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Biomedical subjects

R Stan

Publications and source records attributed to R Stan.

14 recordsLinked to original sources
PubMed2006

In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring.

BACKGROUND: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials. METHODS: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase. RESULTS: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12). DISCUSSION: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.

Antigen-Presenting Cells↗
PubMed2001

[Orbital tuberculoma].

We present a case of orbital tumor with skin lesions at the lateral cantus. CT scan confirmed the orbital tumor and we considered it to be malignant. The histological examination of a piece of the tumor showed tuberculous characteristics. Patient received treatment with antituberculous drugs and steroids. The tumor decreased after two months of treatment.

Aged↗
PubMed2000

DNA helicases, genomic instability, and human genetic disease.

DNA helicases are a highly conserved group of enzymes that unwind DNA. They function in all processes in which access to single-stranded DNA is required, including DNA replication, DNA repair and recombination, and transcription of RNA. Defects in helicases functioning in one or more of these processes can result in characteristic human genetic disorders in which genomic instability and predisposition to cancer are common features. So far, different helicase genes have been found mutated in six such disorders. Mutations in XPB and XPD can result in xeroderma pigmentosum, Cockayne syndrome, or trichothiodystrophy. Mutations in the RecQ-like genes BLM, WRN, and RECQL4 can result in Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively. Because XPB and XPD function in both nucleotide excision repair and transcription initiation, the cellular phenotypes associated with a deficiency of each one of them include failure to repair mutagenic DNA lesions and defects in the recovery of RNA transcription after UV irradiation. The functions of the RecQ-like genes are unknown; however, a growing body of evidence points to a function in restarting DNA replication after the replication fork has become stalled. The genomic instability associated with mutations in the RecQ-like genes includes spontaneous chromosome instability and elevated mutation rates. Mouse models for nearly all of these entities have been developed, and these should help explain the widely different clinical features that are associated with helicase mutations.

Amino Acid Sequence↗
PubMed2000

[The influence of meteorological factors for frequency of Herpes simplex keratitis].

HYPOTHESIS: The variation of the meteorological factors could be a stress for the organism, which could determine the start of some diseases. MATERIAL AND METHOD: We studied 252 cases of herpes simplex keratitis which had been hospitalized in Eye Clinic of Cluj between 1983-1991. The selection criteria were: known diagnosis; day of the rise could be exactly established; disease was taken place in Cluj or in region not longer than 30 km on Somes Valley. The meteorological parameters were collected by specialist from the Meteorological Station of the city and from the Cluj's Airport. The results were verified statistical and were guaranted by p = 0.05. RESULTS: The herpes simplex keratitis are more frequently in days with a temperature higher and an atmospheric pressure lower than the average value of the season. DISCUSSIONS: We drew a possible pathogenic mechanism to explained the reactivation of the virus under the influence of warm weather.

Adult↗
PubMed1999

A role for PML and the nuclear body in genomic stability.

The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells. The Bloom syndrome gene BLM encodes a RecQ DNA helicase, whose absence from the cell results in genomic instability epitomized by high levels of sister-chromatid exchange (SCE) and cancer predisposition. We show here that BLM co-localizes with PML to the NB. In cells from persons with Bloom syndrome the localization of PML is unperturbed, whereas in APL cells carrying the PML-RARalpha oncoprotein, both PML and BLM are delocalized from the NB into microspeckled nuclear regions. Treatment with retinoic acid (RA) induces the relocalization of both proteins to the NB. In primary PML-/- cells, BLM fails to accumulate in the NB. Strikingly, in PML-/- cells the frequency of SCEs is increased relative to PML+/+ cells. These data demonstrate that BLM is a constituent of the NB and that PML is required for its accumulation in these nuclear domains and for the normal function of BLM. Thus, our findings suggest a role for BLM in APL pathogenesis and implicate the PML NB in the maintenance of genomic stability.

Adenosine Triphosphatases↗
PubMed1999

Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes.

BLM is a DNA helicase encoded by a gene which is mutated in persons with Bloom's syndrome. The protein is a member of the RecQ subfamily of helicases and contains a central domain constituted by the seven motifs conserved in all helicases. In contrast, the N-terminal portion of BLM lacks similarity to any other known proteins or motifs. We have expressed the first 431 amino acids of this domain as a fusion to a hexahistidine tag (BLM N431) in Escherichia coli. A method of purification was developed which involves elution from Ni-NTA resin in imidazole and EDTA, followed by treatment with DTT and gel filtration on Sephacryl-300. The treatment with EDTA and DTT prevents and disrupts aggregation of BLM N431. The purified protein appears to form hexamers and dodecamers, suggesting that the N-terminal domain of BLM is involved in the organization of the quaternary structure of BLM.

Adenosine Triphosphatases↗
PubMed1994

Interaction between FKBP12-rapamycin and TOR involves a conserved serine residue.

The yeast TOR1 and TOR2 proteins were previously discovered as putative targets of the immunosuppressive drug rapamycin. Although their cellular function is unknown, they are predicted to be at least 215 kDa in size and possess a C-terminal phosphatidylinositol (PI) kinase-related domain. We previously identified a conserved Ser residue, within the PI kinase-related domain of both yeast TOR proteins (Ser1972 in TOR1; Ser1975 in TOR2), as being the site of missense mutations conferring dominant rapamycin resistance. The Ser1972/1975 residue of yeast TOR is conserved in mammalian TOR homologs. One possibility is that this residue is critical for a direct interaction between TOR and the FKBP12-rapamycin complex. There is very recent biochemical evidence for an interaction between mammalian TOR and FKBP12-rapamycin (Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758; Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994) Cell 78, 35-43). Using the yeast two-hybrid system, we now have obtained genetic proof of a physical interaction between FKBP12-rapamycin and TOR and have demonstrated that this interaction requires the conserved Ser residue. We have found that a small fragment of wild-type yeast TOR2 spanning Ser1975 is capable of interacting with human FKBP12 in the presence of rapamycin, whereas an Arg1975 mutant fails to interact. This effect is dependent upon rapamycin and is antagonized by FK506.

Amino Acid Sequence↗
PubMed1992

Ergosterol levels in two L-methionine-enriched mutants of the methylotrophic yeast Candida boidinii ICCF26.

Two L-methionine-enriched mutants, SN-78 and SE-57, were isolated in a sulphur-deficient medium from the methylotrophic yeast Candida boidinii ICCF26. No significant differences were detected between the L-cysteine pools of the mutants and the wild-type. In mutant strain SE-57, S-adenosylmethionine and ergosterol levels were higher than in the wild-type strain, while in the other mutant, SN-78, the levels were lower. The evidence presented would suggest that, in both the mutants and the wild-type used in this study, S-adenosylmethionine was of key importance for the accumulation of L-methionine and ergosterol.

Candida↗