HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing.

Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.

Associations between six classical HLA loci and rheumatoid arthritis: a comprehensive analysis.

Although the HLA region contributes to one-third of the genetic factors affecting rheumatoid arthritis (RA), there are few reports on the association of the disease with any of the HLA loci other than the DRB1. In this study we examined the association between RA and the alleles of the six classical HLA loci including DRB1. Six HLA loci (HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1) of 1659 Japanese subjects (622 cases; 488 anti-cyclic citrullinated peptides (CCP) antibody (Ab) positive (82.6%); 103 anti-CCP Ab negative (17.4%); 31 not known and 1037 controls) were genotyped. Disease types and positivity/negativity for CCP autoantibodies were used to stratify the cases. Statistical and genetic assessments were performed by Fisher's exact tests, odds ratio, trend tests and haplotype estimation. None of the HLA loci were significantly associated with CCP sero-negative cases after Bonferroni correction and we therefore limited further analyses to using only the anti CCP-positive RA cases and both anti-CCP positive and anti-CCP negative controls. Some alleles of the non-DRB1 HLA loci showed significant association with RA, which could be explained by linkage disequilibrium with DRB1 alleles. However, DPB1*02:01, DPB1*04:01 and DPB1*09:01 conferred RA risk/protection independently from DRB1. DPB1*02:01 was significantly associated with the highly erosive disease type. The odds ratio of the four HLA-loci haplotypes with DRB1*04:05 and DQB1*04:01, which were the high-risk HLA alleles in Japanese, varied from 1.01 to 5.58. C*07:04, and B*15:18 showed similar P-values and odds ratios to DRB1*04:01, which was located on the same haplotype. This haplotype analysis showed that the DRB1 gene as well as five other HLA loci is required for a more comprehensive understanding of the genetic association between HLA and RA than analyzing DRB1 alone.

Chemotherapy of human yolk sac tumor heterotransplanted in nude mice.

The chemotherapeutic effects of cis-diamminedichloroplatinum + vinblastine + bleomycin (PVB) on 3 human yolk sac tumors (YST-1, YST-2, and YST-3) of the ovary, which were heterotransplanted into BALB/c nude mice, were compared with the effects of vincristine + actinomycin D + cyclophosphamide (VAC), the combination currently favored for treatment of yolk sac tumors. PVB and VAC therapies were performed for 3 weeks. Both PVB and VAC significantly reduced the tumor volume of all the treated tumors. The mean weights of tumors in animals treated with PVB or VAC were, in percent of the mean tumor weight in untreated animals: 1.3 and 1.6 for YST-1, 2.5 and 3.3 for YST-2, and 5.5 and 2.7 for YST-3, respectively. A strong correlation was noted between tumor volume and alpha-fetoprotein level in the sera of mice bearing YST-1 or TST-2 tumors.

Correction of the HLA-DQB1*04:01:01 sequence at position 79 in exon 1.

The HLA-DQB1*04:01:01 sequence is corrected at position 79 in exon 1 (G > A, Ala > Thr).

The lca as an onco-fetal gene: its expression in human fetal liver.

The lca-transforming DNA was isolated from human hepatocellular carcinomas. This gene has no homology with known transforming DNA from human sources, and its role in neoplastic tissue formation has been left unanswered. In this communication, we report that RNAs prepared from human fetal livers hybridize to the lca DNA probe. The RNA is 1.8 kilobase in size and appears in the fetal liver only for a limited period during its development, viz. 19 weeks through 24 weeks of gestation. No other tissues carry detectable levels of the lca messenger RNA. Fetal hepatocytes at 5 weeks of gestation showed no transcripts of lca, but upon culturing for 2 more weeks in vitro, the cells became producers of the lca messenger RNA. These results suggest that the lca plays some role in the proliferative stage of the liver.

Heat, drugs, and radiation given in combination is palliative for unresectable esophageal cancer.

From April 1966 to April 1986, 101 men and women with unresectable squamous cell carcinoma of the esophagus were treated in our clinic. Since 1983, 21 were treated with a combination of hyperthermia, chemotherapy, and radiotherapy (group I). Before 1983, for another 80 patients, radiation plus chemotherapy had been prescribed (group II). Nine of 21 patients in group I had an unresectable carcinoma due to an advanced tumor, 9 had an associated severe clinical status, and 3 refused surgery. Out of 80 in group II, 50 had a far advanced tumor, 21 had a poor clinical condition, and 9 refused operation. With regard to staging, for 21 in group I, 6 were classified as Stage I, 5 as Stage II, 7 as Stage III, and 3 as Stage IV. As to the 80 in group II, those in Stage I, II, III, and IV accounted for 8, 22, 39, and 11, respectively. The median doses of each modality, for patients in group I, were 6 times of hyperthermia at 42-45 degrees C for 30 minutes, 40 Gy of X ray and 30 mg of bleomycin. For patients in group II, a median dose of 56 Gy of X ray was given. Response rates determined by esophagograms and endoscopies for the patients in groups I and II were 76.2% (16/21; 4 CRs, 12 PRs) and 39.2% (31/79; 2 Crs, 29 PRs), respectively (p less than 0.001). The effective rates determined by improvement in quality of life (relief of pain and dysphagia) for groups I and II were 61.9% and 37.2%, respectively. A longer survival was obtained for patients in group I (median survival: 9 months vs 6 months). Especially for the patients classified as Stage I, a significantly longer survival was obtained with a combination of hyperthermia, chemotherapy, and radiotherapy (p less than 0.01).

Tumor antigen TA-4: an aid in detecting post-operative recurrences of esophageal carcinoma.

Radioimmunoassay for serum TA-4 was performed in 35 patients with squamous cell carcinoma of the esophagus. The results were positive in 13 of 35 patients (37.1 per cent) in whom squamous cell carcinoma of the esophagus was present at the time of assay. Preoperatively, undetectable TA-4 levels in patients with esophageal carcinoma suggested localized tumor and a good prognosis, whereas, strongly positive TA-4 levels correlated with extensive tumors and a poor prognosis. Postoperatively, a positive result for TA-4 levels indicated the presence of a residual tumor. Negative TA-4 levels, however, did not exclude a residual tumor. Therefore, assessment of periodic TA-4 levels in patients who have undergone resection of esophageal carcinoma may lead to detection of a tumor recurrence.

Problems in esophageal bypass for unresectable carcinoma of the thoracic esophagus.

The clinical evaluation of esophageal reconstruction in both resectable and unresectable carcinoma of the thoracic esophagus is described. Our operative technique for obtaining a long gastric tube and creating and end-to-side anastomosis has been highly successful, and the anastomotic failure rate was remarkably lowered in patients with resectable carcinoma of the esophagus. However, anastomotic leakage remains a serious problem for those undergoing esophageal bypass for unresectable carcinoma of the esophagus. The most likely cause of anastomotic failure after bypass seems to be the tug on the anastomosis with each swallow.

Application of clonal analysis. Differential diagnosis for synchronous primary ovarian and endometrial cancers and metastatic cancer.

Simultaneous involvement of the endometrium and the ovary by carcinoma is a familiar problem in the routine practice of surgical pathology. Such cases may be considered either examples of a single primary carcinoma with metastasis or as synchronous primary neoplasms. The distinction between these two possibilities is made based on clinicopathologic observations, and therefore may not be definitive. In the present study, the authors used molecular techniques to analyze the clonal composition of five cases of concurrent adenocarcinomas of the endometrium and ovary that were clinicopathologically diagnosed as synchronous primary tumors. Patterns of X-chromosome inactivation, mutations in the K-ras gene, mutations or allelic loss of the p53 gene, or human papillomavirus detection were identical in both endometrial and ovarian lesions in three of the cases suggesting that those three cases represented single primary tumors with metastases. In both of the other two cases, the patterns of X-chromosome inactivation clearly demonstrated the presence of independent primary tumors. The application of molecular technology may play an important role for the differential diagnosis between synchronous primary carcinomas and a single carcinoma with metastasis.

Postoperative mechanical ventilation preventing the occurrence of pleural effusion after hepatectomy.

To determine factors leading to pleural effusion after hepatectomy, the frequency of pleural effusion was investigated in 68 patients who underwent hepatic resection, with or without dissection of the right coronary ligament. In 36 of 44 patients (81.8%) who underwent hepatic resection with complete dissection of the right coronary ligament, and in two of 24 (8.3%) who underwent the procedure without dissection of this ligament, pleural effusion developed in the right hemithorax (p less than 0.001). Difference in pressure between the abdominal and thoracic cavities was considered to lead to ascites in the chest via the diaphragmatic triangular area (where there is no parietal peritoneum). While investigating the preventive effect of mechanical ventilation following hepatic resection on the occurrence of pleural effusion in 12 patients, we concluded that effusion could be prevented with use of mechanical ventilation--an approach that retained the intrathoracic pressure in a positive state.

Ultrastructure of molar trophoblast. Observations on hydatidiform mole and chorioadenoma destruens.

The ultrastructure of molar trophoblast is described in hydatidiform mole and chorioadenoma destruens. Villous molar trophoblast exhibits only minor deviations from normal early villous trophoblast. Invasive trophoblast of chorioadenoma destruens shows features of more-active cell growth, multiplication, differentiation, and degradation. Evidence for evolution of highly differentiated syncytial trophoblast via intermediate stages from primitive undifferentiated cytotrophoblast is confirmed. Cytoplasmic inclusions resembling virus-like particles, observed in chorioadenoma destruens, have not been hitherto described in normal or pathologic trophoblast.

Clinical efficacy of the stapled anastomosis in esophageal reconstruction.

We have clinically evaluated Russian and American stapling devices used for esophageal reconstruction and compared the results following stapled anastomosis with those following hand-sutured procedures, both performed by the same surgeon. With the Russian stapler, anastomoses performed in 17 patients with carcinoma of the thoracic esophagus resulted in only one (5.9%) anastomotic leak; in 12 hand-sutured anastomoses and in 11 anastomoses done with an American stapler, no anastomotic complication occurred. Thus, a long gastric tube with good blood supply is of paramount importance, and if the technical details of surgical stapling can be overcome, a stapled anastomosis appears to be as safe as a manual suturing for patients undergoing esophageal reconstruction.

Use of intragastric balloons for weight reduction. An experimental study.

The effect of intragastric balloons as a noninvasive method for weight reduction was observed in nine castrated Yorkshire pigs. None of the animals lost weight. The appetites of the experimental animals were inhibited for only a short period of time. A significant dilatation of the stomach after balloon insertion was noted which resulted from increased food intake and obliterated the effect of the intragastric balloons. A long-standing lower blood glucose level played an important role in the increments in food intake and dilatation of the stomach. At the same time, ulceration was seen in 83.3 percent of the animals that received balloons. We conclude that intragastric balloon therapy may not be an effective approach for managing morbid obesity in human subjects.

Intestinal valve formation with a simple invagination technique.

In 17 fully conditioned mongrel dogs, the small intestine approximately 50 cm proximal to the ileocecal valve was divided. Three centimeters of the proximal bowel was then inserted into the distal bowel and anastomosis was performed using interrupted 3-0 Dexon sutures in the seromuscular layer of the proximal end and through the entire thickness of the distal end. Two to four dogs were then sacrificed 2, 4, 6, 8, and 10 weeks postoperatively. Two dogs died 3 and 4 weeks postoperatively due to mechanical obstruction just proximal to the anastomosis by hair bezoar, and 15 survived without complications. Retrograde contrast radiologic examination of all segments showed no reflux. Microscopically, the serosal surface of the invaginated segment was found to be gradually covered with mucosa, and the whole segment formed a valvelike structure within 10 weeks time.

Experimental study of singly placed staples for an everted intestinal anastomosis.

This study was undertaken to confirm the technical feasibility and efficacy of an intestinal anastomosis created with singly placed staples in dogs. In 12 fully conditioned dogs, two end-to-end small bowel anastomoses were performed in an everted manner. In each dog, one anastomosis was performed with singly placed staples utilizing the Auto Suture 4.8 mm disposable skin stapler, and the other anastomosis was performed with a manual technique using 3-0 Dexon interrupted suture. Operation time was measured for each anastomosis. Dogs were sacrificed 2, 4, and 8 weeks postoperatively. Each anastomosis was examined grossly and microscopically, and its tensile strength was measured. All dogs survived without complications. Gross and microscopic examinations showed satisfactory healing of both types of anastomoses at different postoperative intervals. The stapled anastomosis required significantly shorter time (p less than 0.01), and had significantly greater tensile strength (p less than 0.05) when compared with manual anastomosis.

Subclavian artery aneurysm associated with absence of the ipsilateral internal carotid artery.

A 48-year-old male presented with a rare subclavian artery aneurysm associated with absence of the ipsilateral internal carotid artery. The aneurysm was resected and replaced with a Gore Tex artificial graft. Computed tomography, angiography and cerebral blood flow findings suggest that defect of the right internal carotid artery occurred in the developmental stage. There was no past history of trauma, nor histological evidence of inflammatory or sclerotic changes in the aneurysmal wall. The two vascular lesions may have been influenced by a synchronous causative factor in his developmental stage.

Adjuvant long-term immunochemotherapy for squamous cell carcinoma of the thoracic esophagus.

Between 1965 and 1978, 131 patients with primary squamous cell carcinoma of the thoracic esophagus underwent subtotal esophagectomy. Since 1972 we hae used active therapy involving pre- and postoperative irradiation and postoperative long-term cancer immunochemotherapy. When the surgical results of the active therapy and the historical control group, in which postoperative radiotherapy was used primarily, were compared, active therapy appeared to be high effective. The one, two, and three-year survival rates were 54.7, 36.3, and 27.1 per cent, respectively, in the former, and 32, 14, and 12 12 per cent, respectively, in the latter group. With respect to noncurative cases, the one-year survival rate was 42.2 per cent in the active therapy group, and 20 per cent in the control group.

The healing and tensile strength of the gastroplasty staple line. An experimental study.

Inverted and everted staple lines, with and without division of the anterior gastric wall, were performed in 15 mongrel dogs to simulate reduction gastroplasty staple line. There were no significant differences in tensile strength and healing between staple lines at different postoperative periods. The tensile strength of the inverted staple line seemed to be stronger due to breakage of the adjacent gastric tissue. However, the tensile strength of the everted stapling without division of the gastric wall, which is used clinically, was almost equal to that of normal gastric tissue. In addition, there was satisfactory healing 4 weeks postoperatively in the everted stapling without dividing the gastric wall. These results suggest that the everted staple line without division of stomach is not inherently weaker to any significant extent than if the gastric wall was divided or inverted. The reported failure rate of this procedure may be due to other factors.

Early gastric cancer and a concomitant pregnancy.

Gastric cancer associated with pregnancy is most often associated with a poor prognosis because the cancer stage is usually advanced at the time of diagnosis. The symptoms are frequently masked by factors related to the normal pregnancy and diagnostic approaches are restricted by physical and psychological clinical events. The authors treated a 30-year-old woman at 35 weeks of gestation. She had episodes of hematemesis and a prompt diagnosis of early gastric cancer was made following observations using a gastroduodenal fiberscope. A living normal child was delivered following oxytocin-induced labor and, 18 days later, curative resection for the gastric cancer was done. The patient has been doing well for over 16 months at this writing, with no evidence of recurrence. Based on the author's experience and a review of the literature, they conclude that early gastric cancer detected during pregnancy has a satisfactory prognosis and that use of the gastrofiberscope facilitates an early diagnosis in a pregnant subject with untoward symptoms of the GI tract.

[A case of pituitary mass suggestive of lymphocytic adenohypophysitis].

A 29-year-old woman suffered from frontalgia with vomiting and visual field defects in the 9th month of pregnancy. Two weeks after delivery, a pituitary mass with suprasellar extension was demonstrated on computed tomography (CT). Angiogram revealed elevation of bilateral A1 segments and right persistent primitive trigeminal artery. Repeated CT performed three months after delivery showed that the pituitary mass had undergone a spontaneous regression in size, and her visual impairment improved. The pituitary function tests revealed no response to ACTH, GH and PRL stimulation performed about a year after delivery. Laboratory data showed high titer of antinuclear antibody and low titer of complement. This case suggests that some patients with postpartum hypopituitarism suggestive of lymphocytic adenohypophysitis, and with pituitary mass need not have early surgical intervention but may be closely observed and treated by hormone replacement alone.