Properties of tablets containing granulations of ibuprofen and an acrylic copolymer prepared by thermal processes.

The objective of this study was to investigate the properties of tablets containing granulations of ibuprofen (Ibu) and Ammonio Methacrylate Copolymer, Type B (Eudragit RS PO) prepared by hot-melt processing. Tablets were compressed from granules prepared by hot-melt granulation (HMG) or direct compression (DC). For the hot-melt extrusion (HME) process, tablets were prepared by cutting the extrudate, manually. The physicochemical properties of tablets were investigated using thermal analysis, powder X-ray diffraction analysis, tablet hardness, and drug dissolution. The effect of thermal treatment of tablets on the dissolution characteristics of Ibu was also investigated. The results demonstrated that the Ibu lowered the glass transition temperature (Tg) of the Eudragit RS PO and the softened polymer functioned as a thermal binder in the granulation. Ibu was demonstrated to be an effective plasticizer for Eudragit RS PO in the thermal processes. The efficiency of the granulation process increased with increasing levels of Eudragit RS PO in the powder blend. Higher levels of Eudragit RS PO in the tablets prepared by HMG or HME resulted in a decrease in the dissolution rate of the Ibu. An increase in the amount of Ibu in the tablets prepared by HMG or DC led to a decrease in the initial dissolution rate of the Ibu. Following the thermal treatment of the Ibu tablets prepared by HMG, the dissolution rate was significantly decreased due to structural changes in the tablets that resulted from the fusion and coalescence of plasticized polymer particles, causing a reduction in tablet porosity. The Ibu tablets prepared by HME demonstrated minimal changes in their release properties following thermal treatment even at temperatures higher than the Tg of the polymer. HME was shown to be a novel method to prepare matrix tablets and stable dissolution properties were obtained when tablets were stored at 40 degrees C for 30 days.

Transfer of SV40 temperature-sensitive early gene into human epidermal keratinocytes by the recombinant adenovirus vector.

We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34 degrees C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca++-resistant cells did not proliferate at the nonpermissive temperature (40 degrees C), indicating that they depended on T antigen for their proliferation. The temperature-sensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40 degrees C. The serum/Ca++-resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca++-resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that deltaE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.

An in vivo model of human skin acute graft-versus-host disease: transplantation of cultured human epidermal cells and dermal fibroblasts with human lymphocytes into SCID mice.

The ability of mixed epidermal cell-lymphocyte reactions to detect allogeneic reactivities in an in vivo model was investigated by developing an in vivo model of acute graft-versus-host disease (GVHD), using SCID mice with a C.B-17 background in which human skin structures were generated by transplantation of cultured human epidermal cells (HEC) with dermal fibroblasts (HDFC). Suspensions containing cultured HEC and HDFC from a single donor were mixed with autologous peripheral blood mononuclear cells (PBMNC) or with PBMNC from unrelated individuals, and were injected into the flanks of C.B-17-SCID mice. Ten and 21 days after injection, subcutaneous nodules generated in the mice were examined histologically and immunohistochemically. Cystic structures developing after injection of HEC and HDFC without human PBMNC showed normal epidermislike tissue. Human skin generated in SCID mice injected with HEC and HDFC with auto-PBMNC showed no graft-versus-host reaction (GVHR) histologically, whereas those mice injected with PBMNC from siblings that shared an HLA haplotype showed mild GVHR. Human skin in SCID mice injected with HEC and HDFC with histoincompatible unrelated PBMNC showed moderate to severe GVHR. The severity of GVHR paralleled the dose of unrelated PBMNC, and GVHR was prevented by peroral treatment with cyclosporine A. Immunohistochemically, inflammatory cells infiltrating human cutaneous tissue formed in the SCID mice were stained by an anti-human CD45RO antibody that reacts with human T cells but not with murine lymphocytes, and most T cells were stained by an anti-human CD8 antibody recognizing HLA class I antigens. These findings are similar to those in clinical skin graft-versus host disease (GVHD) observed in patients undergoing allogeneic bone marrow transplantation. This experimental system should be useful as an in vivo model of human skin GVHD.

Preparation and Characterization of Barium-Strontium Hydroxyapatites

Barium-strontium hydroxyapatite solid solutions with different molar ratio Ba/(Ba + Sr) were synthesized by a wet method and characterized by various means. The solid solution particles could be prepared at molar ratios ranging from 0 to 1; however, Ba2+ ions were more difficult to be incorporated into hydroxyapatite crystals compared to Sr2+ ions. With increasing Ba2+ content, the particles grew and finally turned into pure rod-shaped barium hydroxyapatite particles with a size of ca. 0.2 x 2 &mgr;m. The resulting particles were agglomerates consisted of primary fine particles except for strontium hydroxyapatite. The molar ratios (Ba + Sr)/P of all the particles were larger than the stoichiometric ratio of 1.67, suggesting that CO32- ions, OH- ions, and/or H2O molecules substitute for PO43- ions in the crystal lattices. The amount of CO2 adsorbed irreversibly on the particles increased with increasing (Ba + Sr)/P except for strontium hydroxyapatite and fitted a curve with a minimum at a cation/P ratio of ca. 1.56 as well as other HAPs.

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal keratinocytes in immunodeficient mice.

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.

Immortalisation of human oesophageal epithelial cells by a recombinant SV40 adenovirus vector.

We introduced the origin-defective SV40 early gene into cultured human oesophageal epithelial cells by infection of a recombinant SV40 adenovirus vector. The virus-infected cells formed colonies 3-4 weeks after infection in medium containing fetal calf serum. When the cells derived from 'serum-resistant' colonies were then maintained in the serum-free medium with a low calcium ion concentration, some of them passed the cell crisis and kept growing for over 12 months. These cells, regarded as immortalised cells, resembled the primarily cultured oesophageal epithelial cells in morphology and had some of their original characteristics. Treatment of the cells with a high calcium concentration induced phenotypic changes. These cells still responded to transforming growth factor beta. When the immortalised cells were injected into severe combined immunodeficient mice, they transiently formed epithelial cysts, although the typical differentiation pattern of the oesophageal epithelium was not observed. These cysts regressed within 2 months without development into tumours. The results indicated that human oesophageal epithelial cells were reproducibly immortalised by infection with a recombinant SV40 adenovirus vector at relatively high efficiency. The immortalised cells should be useful in studies on oesophageal carcinogenesis and in assessing the cooperative effects with other oncogene products or carcinogens.

Isolation of human CD34+ peripheral blood stem cells using an immunomagnetic positive selection system: prior monocyte depletion using a nylon-wool column.

An immunomagnetic separation system has been used to collect CD34+ cells in mobilized blood after treatment with a nylon-wool column. Cell purities were increased from 2.6% preseparation to 94.6% postseparation, with a mean yield 45.2% (n = 4). Forty percent of CD34+ cells separated by the immunomagnetic procedure formed colonies in the presence of hematopoietic growth factors in a limiting dilution assay. Eleven percent of these clones proliferated to over 10(5) cells and contained megakaryocytes.

Use of cultured human epidermal allografts for the treatment of extensive partial thickness scald burn in children.

In spite of recent progress in burn treatment, the early surgical therapy of partial thickness scald burns in children is still controversial. Early tangential excisions is not easily applicable for these patients because of difficulties in determination of the burn depth and probable physiological derangement after surgery. Hypertrophic scar formation and wound contraction after meshed autografts are other limitations. For these reasons, conservative treatment, not early excision therapy, has been chosen initially for these injuries. We used cultured epidermal allografts for extensive, partial thickness scald burns, during the early post-burn period without escharectomy. Fifty to 100% of the engrafted superficial dermal burns were epithelialized within 7 days. In contrast, untreated identical wounds remained open. Repeated grafting of cultured allografts on unexcised wound granulations of dermal burns also enhanced epithelialization. Long term results showed that hypertrophic scar formation in the mixed superficial and deep dermal burns was reduced when cultured allografts were used. Allografting of the cultured epidermis without surgical excision apparently promoted the rapid regeneration of the partial thickness burns. Procedural complications did not occur. Cultured allografts should be used as an effective and safe biological dressing for partial thickness scald burns in children.

Large-scale culture system of human CD4+ helper/killer T cells for the application to adoptive tumour immunotherapy.

A simple method for the rapid expansion of human CD4+ T cells with both helper and killer functions was established. CD4+ T cells separated from peripheral blood mononuclear cells using immunomagnetic beads were stimulated with immobilised OKT-3 monoclonal antibody (mAb) plus recombinant interleukin 2 (rIL-2) in 96 well culture plates. After 6 day-culture, the CD4+ T cells were restimulated by immobilised OKT-3 mAb for an additional 24 h, then inoculated into concentrated rotary-tissue culture bag and cultured for further 9 days. This procedure yielded a 3000-fold increase in cell number (about 3-5 x 10(9) per bag). Most of the cells (over 96%) continued to express CD4+ antigen and retained their capacity to produce IL-2. The activated CD4+ T cells showed marked cytotoxicity against Fc receptor positive tumour cells in the presence of OKT-3 mAb. Moreover, we succeeded in a specific targeting of the expanded CD4+ helper/killer T cells to c-erb B-2 positive tumour cells by means of anti-CD3 x anti-c-erb B-2 bispecific antibody. These results suggested that our established simple system will be available for the expansion of large number of CD4+ helper/killer T cells which may provide an efficient strategy for adoptive tumour immunotherapy.

[Immunization of healthy children with measles-mumps-rubella trivalent vaccine simultaneously given with varicella vaccine].

Trivalent vaccine, containing measles TD97, rubella TCRB-19 and mumps NK-M46 strains (MMR vaccine) was administered to a total of 116 healthy children of which 50 subjects were simultaneously injected with varicella vaccine in the opposite arm. The seroconversion rates for measles, mumps, rubella, and varicella in those who received both MMR and varicella vaccines (MMR + V group) were 100% (44/44), 91% (39/43), 100% (46/46) and 95% (39/41), respectively. And these rates were comparable to those in subjects receiving only MMR vaccine, namely 100% (64/64) for measles, 95% (57/62) for mumps, and 97% (58/60) for rubella. Fifty-eight children receiving MMR vaccine were seronegative to measles, mumps and rubella before vaccination, and 51 (88%) of them were found to be seropositive against all three viruses at 6 to 8 weeks after vaccination. Among the children injected with MMR and varicella vaccines, 36 subjects had no pre-antibodies to measles, mumps, rubella and varicella. Seroconversion in post-serum to all four viruses were found in 31 cases (86%). Clinical reactions observed in some vaccines were mild fever (17%) and exanthem (5%). There were no complications of lymphadenopathy, swelling in parotid regions, or meningitis. Our results indicate that simultaneous administration of MMR vaccine and varicella vaccine is a safe and effective method for immunizing children against these four infectious diseases.

[Immunization of healthy children with mumps-rubella bivalent live vaccine and simultaneous vaccination with mumps-rubella and varicella vaccines].

Bivalent virus vaccine, containing rubella TCRB-19 strain and mumps NK-M46 strain (MR vaccine), was administered to a total of 95 healthy children who had already received measles vaccine or had been infected with wild measles virus. The seroconversion rates for rubella and mumps viruses in subjects having no antibody to rubella or to mumps virus were 99% (75/76) and 97% (63/65), respectively, at 6-8 weeks after vaccination. The seroconversion rates for both rubella and mumps in vaccinees initially seronegative to both viruses were 95% (56/59). Immune responses after MR vaccine injection were comparable to those after administration of monovalent rubella or mumps vaccine. Clinical reactions observed in some subjects who received MR vaccine were mild fever (3.6%), exanthem (8%), lymphadenopathy (1.8%), and swelling of the parotis region (1.8%). MR vaccine could be simultaneously injected with varicella vaccine at the opposite site producing no adverse effect on immune response. Our results indicate that MR vaccine is a safe and effective vaccine, especially for children who have had wild measles or who have received measles vaccine.

Predominant recognition of human T cell leukemia virus type I (HTLV-I) pX gene products by human CD8+ cytotoxic T cells directed against HTLV-I-infected cells.

We established long-term cell lines of cytotoxic T lymphocytes (CTL) specific for human T cell leukemia virus type I (HTLV-I) from peripheral blood lymphocytes (PBL) of a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an HTLV-I-carrier with Sjögren syndrome, and an asymptomatic HTLV-I-carrier, by repeated stimulation with autologous HTLV-I-infected T cells in vitro. CTL derived from the patient with HAM/TSP expressed CD8 antigen, and their function was restricted by HLA-A2. They showed cytotoxic effects predominantly against the target cells expressing HTLV-I p40tax among the autologous B cell lines infected with vaccinia recombinants containing various HTLV-I genes which served as targets. These data are consistent with the previously reported findings that fresh PBL of HAM/TSP patients contain p40tax-specific CTL activity. Furthermore, CTL derived from the patient with Sjögren syndrome without neurological involvement also demonstrated cytotoxicity predominantly to p40tax. The cytotoxicity to the target cells experimentally expressing p40tax was blocked by unlabeled HTLV-I-infected cells possessing HLA-A2. HTLV-I-specific cytotoxicity was also inhibited by unlabeled B cells bearing p40tax. Thus, HTLV-I p40tax-specific cytotoxicity is mediated by the major CTL population activated by native HTLV-I antigens in patients with HAM/TSP or Sjögren syndrome. In contrast to the CTL of these patients, CTL similarly induced from the asymptomatic HTLV-I-carrier, which were highly cytotoxic to autologous HTLV-I-infected T cells, did not show significant levels of cytotoxicity to autologous B cells expressing p40tax.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of poly(L-lysine-alt-terephthalic acid) microcapsules with fibrinogen.

Interaction of poly(L-lysine-alt-terephthalic acid) microcapsules with fibrinogen was investigated by measuring the degree of microcapsule disintegration, fibrinogen adsorption to microcapsules, and the zeta potential of microcapsules as a function of fibrinogen concentration in phosphate buffer solutions of pH 7.4 at different ionic strengths. A minimum concentration was found to exist for fibrinogen to cause disintegration of the microcapsules at any ionic strength, and at this fibrinogen concentration the fibrinogen adsorption levelled off and the zeta potential exhibited an abrupt change. Increases in the ionic strength of the medium and the surface hydrophobicity of microcapsules produced an increase in the degree of microcapsule disintegration by fibrinogen. These findings were interpreted as showing that the interaction of the microcapsules with the protein is not of an ionic nature but of a hydrophobic nature.

Development and evaluation of the TD97 measles virus vaccine.

The TD97 strain vaccine virus was prepared from the Tanabe strain measles virus by low-temperature passages in primary cell cultures and ultraviolet (UV) mutagenesis. The TD97 strain exhibited the following characteristics: highly temperature sensitive, neither multiplying nor forming any plaques at 40 degrees C in Vero cells; genetically stable, maintaining high temperature sensitivity after ten successive passages in CE cells at 30 degrees C or 35 degrees C; and M proteins of this virus about 1 KD slower in mobility in SDS-PAGE than that of the Tanabe strain. The TD97 strain was further confirmed to be attenuated by an inoculation test into primate brain. In field trials, 752 healthy children were inoculated with a live virus vaccine prepared with this strain, and the following results were obtained: the seroconversion rate was 97% (517/533), and the average HI antibody titer was 2(5.2). An antibody-increasing effect was also observed in children who were initially seropositive. In children who seroconverted, the rates of fever were 15.7% (55/351) for 37.5 degrees C or higher and 4.0% (14/351) for 39 degrees C or higher. The rash rate was 7.7% (27/351), and the incidence of local reaction was 5.4% (19/351). The TD97 strain is thus considered to be suitable in use for an attenuated measles vaccine.

Effects and virulences of recombinant vaccinia viruses derived from attenuated strains that express the human T-cell leukemia virus type I envelope gene.

We constructed recombinant vaccinia viruses (RVVs) that expressed human T-cell leukemia virus type I (HTLV-I) envelope glycoproteins by using attenuated vaccinia viruses (VVs) which have much lower neurovirulence than the WR strain that is extensively used as a vector. The RVV produced from the LC16mO strain, one of the attenuated VVs, elicited a high titer of anti-HTLV-I antibody in rabbits and protected them against HTLV-I infection. The env gene was inserted into the VV hemagglutinin gene. The resultant inactivation of the hemagglutinin gene led to the attenuation of VVs, but the extent of their attenuation depended on the VV strain. The propagation of LC16mO and its RVV in rabbit brain was poorer than that of LO-1, a cloned derivative of Lister strain, and its RVV, although LC16mO replicated in other organs better than did LO-1. Taken together, these results suggest that LC16mO is a good candidate as a vector for vaccination of humans.

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.

(+)-Nortrachelogenin, a new pharmacologically active lignan from Wikstroemia indica.

A new lignan, (+)-nortrachelogenin (I), and a known compound, daphnoretin were isolated from Wikstroemia indica C. A. Meyer (Thymelaeaceae). The structure of (+)-nortrachelogenin was established as 8(R), 8'(R)-4,4',8'-trihydroxy-3,3'-dimethoxylignan-olid(9, 9') on the basis of spectroscopic evidence and comparison with its enantiomer, (-)-nortrachelogenin. (+)-Nortrachelogenin (I) showed effects on the central nervous system producing depression in rabbits.

(+)Nortrachelogenin, a new pharmacologically active lignan from Wikstroemia indica.

A new lignan, (+)-nortrachelogenin (I), and a known compound, daphnoretin were isolated from Wikstroemia indica C.A. Meyer (Thymelaeaceae). The structure of (+)-nortrachelogenin was established as 8(R)8'-4,4',8'-trihydroxy-3,3'-dimethoxylignan-olid, (9,9') on the basis of spectroscopic evidence and comparison with its enantiomer, (-)-nortrachelogenin. +-nortrachelogenin(I) showed effects on the central nervous system producing depression in rabbits.