Mutation frequencies of EXT1 and EXT2 in 43 Japanese families with hereditary multiple exostoses.

Hereditary multiple exostoses (EXT) is an autosomal dominant bone disease characterized by the formation of cartilage-capped prominences. EXT is genetically heterogeneous with at least four chromosomal loci. Among the four loci, the exostosis type 1 gene (EXT1) and type 2 gene (EXT2) have been cloned. Previous studies have shown that disease-type-specific frequency of mutations is different among various ethnic populations. To determine those frequencies in the Japanese, we conducted a large-scale mutation screening on both genes. In 23 of 43 Japanese families examined, we found 21 different mutations, of which 18 are novel. Seventeen (40%) of the 23 families had a mutation in EXT1 and six (14%) had a mutation in EXT2, suggesting that the former mutations are more frequent than the latter in Japanese EXT families. Of the 17 families with EXT1 mutations, 13 had those causing premature termination of the EXT1 protein and four showed missense mutations, whereas five of the six families with EXT2 mutations had those causing premature termination and one showed missense mutation. Interestingly, all four EXT1 missense mutations occurred in an arginine residue at codon 340 (R340) that is known as a critical site for expression of heparan sulfate glycosaminoglycans, suggesting that the region encompassing the arginine residue may play an important role in the function of the EXT1 protein. These results expand our knowledge of the ethnic difference of EXT and the structure-function relationship of the EXT genes.

Spectra of spontaneous mutations at the hprt locus in colorectal carcinoma cell lines defective in mismatch repair.

Spectra of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in colon carcinoma cell lines HCT116 and HCT-15 deficient in mismatch repair and displaying mutator phenotypes were determined. HCT116 and HCT-15 cells, respectively, harbour a mutation in the mismatch repair gene hMLH1 and GTBP. The mutation frequency at the hprt locus in both cell lines was elevated by about two orders, but the microsatellite instability in HCT116 cells was one order higher than in HCT-15 cells. Except for one mutant of HCT-15, all the mutations (114/115) were point mutations; base substitutions of various types and frameshifts (deletions/insertions of less than a few bases, predominantly of +/-1 bp). Base substitutions (57%) and frameshifts (43%) occurred at a comparable rate in HCT116, whereas base substitutions (92%) were the major mutational events in HCT-15. Most frameshifts in HCT116 occurred at sites of monotonous or short tandem repeating sequences, and two of these sites, where there was a run of six Gs and four As, were hot spots. Three hot spot sites of base substitutions were found in HCT-15; two of them at splice acceptor sites, the other at the CpG site shared with HCT116. The distinct mutation spectra of the HCT116 and HCT-15 cell lines may reflect functional differences in the hMLH1 and GTBP gene products in mismatch repair. The gene product GTBP may be involved in the preferential repair of base mismatches, and MLH1 in the repair of both base mismatches and deletions/insertions of less than a few bases. These results suggest that mismatch repair deficiency affects the microsatellite stability as widely reported in colorectal tumour cells, but that it may not severely affect chromosome integrity as the karyotypes of these tumour cells are, unlike other tumour cells, relatively stable.

[192Ir brachytherapy].

Modern high technology has recently brought a precision treatment modality in the field of brachytherapy for cancer patients. Ir-192 manual afterloading (hair pin technique of Ir-192 wire) replaced the technique utilizing Ra-226 needles at our department in 1973. In May 1991, microSelectron-HDR (Ir-192 micro-source of 370 GBq) was installed in Osaka University Hospital. Preliminary analysis of phase I/II study resulted in no significant differences between the incidence of an acute mucosal reaction as well as early tumor response after high and low dose rate interstitial brachytherapy for oral cancer. Since April 1992, a phase III study has been under way to completely eliminate the problem of hospital personnel exposure to radiation in the field of brachytherapy. The introduction of remote after-loading of Ir-192 micro-source has resulted in improvements in elderly patient care during the interstitial brachytherapy for malignancies. The indications for HDR brachytherapy have been expanded, and new technology was developed to improve the local cure of the disease, such as linked double button technique for oral cancer, template interstitial brachytherapy for perineal cancer, and postoperative brachytherapy using intraoperative flexible catheter placement for locally unresectable disease or microscopically residual disease. Through these meticulous efforts, HDR interstitial brachytherapy will soon become a satisfactory substitute for traditional LDR interstitial brachytherapy.

Transforming growth factor-beta-induced inhibition of T cell function. Susceptibility difference in T cells of various phenotypes and functions and its relevance to immunosuppression in the tumor-bearing state.

The present study investigates the nature of humoral component(s) generated in tumor-bearing hosts to induce immune dysfunction of T cells. Cell-free ascitic fluid and culture supernatant (SN) were obtained from the ascites and cultures allowing MH134 hepatoma cells to grow. These ascites and SN samples were tested for their abilities to influence the generation of CTL responses to TNP and alloantigens. The generation of the anti-TNP CTL responses that require self H-2-restricted CD4+ Th cells was markedly suppressed by addition of the ascites or SN under conditions in which these samples did not inhibit anti-allo CTL responses capable of using alternate pathways of allo-restricted CD4+ and CD8+ Th. The activation of CD8+ CTL precursors and CTL activity were also resistant to the ascites or SN. The ascites- or SN-induced suppressive effect to which CD4+ Th were most susceptible was found to be mediated by transforming growth factor-beta (TGF-beta) activity, because: 1) the TGF-beta activity was detected in the MH134 ascites and culture SN; 2) the suppression of CD4+ Th function required for anti-TNP CTL responses was almost completely prevented by addition of anti-TGF-beta antibody to cultures and; 3) rTGF-beta also induced similar patterns of immunosuppression to those observed by ascites or SN. These results indicate that TGF-beta produced by tumor cells induces deleterious effects on T cell, especially on the CD4+ Th subset, and provide an explanation for the molecular mechanism underlying the previously observed CD4+ Th-selective suppression in the tumor-bearing state.

Enhanced production of IL-6 in tumor-bearing mice and determination of cells responsible for its augmented production.

IL-6 is a cytokine secreted in normal individuals by monocytes, fibroblasts, and endothelial cells. We have found increased levels of IL-6 in the sera from MH134 hepatoma- and CSA1M fibrosarcoma-bearing mice. Concerning the capacity of these tumor cells themselves to produce IL-6 in vitro, they exhibited the distinct contrast, i.e., the MH134 tumor cells produced high levels of IL-6 whereas the CSA1M generated a marginal level of IL-6. It was, however, demonstrated that appreciably enhanced IL-6 production was observed in spleen cell culture supernatants from both types of tumor-bearing mice when compared to those obtained from normal mice. More importantly, in contrast to the production of IL-6 by non-T cell compartment of normal spleen cells, enhanced IL-6 production of spleen cells from tumor-bearing mice was ascribed to T cell compartment. Analysis of T cell phenotype has revealed that enhanced IL-6 production was mediated predominantly by Lyt-2+ but not by L3T4+ T cell subset. Thus, these results indicate that increased circulating IL-6 is elicited in the tumor-bearing state and that irrespective of the potential of tumor cells themselves to produce IL-6, T cells, especially Lyt-2+ T cells from tumor-bearing mice are responsible for such a high level of IL-6 production.

Simultaneous bilateral primary diffuse malignant uveal melanoma: case report with pathological examination.

A 50-year-old Japanese housewife became blind owing to bilateral diffuse malignant melanoma of the choroid, ciliary body, and iris. These were histologically proved when enucleation was required because of blind painful eyes with raised intraocular pressures. She presented with reduced visual acuity and night blindness. Small scattered atrophic areas were observed between disc and macula on both sides, which fluorescein angiography showed were due to patchy atrophy of the retinal pigment epithelium. There was reduced amplitude in the electroretinogram. Inferior retinal detachments soon appeared and spread to become total. Pathological examination showed diffusely thickened choroid on both sides, due to infiltration by epithelioid and spindle-shaped malignant melanoma cells, which also affected the thickened ciliary body and iris diffusely. A carcinoma of the uterus successfully treated three years previously and the probably multicentric origin of the malignancy in each eye, and its bilateral occurrence, suggest a tumour-producing tendency in this patient, an interpretation which also applies to one of the other eight reported cases.

[Extrarenal nephroblastoma: report of a case and review of the literature].

A 3-year-old girl with an extrarenal nephroblastoma arising from the right retroperitoneal space is described. She was admitted to our hospital with the chief complaint of abdominal pain. On physical examination, she was found to have a large (12 X 10 cm in size), firm and nontender mass in the right upper quadrant of the abdomen. The mass did not extend beyond the midsagital line. The physical examination did not reveal any particular findings or any congenital anomalies. Urinalysis and hematological data were within the normal limit. Radiological examinations including CT scan showed that the solid tumor was related to the right kidney. Under the diagnosis of right nephroblastoma, 15 micrograms/kg/day of actinomycin D was given intravenously for 5 days from October 18, 1982. The regression rate of the tumor was 78 percent on CT scan after chemotherapy. On November 22, 1982, transperitoneal nephrectomy was performed through a right paramedian incision. The tumor was found to adhere tightly to the upper pole of the kidney. The surgical specimen was 76 g in weight. A section of the surgical specimen showed an extrarenal tumor located completely outside the kidney and separated from the renal cortex by a thickened renal capsule. Histological diagnosis was extrarenal nephroblastoma showing renal capsular invasion by epithelial tumor cells. No teratomatous components were encountered in the tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistologic localization and immune phenotypes of lymphocytes expressing Tac antigen in human lymphoid tissues.

A monoclonal anti-Tac antibody has been identified as a putative antibody against the human interleukin 2 (IL 2) receptor. In the present study, anti-Tac antibody was used to determine the location of cells expressing IL 2 receptors in frozen sections of human lymph nodes and tonsils by means of an immunoperoxidase technique. It was found that a substantial number of lymphoid cells reactive with anti-Tac antibody were present in these tissues. The majority of the Tac-positive cells were located in the paracortical and interfollicular regions of lymph nodes and tonsils, whereas only a few Tac-positive cells were scattered in the mantle zones and germinal centers of the secondary follicles. In contrast, no Tac-positive cells were demonstrated on cytocentrifuge preparations of peripheral blood lymphocytes from some of tissue donors, as evaluated by the same technique. In some experiments, a double-marker immunofluorescence analysis with the use of different fluorochromes, fluorescein isothiocyanate (FITC), and tetramethylrhodamine isothiocyanate (TRITC) was applied to characterize the phenotypes of cells expressing Tac antigen. Double staining with TRITC and FITC, respectively, for the identification of Tac-positive cells and T cells, showed that Tac-positive cells in lymph nodes and tonsils almost exclusively co-expressed a pan-T cell marker, Leu-1 antigen, that probably does not belong to non-T cell lineages. About 80% of Tac-positive cells were Leu-3 (helper/inducer) positive, and 20% of them Leu-2 (suppressor/cytotoxic) positive. These observations imply the plausible notion that an IL 2-mediated immune activation of T cells may actually occur in local lymphoid organs.

Sequential expression of T cell activation (Tac) antigen and Ia determinants on circulating human T cells after immunization with tetanus toxoid.

Recent studies have indicated that a monoclonal antibody, termed anti-Tac, may recognize the receptor sites or closely associated structures for interleukin 2 on activated human T cells. The Tac antigen, definable by anti-Tac antibody and usually found on mitogen- or alloantigen-stimulated T cells, was not expressed to any appreciable extent on normal circulating T cells. In the present study, we showed that an increase in circulating T cells expressing Tac antigen as well as Ia determinants occurred in normal individuals after immunization with tetanus toxoid. The expression of Tac antigen and Ia determinants on T cells was evaluated by the rosette method with Staphylococcal protein A-(SPA) coated bovine red blood cells (BRBC) or the indirect immunofluorescence method with monoclonal anti-Tac and anti-Ia antibodies. An increase in Tac-positive or Ia-positive T cells was more evident with the use of the rosette method with SPA-coated BRBC than with conventional immunofluorescence. The percentage of Ia-positive T cells showed a peak between 24 and 48 hr after toxoid injection, and remained at high levels until 2 wk after immunization. In contrast to Ia-positive T cells, the appearance of Tac-positive T cells was transient and at a rather early period of toxoid immunization. The maximum increase of Tac-positive T cells was apparent around 12 hr after toxoid injection, and Tac-positive T cells disappeared abruptly from circulation by 24 hr after inoculation. Ia-positive T cells were induced in both Leu-2 suppressor/cytotoxic and Leu-3 helper/inducer subsets, whereas Tac-positive T cells were generated only within the Leu-3 subset. The fact that induction of Tac-positive and Ia-positive T cells might occur at different stages of T cell activation and in different subsets of T cells seemed to be important for elucidating their roles in the in vivo T cell proliferation and differentiation.

Cyclosporin A does not prevent expression of Tac antigen, a probable TCGF receptor molecule, on mitogen-stimulated human T cells.

Results of recent studies indicated that a monoclonal anti-Tac antibody might recognize the receptor sites or closely related structures for T cell growth factor (TCGF) on activated human T cells. In the present study, we examined the effect of cyclosporin A (CsA) on the expression of Tac antigen by mitogen-stimulated T cells. CsA inhibited the proliferative response of T cells to Con A and PHA in a dose-dependent manner. Both Con A- and PHA-induced cellular proliferation were decreased to about 10% of controls at 5 micrograms/ml of CsA. When T cells were stimulated with these mitogens, many of them expressed Tac antigen on their surfaces, assessed by the immunoperoxidase method. The appearance of Tac-positive cells occurred earlier than a rise of cellular DNA synthesis. Characteristically, CsA showed no inhibitory effect on the expression of Tac antigen by mitogen-stimulated T cells, even at a relatively high concentration of 5 micrograms/ml, whereas the expression of other "activation" antigens reactive with monoclonal anti-Ia, OKT9, or OKT10 antibodies by T cells was blocked completely by CsA. Morphologically, the majority of Tac-positive cells in culture with mitogens alone showed the characteristics of blastoid cells; Tac-positive cells in the culture containing CsA mainly consisted of medium-sized cells, indicating these cells probably accumulated at a stage of partial activation. T cells, once stimulated with Con A or PHA for 3 days whether in the presence or in the absence of CsA, were able to absorb TCGF activity from TCGF-containing media similarly. In addition, T cells, even stimulated in the presence of CsA with these mitogens for 24 hr, were capable of responding to TCGF with the same grade of proliferation as did T cells stimulated with mitogen alone. CsA showed no appreciable inhibition in a TCGF-dependent proliferation of such prestimulated cells. These functional properties of activated T cells might be correlated with their ability to express Tac antigen. These experimental findings present some evidence that CsA might not prevent the expression of probable functional receptor sites for TCGF in mitogen-dependent activation of human T cells.

Kinetics of expression of T-cell "activation" antigens on in vivo- and in vitro-stimulated T cells.

The in vivo expression of T-cell activation antigens defined by means of an OKT series of monoclonal antibodies and the anti-Tac antibody was evaluated in peripheral T lymphocytes obtained from patients with infectious mononucleosis and in cerebrospinal fluid (CSF) cells collected from individuals with mumps meningitis by the immunoperoxidase method. Although Tac antigen was definitely expressed on about 10% of CSF cells in mumps meningitis, Tac+ T cells could not be identified on any peripheral blood T cells in infectious mononucleosis, which expressed OKIa 1+, OKT10+, and OKT9+ determinants, as a probable sign of in vivo activation. The coexpression of Ia+ antigens on a T-cell functional subset might be important for their functional properties. Our kinetic study and cell cycle analysis of the expression of these activation antigens suggested that Tac antigen might be expressed at the early stage of the G1 phase of the cell cycle; OKT9+ antigen at the late stage of the G1; and Ia+ and OKT10+ determinants might be expressed at the S phase or later on in vitro, probably one after another.

Functional significance of Tac antigen expressed on activated human T lymphocytes: Tac antigen interacts with T cell growth factor in cellular proliferation.

Cultured human T cells (CTC), which are grown in conditioned medium containing T cell growth factor (TCGF), proliferate in response to TCGF. It has been shown that an antigen (Tac) defined by a monoclonal antibody, termed anti-Tac antibody, is expressed on human T cells activated by mitogens or antigens and CTC grown in the presence of TCGF. To elucidate the functional significance of Tac antigen expressed on activated T cells, we studied the effect of anti-Tac antibody on TCGF-dependent proliferation of CTC. The addition of anti-Tac antibody strongly inhibited the proliferation of CTC induced by TCGF. This inhibition was observed only when the antibody was added at the early phase of culture, but not when the addition of the antibody was delayed beyond 24 hr of culture. Seven-day-old PHA-induced T cell blasts, but not fresh peripheral blood lymphocytes, were able to absorb TCGF activity in conditioned medium, as assessed by the DNA synthesis of CTC. When PHA-induced blasts were treated with anti-Tac antibody before absorption, their capacity to absorb TCGF activity was almost completely eliminated. In contrast, absorption of TCGF by PHA-induced blasts was not significantly reduced even when they were pretreated with other monoclonal antibodies (anti-Ia, OKT9, or OKT10) with specificity for antigens expressed on activated T cells. Based on the view that TCGF interacts with activated T cells via specific membrane receptors, these observations suggested that anti-Tac antibody might specifically block the binding of TCGF to the corresponding membrane binding sites, resulting in the inhibition of TCGF-dependent proliferation of CTC. Tac antigen expressed on activated T cells seems to participate in responding process of activated T cells to TCGF.

Discrepancy in expression ability of Tac antigen and Ia determinants defined by monoclonal antibodies on activated or cultured cord blood T lymphocytes.

T lymphocytes from cord blood and adults stimulated with mitogens and alloantigen were analyzed using monoclonal antibodies that detect Ia and Tac antigens associated with lymphocyte activation. Less than 5% of T cells freshly isolated or cultured without stimuli expressed Ia and Tac antigens assessed by the indirect immunofluorescence method. When both T cells from cord blood and adults were cultured with phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), or allogeneic cells, variable proportions of them expressed Tac antigen. The expression of Tac antigen on stimulated T cells increased in parallel with DNA synthesis, and was significantly correlated with the proliferative response of stimulated T cells. In contrast to Tac antigen, the expression of Ia antigens appeared to show some time lag behind the proliferative response. Although expression of Ia antigens was too weak to assess in PHA- or Con A-stimulated cultures, adult T cells were stimulated by PWM or allogeneic cells to express strongly Ia antigens in culture. However, the percentage of Ia+ cells in cord blood T cells stimulated with PWM or allogeneic cells was markedly lower as compared with that of adults. In addition, cord blood T cells grown in the presence of T cell growth factor showed the same degree of Tac antigen as adult ones did, whereas their expression of Ia antigens was negligible as compared with adult controls. These observations suggested that T cells in cord blood might be deficient by nature in their ability to express Ia antigens on activation. The inability to develop Ia+ T cells seemed to be characteristic of functional immaturity of T cells in early human ontogeny.