Establishment of cytotoxic CD4+ T cell clones from cancer patients treated by local immunotherapy

We have previously reported that the antitumor effect of OK-432, aStreptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (Cancer, 69: 636–642, 1992). In order to elucidate the mechanism of the antitumor effects, we established T cell clones from regional lymph nodes of colorectal cancer patients who received this local immunotherapy. By culture of lymph node lymphocytes, in the presence of IL-2 and OK-432, 4 clones of T cells were established from 4 patients treated by local immunotherapy. These clones had a helper T cell phenotype (CD3⁺, CD4⁺, CD8^–, CD56^–, WT31⁺) and were successfully maintained for several months. The cells strongly expressed CD25 when stimulated with OK-432 and exhibited a high level of cytotoxic activity in part explained by the increased expression of ICAM-1 and LFA-1, and the release of TNF. These results suggest that the CD4⁺ T cells play a role in the antitumor mechanism of local immunotherapy.     

Induction of tumor-specific T lymphocyte responses in vivo by prothymosin α

We have recently reported that administration of Pro T to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunother 38:281]. In this report we demonstrate that Pro T , when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumorspecific CD8⁺ cytotoxic T lymphocytes (CTL). The Pro T -induced CD8⁺ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2K^d allelic product could inhibit the cytotoxic response. Mice receiving only Pro T developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving Pro T and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The Pro T -induced CD8⁺ CTL activity was regulated by Pro T -induced L1210-specific syngeneic CD4⁺ cells. This was shown in two different ways: first, CD8⁺-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4⁺ cells and, second, CD4⁺ cells from mice that had received both Pro T and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8⁺ cells from mice that had received only L1210 cells. Our data suggest that Pro T is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since Pro T may prove to be useful in clinical protocols aimed at cancer immunotherapy.This work was supported by a CEC grant to Dr. M. Papamichail     

Phenotypic and functional analysis of lymphocytes infiltrating paediatric tumours,with a characterization of the tumour phenotype

Summary Tumour-infiltrating lymphocytes (TIL) of paediatric tumours obtained from 37 lesions of different histo-type (12 osteosarcomas, 5 Wilms' tumours, 7 soft-tissue sarcomas, 5 neuroblastomas and 8 miscellaneous) were studied to establish their potential for therapy. Fresh isolated TIL were cultured for the first 2 weeks with low doses of interleukin-2 (IL-2) (20 Cetus U/ml) to select for tumour-specific lymphocytes potentially present in the neoplastic lesion, followed by culture with high doses of IL-2 (1000 Cetus U/ml) to achieve TIL expansion. TIL were grown with more than 10-fold expansion in only 9 cases (mean expansion: 58-fold, range 13.5–346). In 17 cases no viable cells were obtained. After 30 days of culture with IL-2 the proliferative ability of TIL declined sharply in the majority of cases and TIL became refractory to any further stimulus, including addition of IL-4, tumour necrosis factor (TNF) or interferon , and activation with OKT3 in solid phase. In 20 out of 37 cases TIL were available for phenotypic and functional analysis. TIL after long-term culture were predominantly CD3⁺ but 2 cases of osteosarcoma showed a predominance of CD3⁺TcR / cells. The CD4/CD8 ratio was more than 1 in 10 cases, without correlation with tumour histology, site of lesion or TIL growth. The number of CD16⁺ and CD25⁺ lymphocytes decreased progressively during culture, the latter concomitantly with a reduction of TIL growth rate. The lytic pattern of TIL against allogeneic and autologous tumour (Auto-Tu) cells was variable, but specific lysis of Auto-Tu was seen in only one case (Wilms' tumour) after culture with TNF and irradiated Auto-Tu cells. The immunohistochemical analysis of tumour lesions revealed a limited lymphocyte infiltrate, a low expression of histocompatibility leukocyte antigens (HLA) class I and of the adhesion molecules ICAM1, LFA3, and a significant production of transforming growth factor (TGF). These data indicate that TIL obtained from paediatric patients are difficult to expand at levels required for immunotherapy and lack a significant number of tumour-specific T lymphocytes. A low expression of immunomodulatory molecules on tumour cells or the production of suppressive factors may prevent activation and expansion of TIL in paediatric tumours.     

T-cell receptor gene rearrangements and expression in normal human large granular lymphocytes (LGL) and their pathological expansions

The lineage to which normal large granular lymphocytes/natural killer (LGL/NK) cells belong is controversial; in fact they share some surface markers and functional activities with monocytes, but also with T lymphocytes. The relationship of LGL to the T cell lineage by analysis with the T cell receptor (T-rec) gene has been investigated. Pure preparations of human LGL and their CD11⁺ CD8^- and CD11^- CD8⁺ subsets had the T gene in its unrearranged germline configuration. Expression of T and T genes was not detectable. The organization of T gene, which is of particular importance because it occurs early in T cell ontogeny, was also found in its germline configuration.A rare type of lymphoproliferative disorder, termed T-LPD, is characterized by expansion of cells very similar to LGL for morphology, phenotype, and functional activity. Of 17 patients with T-LPD studied for T-rec rearrangement, 15 displayed rearrangement of T and T loci and were CD3+ (14/15 had monoclonal rearrangement), while 2 cases were in germline configuration and were CD3–. Similarly to very small subsets of CD3+ LGL recently described, most T-LPD cases are CD3+ and have T-rec genes rearranged. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage; they also demonstrate that, in contrast to previous views, most TLPD are monoclonal, presumably neoplastic, lymphoproliferative disorders.Abbreviations LGL large granular lymphocytes - NK natural killer - T-rec T-cell receptor - TLPD T lymphoproliferative disease     

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

Tumor-specific expression of Qa-2^k antigen coded by the Q5^k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2^– mice. Lymphocytes stimulated in vivo withP. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (L^Q7b/Kb), which expressed the 1 and 2 domains of the Qa-2 antigen (Q7^b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5 half of the Q7^b gene and the 3 half of the H-2K^b gene (pQ7^b/K^b). Using L^Q7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes fromP. acnes-stimulated (C3H/He × B6.K1)F1 mice (H-2^k, Qa-2^–) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the 1/2 region of the Q7^b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the / type (TCR/). The (C3H/He × B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2^k+) derived from AKR mice (Qa-2^–, H-2^k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed L^Q7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the 1/2 region of the Qa-2^k tumor antigen by TCR/.     

Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Enhancement of the antibody-dependent cellular cytotoxicity of human peripheral blood lymphocytes with interleukin-2 and interferon α

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3^–, CD56⁺, CD16⁺ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon (rIFN) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFN induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15–30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20–50 U/ml. We now show that activation of the NK cells with a combination of rIL-2and rIFN induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFN was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of FcRIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this FcR was essential for ADCC of the human effector cells.Supported by a grant from the Dutch Cancer Society (grant NKI-84-14)     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Effect of β-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-El cells: Increase in osteocalcin,insulin-like growth factor-I and transforming growth factor-β

The effect of -alanyl-L-histidinato zinc (AHZ) on protein components in osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 3 days at 37°C in CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further 3 or 6 days. The homgenate of cells was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of AHZ (10^–7 to 10^–5 M) caused an appreciable increase of many protein components in cells. Especially, the 67 killo-dalton (kDa) and 44 kDa proteins which are the major components from control cells were clearly increased by the presence of AHZ. Furthermore, the concentrations of osteocalcin, insulin-like growth factor-I and transforming growth factor- in the culture medium secreted from osteoblastic cells were markedly increased by the presence of AHZ (10^–6 and 10^–5 M). The effect of AHZ was a greater than that of zinc sulfate (10^–6 and 10^–5 M). The present findings suggest that AHZ can increase many proteins which are involved in the stimulation of bone formation and cell proliferation in osteoblastic cells.     

Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies?

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn₇–MT 1 and corresponding Zn₄-MT 1 and Zn₃-MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn₇–MT 1, Zn₄–MT 1 and Zn₃-MT 1 with AgClO₄ at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn₇–MT 1 at pH 7.5 has revealed the subsequent formation of Ag₄Zn₅–MT, Ag₇Zn₃–MT, Ag₈Zn₃–MT, Ag₁₀Zn₂–MT, Ag₁₂Zn₁–MT, Ag_x–MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag_x–MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the _xZn_y–MT (0y<7) species found, only M^I₄Zn₅–MT [(Zn₄)(₄Zn₁)] and M^I₇Zn₃–MT [(₃Zn₂)(₄Zn₁)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Summary Expression of the red ⁺ and gam ⁺ genes of bacteriophage in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red ⁺ or gam ⁺ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam ⁺ or red ⁺ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red ⁺ products, protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD^– Exol^– cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3 singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.Abbreviations PLM plasmid linear multimers - BUDR 5-bromo-2-deoxyuridine - bp base pair     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.     

Effects of natural interferon α, natural tumor necrosis factor α and their combination on human mesothelioma xenografts in nude mice

Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 10⁷ or 2 × 10⁸ IU kg^–1 day^–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 10⁷ or 2 × 10⁸ U kg^–1 day^–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.     

Glucose-induced reduction of the sodium content in β-cell-rich pancreatic islets

The sodium contents of -cell-rich pancreatic islets fromob/ob-mice were measured with an integrating flame photometer. After washing to an apparent steady state with different types of ice-cold media, islets incubated in the absence of glucose contained 79–108 mmol sodium kg^–1 dry weight. Exposure to glucose resulted in 25 % reduction of the islet content of sodium. This effect became manifest in the presence of 5 mM glucose, there being no additional reduction with a further increase of glucose to 20 mM. Depression of Na⁺ activity may partially explain why glucose, under certain conditions, can lower cytoplasmic Ca²⁺ and even inhibit insulin release.     

Typing of hepatic nonparenchymal cells using fibulin-2 and cytoglobin/STAP as liver fibrogenesis-related markers

Fibulin-2 and cytoglobin/stellate cell activation-associated protein (Cygb/STAP) are considered to be markers of hepatic myofibroblasts (MFs) and stellate cells (HSCs), respectively. The aim of the present study was to characterize the nonparenchymal cells (NPCs) of normal rat livers and carbon tetrachloride-induced fibrotic rat livers with respect to the expression of these two proteins. NPCs in normal (Glissons capsules) and fibrotic (fibrotic septa) connective tissues were immunohistochemically categorized into four cell types in terms of the expression of fibulin-2 and Cygb/STAP: fibulin-2 and Cygb/STAP double-positive (Fib⁺/STAP⁺); fibulin-2-positive and Cygb/STAP-negative (Fib⁺/STAP^–); Fib^–/STAP⁺; and Fib^–/STAP^–. The Glissons capsules had Fib⁺/STAP⁺ and Fib^–/STAP^– cell occupancy rates of 45.5% and 54.5%, respectively, but did not contain Fib⁺/STAP^– or Fib^–/STAP⁺ cells. On the other hand, the fibrotic septa contained Fib⁺/STAP⁺, Fib^–/STAP⁺, and Fib^–/STAP^– cells at occupancy rates of 35.0%, 50.5%, and 9.1%, respectively, but did not contain Fib⁺/STAP^– cells. Thus, fibrosis is characterized by a dramatic increase in Fib^–/STAP⁺ NPCs, and a dramatic decrease in Fib^–/STAP^– NPCs. Fib⁺/STAP⁺ NPCs are located uniformly in Glissons capsules and peripherally in fibrotic septa. The present study strongly suggests that Fib⁺/STAP⁺ and Fib^–/STAP⁺ NPCs correspond to MFs and activated HSCs, respectively, both of which may contribute to liver fibrogenesis.