Identification of Lck-derived peptides capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients with distant metastases.

The Lck protein (p56(lck)), a src family tyrosine kinase essential for T cell development and function, is aberrantly expressed in various types of cancers. We revealed recently that Lck can be a tumor antigen recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs) of cancer patients with metastases. In this study, we tried to identify Lck-derived epitopes capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients. The tumor-infiltrating lymphocytes (TILs) from 2 HLA-A2 cancer patients were found to respond to COS-7 cells when co-transfected with the lck gene and either HLA-A0201, -A0206, or A0207 cDNA. These TILs contained CTLs capable of recognizing either the Lck(61-69), the Lck(246-254), or the Lck(422-430) peptide among 24 different peptides, all of which were prepared based on the HLA-A2 binding motif. Importantly, in vitro sensitization with the latter 2 peptides induced tumor-specific CTLs in HLA-A2(+) cancer patients with metastases, but not in those without metastases. Overall, the Lck(246-254) and Lck(422-430) peptides could be useful for specific immunotherapy of HLA-A2(+) cancer patients, especially with distant metastases.

Identification of cyclophilin B-derived peptides capable of inducing histocompatibility leukocyte antigen-A2-restricted and tumor-specific cytotoxic T lymphocytes.

We recently suggested that cyclophilin B (Cyp-B) is a tumor antigen recognized by histocompatibility leukocyte antigen (HLA)-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we tried to identify Cyp-B-derived epitopes, which can induce HLA-A2-restricted and tumor-specific CTLs in cancer patients. The tumor-infiltrating lymphocytes (TILs) from an HLA-A0207 patient with colon cancer were found to respond to COS-7 cells when co-transfected with the Cyp-B gene and either HLA-A0201, -A0206, or -A0207 cDNA. These TILs contained CTLs capable of recognizing either the Cyp-B(129 - 138) or the Cyp-B(172 - 179) peptide among 28 different peptides, all of which were prepared based on the HLA-A2 binding motif. Both Cyp-B peptides possessed the ability to induce tumor-specific CTLs in HLA-A2(+) cancer patients. Cyp-B(172 - 180 (V)), which is a 9-mer peptide with valine added at the C terminus, showed no clear superiority over the parental Cyp-B(172 - 179) peptide in an in vitro sensitization experiment. In vitro-sensitized T cells with these peptides responded to cancer cells in an HLA-A2-restricted manner. These two Cyp-B peptides could be useful for specific immunotherapy of HLA-A2(+) cancer patients.

Molecular basis of T cell-mediated recognition of pancreatic cancer cells.

Pancreatic cancer continues to be a major unsolved health problem in the world. The prognosis of pancreatic cancer is extremely poor with a median survival of 3-4 months and the 5-year survival being 1-4%. This poor prognosis is primarily because of a lack of effective therapies, and thus development of new treatment modalities is needed. One of these treatments could involve specific immunotherapy, for which elucidation off the molecular basis of T cell-mediated recognition of cancer cells is required. We report here six different genes and 19 immunogenic epitopes from pancreatic adenocarcinoma cells and T-cell receptor beta usage of HLA-A2-restricted CTL clones reacting to some of these epitopes. Sixteen of 19 epitopes were found to possess the ability to induce HLA-A2-restricted CTL activity in the peripheral blood lymphocytes of patients with pancreatic and also colon adenocarcinomas. These results should provide a scientific basis for the development of specific immunotherapy for pancreatic and colon cancer patients.

Recognition of the Lck tyrosine kinase as a tumor antigen by cytotoxic T lymphocytes of cancer patients with distant metastases.

The Lck protein (p56(lck)), a src family tyrosine kinase that is essential for T cell development and function, is aberrantly expressed in metastatic colon cancers. p56(lck) seems to facilitate the malignant transformation of epithelial cells through initiation of anchorage-independent proliferation. We demonstrate that the lck gene encodes antigenic epitopes recognized by the HLA class I-restricted and tumor-specific CTL of metastatic cancer patients. Lck peptides augmented CTL activity in peripheral blood mononuclear cells (PBMC) of colon and other epithelial cancer patients with distant metastases, but not those without distant metastases. CTL precursors recognizing the Lck peptide were identified in freshly prepared PBMC of patients with distant metastases, and their frequency was significantly augmented by stimulation with the peptide. Thus, Lck peptides could be useful in developing a specific immunotherapy for cancer patients with distant metastases.

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer.

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer.

Molecular cloning of a glycosylphosphatidylinositol-anchored molecule CDw108.

CDw108, also known as the John-Milton-Hagen human blood group Ag, is an 80-kDa glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that is preferentially expressed on activated lymphocytes and E. The molecular characteristics and biological function of the CDw108 were not clarified previously. In this manuscript, we identify the cDNA clone containing the entire coding sequence of the CDw108 gene and report its molecular characteristics. The 1998-base pairs of the open reading frame of the cloned cDNA encoded a protein of 666 amino acids (aa), including the 46 aa of the signal peptide and the 19 aa of the GPI-anchor motif. Thus, the membrane-anchoring form of CDw108 was the 602 aa, and the estimated molecular mass of the unglycosylated form was 68 kDa. The RGD (Arg-Gly-Asp) cell attachment sequence and the five potential N-linked glycosylation sites were located on the membrane-anchoring form. Flow cytometric and immunoprecipitation analyses of the CDw108 cDNA transfectants confirmed that the cloned cDNA encoded the native form of CDw108. The CDw108 mRNA was expressed in activated PBMCs as well as in the spleen, thymus, testis, placenta, and brain, but was not expressed in any other tissues tested. Radiation hybrid mapping indicated that the CDw108 gene was located in the middle of the long arm of chromosome 15 (15q23-24). This molecular information will be critical for understanding the biological function of the CDw108 Ag.

Sequence variation of allele 27 at the D1S80 locus.

In 180 unrelated Japanese individuals 18 examples of allele 27 were detected at the locus D1S80 (MCT118). On 6% polylacrylamide gels 5 out of these 18 alleles were found to migrate between allele 26 and allele 27, but closer to allele 27, and thus were labelled variants of allele 27. All 18 examples of allele 27 were sequenced and the results were compared. Although all had the same number of base pairs (578 bp) the five variants could be subdivided into three types. V1, V2 and V3. The variants and the standard allele were composed of the same kinds of repeat units, but the order of arrangement was different. We investigated whether it was possible to distinguish the standard allele 27, and the variants V1, V2, and V3 by PCR-RFLP. EcoRII and MspI which have restriction sites within the repeat units were adopted as restriction enzymes. The variants could be discriminated from each other after treatment of the PCR fragments with EcoRII or MspI, followed by PAG electrophoresis.

ACTBP2 gene frequency distribution and sequencing of the allelic ladder and variants in the Japanese and Chinese populations.

The human beta-actin related pseudogene H-beta-Ac-psi-2 (ACTBP2) gene frequency distributions in the Japanese and Chinese Han populations were investigated and compared. Analysis was carried out by applying fluorescently labeled samples and a differently labeled sequenced allelic ladder within the same lanes in denaturing gels, followed by laser detection and automated analysis using Genescan software 672. The discrimination index and the heterozygosity index were calculated to be 0.993 and 0.916 in the Japanese population, and 0.993 and 0.944 in the Chinese Han population, respectively. No deviation from Hardy-Weinberg equilibrium was observed in these two populations. The allelic ladder, which ranged from 233 bp to 319 bp, was constructed from a combination of 23 regularly occurring alleles. The allelic ladder and 12 variants observed in 24 individuals in these two populations were sequenced. The variants could be divided into three types according to their structural variation characteristics. These variants differed from the alleles of the same repeats in the allelic ladder by the presence or absence of hexanucleotides in the central repeat regions, base deletions in the flanking regions, and base insertions in the repeat units.

The allele frequency distribution at VNTR locus D17S5 (YNZ22) in a Japanese population sample.

The allele frequencies for the YNZ22 locus were determined in a Japanese population sample (n = 164) using the polymerase chain reaction (PCR). We observed 10 alleles and 39 genotypes. Allele distributions in our data showed a different pattern from those reported in the literature for European Caucasians. No deviation from Hardy-Weinberg equilibrium was found. The observed heterozygosity was 83.5%. In Japan the YNZ22 is one of useful genetic markers for paternity testing and identify testing, with a chance of exclusion (CE) value of 68% and a power of discrimination (PD) value of 89%.

Allele distribution of a VNTR polymorphism at D4S43 locus in the Japanese population.

Allele frequencies for the VNTR locus D4S43 were determined in a Japanese population sample by the polymerase chain reaction. Nine different alleles containing 1-15 repeats of the basic 14 bp unit were observed in 156 unrelated Japanese. The most common allele was one repeat unit (62.82%), and the next common alleles were those with 11 (14.42%) and 7 repeat units (12.50%). We estimated that D4S43 locus has a heterozygosity index of 0.56, a mean exclusion chance of 0.32, a discrimination rate of 0.76, and a polymorphic information content of 0.52. No evidence of significant deviations from the Hardy-Weinberg equillibrium was found in the Japanese population data.

Chinese Han population study of the D1S80 (pMCT118) locus polymorphisms.

The Chinese Han population was studied for the D1S80 (pMCT118) locus. Bloodstain samples of 216 unrelated individuals were tested through Chelex extraction and the polymerase chain reaction (PCR). After amplification, the genotypes were separated by polyacrylamide gel (PAG) electrophoresis and stained with ethidium bromide. A total of 70 genotypes and 25 alleles were distinguished. Alleles 18, 24 and 30 were the most common alleles in Chinese population. The distribution of the observed genotypes conformed to the Hardy-Weinberg equilibrium for the D1S80 (pMCT118) system. The chance of exclusion, the discriminating power (DP), and the heterozygosity for this system were 0.71, 0.90, and 0.85, respectively, in Chinese population.

Automated DNA typing of ACTBP2: a comparison of allelic ladder with GS 2500 as internal marker.

A comparison was made between the size values of the alleles measured by the internal size marker of Genescan (GS 2500) and the size values measured by an allelic ladder constructed by our laboratory in the application of Genescan software for the typing of the ACTBP2 system. The measurement by GS 2500 showed a greater than 2 bp divergence (SD = 0.893) while the measurement by the allelic ladder showed only 0.1 bp divergence (SD = 0.008). Thus in genotyping of ACTBP2 by Genescan software, the method presented here of using the allelic ladder as the size marker instead of GS 2500 is easier and more reliable.

Nested amplification of COL2A1 3' variable region in skeletal remains.

We identified skeletal remains of a highly decomposed body by amplifying the COL2A1 3' variable region. We extracted degraded and contaminated DNA from teeth and bone fragments and amplified the hyper variable regions of COL2A1 by nested PCR. This method is 2,000 times more sensitive than the 27 cycle standard PCR. The sensitivity and specificity of the amplification was extremely improved using nested primer pairs. Using 47 cycles of total amplification by nested PCR, we were able to effectively amplify the target fragment. A total 47 cycles of dual PCR using one pair of primers was not as sensitive as nested PCR, which enabled us to amplify and detect the fragment from 5 pg of template DNA by agarose gel electrophoresis with ethidium bromide staining. We found that the hypersensitive nested PCR was suitable for the forensic identification of old, unidentifiable skeletal remains.

Effect of prior administration of succinylcholine on duration of action of vecuronium during enflurane anaesthesia.

The effects of succinylcholine, which was given to facilitate tracheal intubation on the duration of action of subsequently administered vecuronium bromide, were evaluated in 54 adult patients who underwent abdominal surgeries under enflurane anaesthesia. The electromyographic response to train-of-four ulnar nerve stimulation was measured. Twenty-seven patients received 1 mg.kg-1 of succinylcholine, followed by 0.15 mg.kg-1 of vecuronium when the electromyographic response recovered to 50% of control after succinylcholine-induced neuromuscular blockade. The other 27 patients served as the control group, receiving 0.15 mg.kg-1 of vecuronium without prior administration of succinylcholine. In both groups, administration of supplemental 0.04 mg.kg-1 of vecuronium was repeated whenever the electromyographic response recovered to 25% of control during surgical procedures. The duration of blockade induced by the initial 0.15 mg.kg-1 of vecuronium was 56.5 +/- 12.8 (mean +/- s.d.) min for the group with succinylcholine, and 58.5 +/- 21.5 min for the control group. In both groups, the average duration of four consecutive supplemental doses of vecuronium was approximately 35 min. No significant differences between groups were found in the duration of neuromuscular blockade induced by initial and supplemental doses of vecuronium.

[The effects of postoperative continuous epidural analgesia on the early recovery from lower abdominal surgery].

This study was conducted to evaluate the effects of continuous epidural analgesia (CEA) on the incidence of postoperative complications and the early recovery after lower abdominal surgery. A total of 109 patients who had received elective lower abdominal surgery were investigated retrospectively by separating them into two groups. Compared to 35 patients who had received standard analgesic techniques without epidural analgesia, 74 patients who had been administered CEA with buprenorphine, mepivacaine and droperidol for 24 hrs after surgery could sit on the bed significantly earlier. But the patients with CEA could not stand on the floor and could not walk significantly earlier than the patients without CEA. The overall postoperative complication rate was not significantly different between the patients with and without CEA. These results show that postoperative CEA exerts a beneficial effects on the early recovery after the lower abdominal surgery, but the effect is not so strong as in upper abdominal surgery group. The results also suggest that CEA does not decrease the incidence of postoperative complications.

[Prostaglandin E1 (PGE1) prevents the pressure responses to tracheal intubation in hypertensive patients].

Forty-five hypertensive patients for elective abdominal surgery were investigated regarding the effects of PGE1 on the cardiovascular responses to tracheal intubation. Administration of PGE1 at the dose of 0.10 or 0.20 micrograms.kg-1.min-1 for 10 minutes before tracheal intubation significantly reduced the blood pressure responses immediately after the intubation and 2 minutes later. The increases in heart rate were not altered with and without the administration of PGE1. So the increases in rate pressure products were markedly reduced with PGE1 compared with the control values. Plasma concentration of catecholamines was measured before and after tracheal intubation. Norepinephrine was elevated markedly immediately after the intubation and this change was not affected by the infusion of PGE1. These results demonstrate that PGE1 ameliorates the pressure responses by the release of norepinephrine and thus reduces the increases in rate pressure products immediately after tracheal intubation.