Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.     

Origin of the increased activity of beta-glucuronidase in the soluble fraction of rat mammary tumors during ovariectomy-induced regression

The activity of beta-glucuronidase in methyl-N-nitrosourea-induced rat mammary tumors regressing after ovariectomy was studied. beta-Glucuronidase, acid phosphatase, and beta-hexosaminidase were similarly increased in the lysosome-rich fraction of regressing compared to growing tumors, whereas in the soluble fraction, a 5-fold increase in the activity of beta-glucuronidase midway regression was not paralleled by an increase in the specific activity of the other two enzymes. Results of sedimentability studies indicated an equal stability of the lysosomes at the two tumor conditions. The elevated beta-glucuronidase activity in the soluble fraction was completely precipitable by rabbit monospecific antisera against beta-glucuronidase from other species. The activity in the soluble and in the lysosome-rich fraction showed a similar pH dependence and electrophoretic mobility of immunoreactive subunits, and only minor differences in affinity towards concanavalin A:Sepharose. Thus, the increased "soluble" beta-glucuronidase activity is neither tumor nor cytosol specific. Cell death during tumor regression is believed to occur according to a controlled sequence of event, called apoptosis. Dying cells become apoptotic bodies which are further degraded during phagocytosis by neighboring tumor cells. We discuss that breakage of these bodies during homogenization of the tumor is the cause for the observed increase in soluble beta-glucuronidase activity, while the lysosomes of the ingesting tumor cells remain intact. Physiologically, the enhanced intratumoral availability of beta-glucuronidase and other lysosomal enzymes might facilitate the hydrolysis of conjugates of a variety of xenobiotics and, possibly, also of steroid hormones.     

Interleukin-10 promoter polymorphisms in rheumatoid arthritis and Felty's syndrome

OBJECTIVE: To examine whether promoter polymorphisms associated with variation in interleukin-10 (IL-10) production are relevant to the development of rheumatoid arthritis (RA) or Felty's syndrome (FS). METHODS: DNA was obtained from 44 FS patients, 117 RA patients and 295 controls. The promoter region between -533 and - 1120 was amplified by polymerase chain reaction, and polymorphisms detected by restriction enzyme digest or sequence-specific oligonucleotide probing. RESULTS: We found no significant difference in allele or haplotype frequencies between the groups. CONCLUSION: There is no association between FS or RA and these recently identified IL-10 promoter polymorphisms. Other genetic or environmental factors could explain the alterations in IL-10 levels seen in these conditions.      

Purging of acute myeloid leukemic cells by ether lipids and hyperthermia

Ether lipids (EL) and hyperthermia have been shown to possess a relatively selective cytotoxicity to leukemic cells. In this study, the combined effects of EL (ET-18-OCH3, ET-16-NHCOCH3, or BM 41.440) and hyperthermia on the growth of hematopoietic progenitors, myeloid leukemic cell lines, and leukemic cells obtained from patients with acute myeloid leukemia (AML) were examined to determine if this combination resulted in a greater selective killing of leukemic cells than that achieved by either EL or heat alone. When the cells were treated simultaneously with EL (50 micrograms/mL) and hyperthermia (42 degrees C) for one hour, the killing of leukemic cell line cells was enhanced considerably. Among the three EL, however, the combination of ET-18-OCH3 and heat seemed to be the most cytotoxic to leukemic cell line cells with no effect on the growth of hematopoietic progenitors. An increase in the duration of treatment with ET-18-OCH3 to four hours with heat added during the last hour resulted in a further reduction of leukemic cell line cells while sparing 50% of hematopoietic progenitors after cryopreservation. The combined treatment with ET-18-OCH3 and heat also inhibited the growth of leukemic progenitors obtained from AML patients by 97% to 100%. These data indicate that the combined treatment with EL and hyperthermia might offer an efficient means to eliminate myeloid leukemic cells in vitro.     

完全腹腔镜Roux—en—Y吻合术式应用于远端胃癌根治术20例

目的探讨完全腹腔镜Roux．en—Y吻合术式应用于远端胃癌根治术的安全性和可行性。方法回顾性分析福建省肿瘤医院腹部外科2012年8月至2013年3月对腹腔镜胃大部切除术后实施完全腹腔镜Roux．en—Y吻合术的20例胃癌患者的术中和术后临床资料。结果20例患者均成功实施完全腹腔镜远端胃癌根治术,无一例中转开腹或中转腹腔镜辅助手术。手术时间（190．8±53．6）min,术中出血量（122．4±57．7）ml,淋巴结清扫数（31．2±5．7）枚,术后病理切缘均为阴性。术后排气时间为（2．6±1．6）d,住院时间为（8．1±2．0）d。有1例术后出现肺部感染,但无吻合术相关并发症发生。结论完全腹腔镜Roux—en—Y吻合术式应用于远端胃癌根治术安全且可行。     

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1- stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.