbed DPT vaccine and acellular vaccine, were tested for mutagenicity. The doses of the preparations covered the range 1-100 ED50. Ames' test and the metaphase analysis of marrow cells of C57BL/6J mice were used. The acellular preparation was also tested on thymectomized mice, taking into consideration chromosomal aberrations in marrow metaphases. Whole-cell and acellular pertussis vaccines did not induce mutations in Salmonella typhimurium and chromosomal aberrations in mouse marrow cells.

[The effect of different Yersinia pestis antigens on the cellular link in immunity]. / Izuchenie vliianiia razlichnykh antigenov Yersinia pestis na kletochnoe zveno immuniteta.

Immunization with live plague vaccine has been shown to give no protection to thymectomized mice from subcutaneous challenge with Y. pestis virulent strain. Under the action of the vaccine or individual Y. pestis antigens (fraction I) the functional and morphological activation of thymocytes and macrophages is observed, more pronounced in C57BL/6 mice and less pronounced in CBA mice. Y. pestis antigenic preparations (fractions I and II, pesticin) act as T-cell mitogens and are thus capable of inducing the in vitro proliferation of thymocytes. At the same time the in vivo action of fraction II induces a decrease in the level of lymphocytes in the peripheral blood of mice and the destruction of lymphocytes in their thymus and spleen.

[Comparative study of platinum complexes in athymic mice with human tumors]. / Sravnitel'noe izuchenie kompleksov platiny na bestimusnykh myshakh s opukholiami cheloveka.

Tumoricidal activity of Soviet-synthesized oxoplatinum and cycloplatam was shown to influence human tumor strains (melanoma, cancer of the kidney, Burkitt's lymphoma) transplanted to nude mice. Their therapeutic effect was associated with lymphopenia; however, they did not suppress the chemically determined activity of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase.

[Effects of plant polysaccharide paliustran on the growth of human tumor transplants in athymic mice]. / Vliianie rastitel'nogo polisakharida paliustrana na rost transplantatov opukholei cheloveka u bestimusnykh myshei.

Plant polysaccharide palyustran inhibited the growth of human lung carcinoma P-1 transplanted to athymic mice by 60% but failed to do so in human rhabdomyosarcoma. Treatment with palyustran was followed by a 2-fold decrease in lympho- and granulocyte levels, selective inhibition of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase activity in tumor cells and--in single cases--metastatic involvement of the liver.