A novel subset of human lymphocytes with a T cell receptor-gamma complex.

We have previously characterized a CD3+ T cell receptor (TCR) alpha/beta- human fetal cloned cell line, termed F6C7, which surface-expresses a CD3-associated gamma chain identified by anti-NKFi, an mAb with a restricted clonotypic reactivity. Here, we have produced an additional antibody, anti-Ti-gamma A, which recognizes a public epitope of the gamma molecule defined by anti-NKFi. Ti-gamma A is present on approximately 3% of circulating lymphocytes with a wide range (1-15%) among 30 healthy individuals tested. Two-color immunofluorescence experiments performed with anti-Ti-gamma A and BMA 031 mAb (a reagent specific for the TCR-alpha/beta receptor) showed that surface expression of Ti-alpha/beta and Ti-gamma A is mutually exclusive. Moreover, it was found that most Ti-gamma A+ cells are CD2+, CD3+, CD4-, CD5+, NKH1-, HLA class II-negative. In contrast, the expression of the CD8 molecule on these T lymphocytes appears to be variable from one individual to another. Finally, we found that Ti-gamma A+ cells represent a majority of peripheral lymphocytes that express CD3 proteins but not the TCR-alpha/beta heterodimer. The delineation of this unique lymphocyte subset should help further studies on the biology of cells with a CD3-associated gamma complex.

A unique V-J-C-rearranged gene encodes a gamma protein expressed on the majority of CD3+ T cell receptor-alpha/beta- circulating lymphocytes.

We have recently described an mAb, anti-Ti gamma A, that recognizes an antigenic determinant carried by a TCR gamma chain. This antibody binds to approximately 3% of human PBLs and delineates a CD2+, CD3+, TCR-alpha/beta-, CD4-, CD8+/-, CD5+, NKH1-, and HLA class II- subset. The present study was designed to identify the gene encoding the Ti gamma A epitope. A first analysis was carried out on a previously characterized TCR gamma + fetal-cloned cell line termed F6C7. It was found that F6C7 cells have one gamma rearrangement on each chromosome: one joins V gamma 3 to J gamma 1, and the second joins V gamma 9 to J gamma P. Because only the latter allele appeared to be transcribed in the F6C7 lymphocytes, these data strongly suggested that anti-Ti gamma A mAb is specific for either a V gamma 9 or a V gamma 9-J gamma P-encoded peptide. To confirm this point, we studied an additional series of 13 randomly selected Ti gamma A+ cloned cells derived from peripheral blood of three distinct adult individuals. Each one of these lymphocytes was shown to both possess and transcribe a V gamma 9-J gamma P-C gamma 1-rearranged gene. It is therefore concluded that a predominant subpopulation of CD3+ TCR-alpha/beta- human circulating T lymphocytes (namely, the subset defined by anti-Ti gamma A mAb) surface expresses a gamma protein with a limited potential of variability from one cell to another.

Further analysis of the T cell receptor gamma/delta+ peripheral lymphocyte subset. The V delta 1 gene segment is expressed with either C alpha or C delta.

In the present study, we have characterized the reactivity of two mAbs that are directed at the human TCR-gamma/delta. These reagents, designated anti-A13 and anti-TiV delta 2, were found to recognize antigenic determinants encoded by the TCR V delta 1 and V delta 2 gene segments, respectively. Immunofluorescence analyses performed with the antibodies confirmed that, in the TCR-gamma/delta+ cell subpopulation, the expression of V delta 2+ delta chains is largely predominant, as compared with the V delta 1+ counterparts. However, these experiments led to an apparently discrepant finding. Indeed, the total number of cells recognized by the anti-A13 plus the anti-TiV delta 2 antibodies was often greater than that detected with anti-TCR-delta 1, a reagent specific for a constant epitope of the human delta chain. Further investigation showed that the presence of a sizeable peripheral lymphocyte subset coexpressing the BMA031 and the A13 epitopes. Because the former antibody is known to recognize an invariant antigenic determinant of the TCR-alpha/beta dimer, these results suggested that the V delta 1 gene segment may be expressed with either C delta or C alpha. This hypothesis was confirmed using T2, an IL-2-dependent BMA031+ A13+ polyclonal cell line developed from peripheral blood of a healthy adult donor. Indeed, T2 cells were found to have productively rearranged the V delta 1 gene. Together, results of Northern blot analysis and cDNA cloning indicated that V delta 1 was expressed in these cells as part of a 1.6-kb full-length message including J alpha-C alpha segments.

Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.

The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.

TCT.1, a target molecule for gamma/delta T cells, is encoded by an immunoglobulin superfamily gene (Blast-1) located in the CD1 region of human chromosome 1.

We have recently generated a series of gamma/delta T cell clones able to kill, after in vitro immunization, an Epstein-Barr Virus-transformed B cell line (designated E418) in a non-major histocompatibility complex-requiring fashion. A monoclonal antibody, termed anti-10H3, produced against E418 was selected by its ability to block these cytotoxic interactions. Further analysis indicated that the inhibitory effects of anti-10H3 were highly selective (i.e., no blocking activity with multiple control clones used as effector cells; no alteration of the natural killer-like function mediated by the relevant gamma/delta clones against 10H3+ tumor cells such as Rex). The molecule immunoprecipitated by anti-10H3, termed TCT.1, was characterized as a 43-kD protein broadly distributed in the hematopoietic system. The TCT.1 molecule has been further studied here by protein microsequencing. Results show that the TCT.1-derived peptide sequences are virtually identical to corresponding regions of Blast-1, a previously described surface protein with unknown function. The likely identity of the two molecules has been strengthened by analyzing the susceptibility of TCT.1 to phosphatidylinositol-specific phospholipase C digestion in light of the known anchorage of Blast-1 to the cell membrane through a glycosyl-phosphatidylinositol-containing lipid. The TCT.1/Blast-1-encoding gene is well characterized; it belongs to the immunoglobulin gene superfamily and it is located in the same band of chromosome 1 as the CD1 gene cluster. Together, these data further support the view that proteins distinct from the conventional class I/II histocompatibility molecules are involved in specific T cell recognition.

LAG-3, a novel lymphocyte activation gene closely related to CD4.

We have identified a novel human gene of the Ig superfamily, designated LAG-3. Expression of this gene is undetectable in resting PBL, while it is found (a 2-kb message) in activated T and NK cells. The LAG-3 gene includes eight exons; the corresponding cDNA encodes a 498-amino acid membrane protein with four extracellular IgSF domains. The first one belongs to the V-SET; it is particular since it includes an extra loop in the middle of the domain and an unusual intrachain disulphide bridge. The three other domains belong to the C2-SET. Strong internal homologies are found in the LAG-3 molecule between domains 1 and 3, as well as between domains 2 and 4. It is therefore likely that LAG-3 has evolved by duplication of a pre-existing gene encoding a two IgSF-domain structure. The compared analysis of LAG-3 and CD4, with respect to both their peptidic sequence as well as their exon/intron organization, indicated that the two molecules are closely related. This point is strengthened by the finding that both genes are located on the distal part of the short arm of chromosome 12.

Cloning and expression of a lymphocyte activation gene (LAG-1).

Using subtractive hybridization of a cDNA library we have identified a human gene, termed LAG-1 (for "Lymphocyte Activation Gene-1"). This cDNA codes for a 69 amino-acid polypeptide which belongs to a new class of recently described proteins secreted by activated lymphocytes and/or monocytes. The LAG-1 gene was cloned, sequenced and its chromosomal location assigned to chromosome 17 (q21 band). The promoter region of the LAG-1 gene was shown to include a GM-CSF-like decanucleotide sequence. Using a baculovirus vector expression system, we found that a 10 kDa recombinant LAG-1 protein is secreted by AcNPV infected SF9 cells, as determined in Western blot experiments by the reactivity of specific anti-peptidic heteroantibodies. Finally the natural LAG-1 protein was precipitated from the supernatant of internally labeled activated Nk cells and shown to migrate as a single entity of 14 kDa in SDS-PAGE analysis.

Bromodeoxyuridine and deoxyribonucleic acid bivariate analysis in human renal cell carcinoma. Does flow cytometric determination predict malignant potential or prognosis of patients with renal cell carcinoma?

Sixty-one renal cell carcinomas were analyzed by simultaneous flow cytometric bivariate measurements of deoxyribonucleic acid (DNA) and bromodeoxyuridine. Bromodeoxyuridine in vitro labeling was performed by sample incubation with bromodeoxyuridine under hyperbaric oxygen. DNA ploidy status statistically correlated only with the presence or absence of distant metastasis. When the mean bromodeoxyuridine labeling index (LI) was accordingly compared with each histologic feature, architecturally solid, grade 3, high-stage (beyond pT3a) tumor, and the presence of distant metastasis showed significantly higher LI. When LI were compared with DNA ploidy, diploid tumors showed a 6.3% +/- 4.9% LI, whereas aneuploid tumors exhibited a 7.6% +/- 5.4% LI. The difference was not statistically significant. Five patients with a mean survival time of 10.2 +/- 4.1 months who had a mean LI of 11.5% +/- 7.7% died, whereas 56 patients with a mean LI of 6.7% +/- 4.8% survived. The difference was significant (P less than 0.05). However, the results did not necessarily correlate with the histologic features of the malignant potential or with the prognosis in individual patients. These data indicate that flow cytometric DNA/bromodeoxyuridine bivariate analysis remains inconsistent for predicting the malignant potential or prognosis of renal cell carcinoma.

Flow cytometry based on heterogeneity index score compared with urine cytology to evaluate their diagnostic efficacy in bladder tumor.

Flow cytometric (FCM) examination of DNA distribution based on heterogeneity index score (HIS) and ploidy pattern and of bladder irrigation specimens were compared with conventional urine cytologic examinations to evaluate the diagnostic efficacy of each method. Of 56 patients with histologically proved bladder tumors, 24 (43%) had positive urine cytology, 41 (73%) had positive FCM, and 45 (80%) had positive urine cytology and/or positive FCM. When bladder tumors were graded histologically, 8 of 11 patients (73%) with grade I bladder carcinoma, 22 of 28 patients (79%) with grade II, and 15 of 17 patients (88%) with grade III had positive urine cytology and/or positive FCM. Of the 28 patients with grade II, 12 with positive urine cytologic results had higher mean HIS (117.5) when compared with 16 (61.1) in whom urine cytology was negative. Of those who continued to have positive FCM in the face of negative findings on cystoscopic and urine cytologic examinations during follow-up, 3 patients eventually were found to have tumors (2 distal ureter, 1 bladder). These results indicate that FCM examinations for DNA distribution of bladder irrigation specimens are as useful as conventional urine cytology in the management of bladder tumors, can be more sensitive for detection and monitoring of the disease, and can contribute further to accurate diagnosis of the disease when combined with conventional urine cytology.

Transitional cell carcinoma of kidney extending into renal vein and inferior vena cava.

A case involving transitional cell carcinoma of the renal pelvis with extension to the main renal vein and inferior vena cava as well as massive involvement of the kidney and ureter is reported. A review of the literature on renal pelvic tumors and patterns of tumor growth is presented.

Renal devitalization with ethanol injection in management of patients with advanced hypernephroma.

Transcatheter injection of ethanol into the renal artery was utilized in 11 patients with hypernephromas, clincally staged C or D, in an attempt to produce renal devitalization. Complete disappearance of the tumor neovascularity was achieved in 9 of 11 patients. Renal divitalization with ethanol injection is safer and more effective than the embolizing technique utilized hitherto and should be included in the primary treatment of hypernephroma in selected cases or in conjunction with other procedures to induce chemical nephrectomy.

One-stage total cystectomy and ileal loop diversion in patients over eighty years' old with bladder carcinoma. Pre- and postoperative functional reserve of various organs.

During the period from 1976 to 1981, 364 patients with bladder carcinoma were seen at the Keio University Hospital. Extensive preoperative investigation of pulmonary, cardiovascular, and renal function was obtained in all patients. Of the 12 patients studied, 9 underwent a one-stage total cystectomy and ileal loop diversion and the remaining 3 a two-stage procedure. Of the 9 patients, decreased FEV 1.0 per cent by spirometry was noted in 5, ECG abnormality such as bundle branch blocks in 8, and diminished creatinine clearance ranging from 28 to 68 ml/min were observed in all 9. Major postoperative complications included pyelonephritis in 2 patients, pneumonia in 1, pelvic abscess in 2, renal insufficiency in 3, and paralytic ileus in 2. There was no immediate postoperative death. In these elderly patients, functional reserve of the lung, heart, and kidney is less than optimal and is further decreased by major surgical procedures. Therefore, total cystectomy in the elderly patients is justifiable only in a selected group of patients, when functional status of the vital organ is thoroughly worked up and prophylactic and therapeutic measures are instituted promptly if indicated.

Immunologic study of human recombinant interleukin-2 (low-dose) in patients with advanced renal cell carcinoma.

Immunologic and antitumor effects of human recombinant interleukin-2 was studied in patients with advanced renal cell carcinoma. Intravenous drip infusion for inpatients or subsequent subcutaneous injection for outpatients at a daily dose of 1 x 10(6) units of interleukin-2 was given over a period varying from twenty-one to two hundred forty days to 13 patients. Natural killer cell activity increased 20 percent or more in 11 of the 13 patients, and lymphokine-activated killer cell activity increased 40 percent or more in 9 of the patients tested. Interleukin-2-receptor positive cells and HLA-DR positive cells in peripheral blood lymphocytes increased in 12 and 11 patients, respectively. Two patients achieved complete response; 1 had a partial response; 6 had no change; and 2 had progressive disease. Two patients were not evaluated because the therapy had been discontinued in less than four weeks. We conclude that low-dose interleukin-2 enhances patients' immunity and has a potential antitumor activity against renal cell carcinoma.

Studies on the mode of bacterial contamination of an operating theatre corridor floor.

Bacterial contamination of the floor of a corridor leading into an operating theatre suite was studied in relation to the site chosen for changing footwear. Recovery of bacteria showed a peak at the site of exchange of footwear, and decreased with increasing distance from the site. When the site of exchange was moved further away from the clean area, the peak of contamination moved to the new site and bacterial contamination decreased in the clean area. These results indicate that exchange of footwear should occur as far from the operating theatre as possible.

Studies on the bacterial permeability of non-woven fabrics and cotton fabrics.

The permeability of cotton and non-woven fabrics to bacteria, air and water was studied. Non-woven fabrics, even when wet, showed low resistance to air, and high resistance to permeation of water and bacteria. Water-repellent cotton fabrics were resistant to permeation of water, air and bacteria, but these properties decreased on washing. Non-water-repellent cotton fabrics were poor bacterial barriers even when new.

[A case of everting ectopic ureterocele in child--controversy in the surgical treatment].

A 4-year-old girl with the history of repeated urinary tract infections was referred from the pediatric department to our department on January, 1987. Under the diagnosis of right ectopic ureterocele of everting type with complete duplication of renal unit, the one-stage operation, that is, right hemi-nephroureterectomy with complete excision of the ureterocele and ureteral stump was carried out on January 27, 1987. Reimplantation of the orthotopic mate ureter was done because of injury to lower part of the mate ureter during operation, although reflux of the mate ureter did not exist. After the operation, the voiding symptom became better except for large residual urine volume (300 ml). In order to protect renal function, intermittent catheterization was carried out. Two years and 8 months after that operation, residual urine volume was reduced to 20 ml. Judging from the literature and our experience, if the patient is not in a severe condition, we recommend one-stage operation, that is, total correction (a single operation designed to correct abnormalities of the upper and lower urinary tract) for ectopic ureterocele in children.

[A case of scrotal elephantiasis 30 years after treatment of penile carcinoma].

A 67-year-old man visited our hospital with complaints of scrotal swelling associated with occasional febrile episodes. Physical examination disclosed a huge scrotal mass, approximately the size of a child's head, with numerous papillomatous lesions on its surface. His past medical history was significant in that he was diagnosed with penile carcinoma at the age of 35 years old and was treated with partial penectomy followed by radiation and chemotherapy at other hospital. During this admission tumor marker squamous cell carcinoma (SCC) and microbiological tests for mcroflariae were both negative. Ultrasound (US), computed tomographic (CT) scan and magnetic resonance imaging (MRI) revealed markedly thickened scrotal skin and small hydrocele with no evidence of local recurrence of the previous penile carcinoma. A percutaneous cystostomy was created because of chronic urinary retention and possible urine extravasation into the scrotum. Histopathological examination of the biopsy specimen from the scrotal mass demonstrated lymphangiectasia consistent with elephantiasis of the scrotum. Surgical excision of this huge scrotal mass was performed in August 1997. The resected tissue weighed 1,400 g. Convalescene was uneventful. He subsequently underwent perineal urethrostomy in place of the suprapubic cystostomy.

[A case report of extensively calcified renal cell carcinoma].

A 73-year-old woman visited another hospital with complaints of occasional episodes of gross hematuria. A computed tomographic (CT) scan revealed extensive calcification in the left kidney, and she was referred to our hospital for further examinations. The physical examination was unremarkable other than mild back pain on her left side. Blood chemistry was normal. The CT scan revealed a mildly enhanced tumor of 6 cm in diameter accompanied by extensive calcification in the left kidney. Angiography revealed a hypovascular tumor in the left kidney. A left radical nephrectomy was carried out. Gross appearance of the specimen showed extensive calcification from the renal pelvic to the parenchyma of the lower pole of the left kidney. The histopathological diagnosis was renal cell carcinoma, alveolar type, mixed subtype, pT2pN0pM0.

[Rupture of an infected urachal cyst causing generalized peritonitis: a case report].

A 68-year-old man visited our hospital with complaints of abdominal pain and fever. Physical examination disclosed findings consistent with acute abdomen. Computed tomographic (CT) scan revealed a 5 cm cystic mass contiguous with the dome of the bladder and fluid collection in the peritoneal cavity. Cystogram demonstrated deformity of the bladder and no communication between the mass and the bladder. A diagnosis of generalized peritonitis either due to the infected urachal cyst or ruptured bladder was made, and emergency exploratory laparotomy was carried out. Based upon findings consistent with an infected urachal cyst associated with its intraperitoneal perforation, resection of the entire urachal remnant including the dome of the bladder was performed. Pathologic examination showed an acutely inflammed urachal cyst and chronic inflammation of the bladder wall.

[Percutaneous removal of renal and upper ureteral stones: clinical results and complications of 103 cases].

We report the results and complications of 103 consecutive patients who underwent percutaneous removal of renal and ureteral stones. The overall clinical success rate was 80.6%. For the recent 33 cases in which UL-arm fluoroscopy was used, however, the success rate was as high as 87.9%, which was considered to be due to easier establishment of percutaneous direct access. The most common complications were bleeding (18.5%), extravasation (15.5%) and fever (9.7%). Four cases with significant bleeding required arteriography, but there were no sign of arteriovenous fistula nor pseudoaneurysms in any cases. To study renal parenchymal damage in the percutaneous procedures, plasma renin activities (PRA) were compared in 54 cases after six months. However, significant elevation of PRA did not occur in any case.