The origin of ß-strand bending in globular proteins.

BACKGROUND: Many ß-strands are not flat but bend and/or twist. However, although almost all ß-strands have a twist, not all have a bend, suggesting that the underlying force(s) driving ß-strand bending is distinct from that for the twist. We, therefore, investigated the physical origin(s) of ß-strand bends. METHODS: We calculated rotation, twist and bend angles for a four-residue short frame. Fixed-length fragments consisting of six residues found in three consecutive short frames were used to evaluate the twist and bend angles of full-length ß-strands. RESULTS: We calculated and statistically analyzed the twist and bend angles of ß-strands found in globular proteins with known three-dimensional structures. The results show that full-length ß-strand bend angles are related to the nearby aromatic residue content, whereas local bend angles are related to the nearby aliphatic residue content. Furthermore, it appears that ß-strands bend to maximize their hydrophobic contacts with an abutting hydrophobic surface or to form a hydrophobic side-chain cluster when an abutting hydrophobic surface is absent. CONCLUSIONS: We conclude that the dominant driving force for full-length ß-strand bends is the hydrophobic interaction involving aromatic residues, whereas that for local ß-strand bends is the hydrophobic interaction involving aliphatic residues.

Myeloid suppressor cell-associated immune dysfunction in CSA1M fibrosarcoma tumor-bearing mice.

CSA1M tumor-bearing mice exhibited a severe immune dysfunction but the underlying mechanism remained unclear. In this study, we demonstrated that the myeloid suppressor cell (Mac-1(+)Gr-1(+) cells)-(MSC) related T cell immunosuppression in this tumor-bearing model. In mice at the late stage of CSA1M tumor-bearing (Late TB [8-10 weeks after cell inoculation in male BALB/c mice]), the percentages for CD4(+) and CD8(+) T cells decreased but Mac-1(+) cells increased in spleens with severe splenomegaly. There was no deficit for concanavalin A-induced CD4(+) and CD8(+) T cell proliferation, interferon-gamma (IFN-gamma) and interleukin (IL)-4 production, but delayed-type hypersensitivity reaction were attenuated. Analysis of cytokine production in unfractionated spleen cells showed a significant reduction of IFN-gamma and a marked increase of IL-10 and IL-4. In Late-TB mice, splenic MSC number intensively accumulated; the mRNA expressions of the signal transducer and activator of transcription 1, interferon regulatory factor 1 (IRF-1), and inducible nitric-oxide synthase (iNOS) were enhanced in MSC; the nitric oxide production and arginase enzyme activity increased in MSC as well. Furthermore, the concanavalin A-induced T cell proliferation was inhibited in the presence of lipopolysaccharide- or IFN-gamma-activated MSC from Late-TB mice, which could be reversed by the iNOS specific inhibitor L-NMMA. iNOS seemed to be required more than arginase for the suppressive activity of MSC. Taken together, our results suggest that the immune dysfunction in tumor-bearing mice might be causally associated with the accumulation of MSC and its tumor-favoring property.

(5R)-5-hydroxytriptolide attenuated collagen-induced arthritis in DBA/1 mice via suppressing interferon-gamma production and its related signaling.

(5R)-5-Hydroxytriptolide (LLDT-8) displays strong immunosuppressive activities both in vitro and in vivo in our previous studies. This study aims to investigate whether LLDT-8 has antiarthritic potential in a murine model of type II bovine collagen (CII)-induced arthritis (CIA) and to show the mechanism(s) of LLDT-8 action. DBA/1 mice were immunized with CII to induce arthritis and administered with LLDT-8. The severity of arthritis was evaluated according to the clinical score and joint damage. The effects of LLDT-8 on immune responses were determined by measurement of serum antibody levels, lymphocyte proliferation assay, cytokine assay, nitric oxide (NO) production, arginase activity assays, fluorescence-activated cell sorting analysis of splenic Mac-1+ cells, as well as polymerase chain reaction analysis for interferon-gamma (IFN-gamma)-related gene expression. We showed that LLDT-8 treatment significantly reduced the incidence and severity of CIA. The preventive and therapeutic effects of LLDT-8 are associated with 1) reduction of serum anti-CII immunoglobulin (Ig) G, IgG2a, and IgG1 levels; 2) inhibition of CII-specific lymphocyte proliferation, IFN-gamma and interleukin-2 production; 3) blockade of gene expressions in IFN-gamma signaling, including IFN-gamma production pathways [signal transducer and activator of transcription (STAT) 1, T-box transcription factor, interleukin 12Rbeta2, and STAT4] and IFN-gamma-induced chemokine transcription [macrophage inflammatory protein (Mip)-1alpha, Mip-1beta, regulated on activation normally T cell expressed and secreted, and inducible protein 10]; and 4) retardation of the abnormal increase of NO via IFN-gamma/STAT1/interferon regulatory factor 1/inducible nitric-oxide synthase pathway and arginase activity. Moreover, the mRNA transcription of chemokine receptors was also suppressed [including C-C chemokine receptor (CCR) 1, CCR5, and C-X-C chemokine receptor 3]. In conclusion, our data suggest that the antiarthritic effect of LLDT-8 is closely related to the blockade of IFN-gamma signaling. LLDT-8 may have a therapeutic value in the treatment of rheumatoid arthritis.

Influence of the polyol pathway on norepinephrine transporter reduction in diabetic cardiac sympathetic nerves: implications for heterogeneous accumulation of MIBG.

PURPOSE: Cardiac scintigraphic studies using 123I-labeled metaiodobenzylguanidine ([123I]MIBG) have demonstrated heterogeneous myocardial accumulation of MIBG in diabetes. The accumulation has been found to correlate with a heterogeneous decrease in the expression of norepinephrine transporter (NET). In diabetic peripheral nerve tissue, polyol pathways are activated and cause nerve dysfunction and degeneration. However, there has been little research on the polyol pathway and cardiac sympathetic nerves. Therefore, to assess the influence of the polyol pathway on cardiac sympathetic nervous function, we investigated the regional accumulation of MIBG and NET protein expression in diabetic model rats treated with aldose reductase inhibitor (ARI) for the blockade of polyol pathways. METHODS: Rats were given a single intravenous injection of streptozotocin (n=76, STZ-D rats). Starting the day after STZ injection, ARI was administered daily to 42 of the rats for 4 weeks (ARI-D rats). To assess the cardiac sympathetic nervous function, [125I]MIBG autoradiographic experiments were carried out. Finally, NET protein expression was assessed with a saturation binding assay. RESULTS: The myocardial sorbitol concentration was significantly higher in STZ-D rats than in ARI-D rats. There was no heterogeneous accumulation of MIBG in ARI-D rats. There was a heterogeneous decrease of NET expression in STZ-D rats, but not in ARI-D or control rats. CONCLUSION: The gathered data indicate that the enhanced polyol pathway correlates with the decrease in regional cardiac sympathetic nervous function, and this impairment may lead to the reduction of NET protein in cardiac sympathetic nerves of the diabetic inferior wall.

HIV entry inhibitor TAK-779 attenuates atherogenesis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: HIV combination therapy using protease inhibitors is associated with elevated plasma levels of atherogenic lipoproteins and increased risk for atherosclerosis. We investigated whether the HIV entry inhibitor TAK-779 affects lipoprotein levels and atherogenesis in low-density lipoprotein receptor-deficient mice. TAK-779 is an antagonist for the chemokine receptors CCR5 and CXCR3, which are expressed on leukocytes, especially T-helper 1 cells, and these receptors may be involved in recruitment of these cells to atherosclerotic plaques. METHODS AND RESULTS: TAK-779 treatment of low-density lipoprotein receptor-deficient mice did not elevate the levels of atherogenic lipoproteins, whereas it dramatically reduced atherosclerosis in the aortic root and in the carotid arteries. The number of T cells in the plaque was reduced by 95%, concurrently with a 98% reduction in the relative IFN-gamma area. TAK-779-treated animals showed a decreased percentage of CD4+ and CD8+ T cells in peripheral blood and in mediastinal lymph nodes compared with control-treated animals. CONCLUSIONS: TAK-779 not only suppresses HIV entry via blockade of CCR5 but also attenuates atherosclerotic lesion formation by blocking the influx of T-helper 1 cells into the plaque. TAK-779 treatment may be especially beneficial for young HIV patients as they face lifelong treatment, and this drug impairs atherogenesis.

Induction of surface CCR4 and its functionality in mouse Th2 cells is regulated differently during Th2 development.

T helper cell type 1 (Th1) and Th2 cells express distinct sets of chemokine receptors. In contrast to Th1 chemokine receptors, it is largely unknown how Th2 chemokine receptors such as CC chemokine receptor 4 (CCR4) are induced during Th2 differentiation. Here, we investigated the induction of CCR4 surface expression and ligand responsiveness evaluated by functional assays such as chemokine binding and chemotaxis. This was done in comparison with those of a Th1 chemokine receptor, CXC chemokine receptor 3 (CXCR3). Resting T cells expressed neither CXCR3 nor CCR4. CXCR3 expression and ligand responsiveness were observed when resting T cells were stimulated with anti-CD3 plus anti-CD28 in the presence of [interleukin (IL)-12+anti-IL-4] and then recultured without T cell receptor (TCR) stimulation. Unlike CXCR3, CCR4 was induced immediately after anti-CD3/anti-CD28 stimulation in the presence of (IL-4+anti-interferon-gamma+anti-IL-12). However, these CCR4-positive cells failed to exhibit chemokine binding and chemotaxis. Although the levels of surface CCR4 expression were not increased after the subsequent reculture in the absence of TCR stimulation, CCR4 responsiveness was induced in this stage of Th2 cells. The induction of CCR4 expression and the acquisition of CCR4 responsiveness did not occur in IL-4-deficient (IL-4(-/-)) and signal transducer and activator of transcription (STAT)6(-/-) T cells. CCR4 expression and functionality were regained in IL-4(-/-) but not in STAT6(-/-) T cells by the addition of recombinant IL-4. Although surface expression and functionality of CCR4 are induced depending on the IL-4/STAT6 signaling pathway, the present results indicate that the functionality of CCR4 does not correlate with CCR4 expression but emerges at later stages of Th2 differentiation.

B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors.

The B7/CD28 co-stimulatory pathway plays a critical role in T cell activation and differentiation. Our previous study demonstrated that administration of B7.2-Ig fusion proteins to tumor-bearing mice elicits IL-4-dependent, CD8+ T cell-mediated tumor regression. Here, we investigated whether B7.2-Ig stimulation of tumor-sensitized CD8+ CTL precursors during in vitro antigen re-sensitization actually results in their differentiation into mature CTLs and if so, whether such a process depends on IL-4 signals. Splenocytes from tumor-sensitized (tumor-bearing or tumor-immunized) mice exhibited low levels of anti-tumor CTL responses upon culturing alone, but induced strikingly enhanced CTL responses when stimulated in vitro with B7.2-Ig fusion proteins. Because CTLs were not generated from normal splenocytes even by B7.2-Ig stimulation, the expression of the B7.2-Ig effect required the in vivo tumor sensitization of CD8+ CTL precursors. Administration of anti-CD4 or anti-CD40 ligand (CD40L) to mice before tumor sensitization resulted in almost complete inhibition of CTL responses generated in the subsequent culture containing B7.2-Ig. In contrast, anti-IL-4 did not influence in vivo tumor sensitization required for CTL induction. However, B7.2-Ig stimulation of tumor-sensitized splenocytes enhanced IL-4 production and neutralization of this IL-4 with anti-IL-4 potently down-regulated CTL responses. These results indicate that B7.2-Ig enhances IL-4-dependent differentiation of anti-tumor CD8+ CTL precursors that can be sensitized in vivo depending on collaboration with CD4+ T cells involving CD40L function.

Type I interferons inhibit maturation and activation of mouse Langerhans cells.

Type I interferons (IFN) have an essential role in antiviral defense, and they are produced upon viral infection in a variety of cells. IFN-alpha/beta treatment of immature dendritic cells (DC) is known to induce their phenotypic and functional maturation, but it remains unclear whether stimulation by this cytokine family influences the functions and maturation of Langerhans cells (LC). We used highly enriched (>95%) LC directly isolated from BALB/c mouse skin and addressed this issue, comparing LC with splenic CD11c(+) DC. Type I IFN-treated LC exhibited impaired ability to produce IL-12 and inflammatory cytokines, IL-6 and TNF-alpha, whereas IL-10 production was not augmented. In splenic DC, the production of inflammatory cytokines was rather enhanced by type I IFN treatment. With regard to chemokines, in both LC and splenic DC, type I IFN upregulated the production of inflammatory chemokines, such as CXCL10, CXCL11, CCL3, CCL4, and CCL5. Strikingly, IFN-beta treatment reduced the expression of CD40, CD54, CD80, and CD86 on LC, whereas IFN-beta-treated splenic DC showed enhanced expression of these molecules. Furthermore, IFN-beta-treated LC had impaired costimulatory activity for anti-CD3-induced proliferation of T cells. Finally, treatment with IFN-alpha/beta reduced the migratory capacity of LC to CCL21. These results indicate that type I IFN inhibit maturation and activation of LC in a direct manner. Our observations may provide a novel explanation for the reported inability of LC to act as potent antigen-presenting cells in cutaneous and mucosal viral infection.

Histiocytoid breast carcinoma: solid variant of invasive lobular carcinoma with decreased expression of both E-cadherin and CD44 epithelial variant.

Histiocytoid breast carcinoma (HBC) is a rare type of breast carcinoma with morphologic characteristics resembling those of histiocytes. Described herein are cytological and histological findings in a case of HBC. Fine-needle aspiration cytology revealed numerous loosely cohesive tumor cells with abundant foamy to granular cytoplasm and bland-appearing nuclei. The resected tumor exhibited a solid growth pattern instead of classic invasive lobular patterns observed in most reported cases of HBC. However, distinct intracytoplasmic lumina and Pagetoid extension to ducts suggested that this tumor was a variant of invasive lobular carcinoma. To determine the cause of the loose cellular cohesiveness of this HBC, its expression of the epithelium-related cell adhesion molecules E-cadherin and CD44v8-10 (CD44 epithelial variant) was examined. Immunohistochemically, E-cadherin was not detected, similar to most lobular carcinomas. Furthermore, competitive reverse transcription-polymerase chain reaction (RT-PCR) analyses among alternatively spliced variants of CD44 revealed that the ratio of expression of CD44v8-10 to that of CD44v10 (dominant variant in leukocytes) was lower than that for the reference breast carcinoma samples. It is concluded that the present case of HBC was a solid variant of invasive lobular carcinoma exhibiting foamy to granular cytoplasmic change. Decreased expression of both E-cadherin and CD44 epithelial variant may be responsible for the loose cellular cohesiveness observed in HBC.

The involvement of neutrophils in the resistance to Leishmania major infection in susceptible but not in resistant mice.

To understand the immunomodulatory roles of neutrophils in Leishmania major infection, we examined the expression of cytokine and chemokine mRNAs from neutrophils of the infected resistant C3H/HeJ and susceptible BALB/c mice. We also examined the effects of neutrophil depletion on the expression of cytokine by peritoneal macrophages and draining lymph node cells and on the footpad lesions and parasite burdens in these mice. Neutrophils from resistant C3H/HeJ but not from susceptible BALB/c mice expressed mRNAs for IL-12p40, IFN-gamma,TNF-alpha and monokine induced by IFN-gamma(MIG). Neutrophil depletion of the resistant mice reduced the expression of IFN-gammaandTNF-alpha in peritoneal macrophages but did not affect the expression of IL-12p40 and IFN-gamma in draining lymph node cells and the growth of footpad lesions. On the other hand, neutrophil depletion of susceptible BALB/c mice did not affect the expression of TNF-alpha and monocyte-derived chemokine (MDC) in peritoneal macrophages but induced the early stage expression of IL-4 in draining lymph node cells and exacerbated the footpad lesions and increased the parasite burden. The exacerbation of footpad lesions induced by neutrophil depletion was abolished by rIL-12 treatment. Our results suggest that even in susceptible BALB/c but not in C3H/HeJ mice there is a certain resistance requiring neutrophils at the early stage of infection.

A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d-galactosamine.

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-alpha) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (alpha-GalCer), an NKT-cell stimulant, into D-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and Valpha14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with alpha-GalCer. The lethal effect was suppressed in TNF-alpha KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN-gamma KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF-alpha secretion and direct cytotoxicity of alpha-GalCer-activated NKT cells.

Influence of the polyol pathway on norepinephrine transporter reduction in diabetic cardiac sympathetic nerves: implications for heterogeneous accumulation of MIBG.

PURPOSE: Cardiac scintigraphic studies using (123)I-labeled metaiodobenzylguanidine ([(123)I]MIBG) have demonstrated heterogeneous myocardial accumulation of MIBG in diabetes. The accumulation has been found to correlate with a heterogeneous decrease in the expression of norepinephrine transporter (NET). In diabetic peripheral nerve tissue, polyol pathways are activated and cause nerve dysfunction and degeneration. However, there has been little research on the polyol pathway and cardiac sympathetic nerves. Therefore, to assess the influence of the polyol pathway on cardiac sympathetic nervous function, we investigated the regional accumulation of MIBG and NET protein expression in diabetic model rats treated with aldose reductase inhibitor (ARI) for the blockade of polyol pathways. METHODS: Rats were given a single intravenous injection of streptozotocin (n=76, STZ-D rats). Starting the day after STZ injection, ARI was administered daily to 42 of the rats for 4 weeks (ARI-D rats). To assess the cardiac sympathetic nervous function, [(125)I]MIBG autoradiographic experiments were carried out. Finally, NET protein expression was assessed with a saturation binding assay. RESULTS: The myocardial sorbitol concentration was significantly higher in STZ-D rats than in ARI-D rats. There was no heterogeneous accumulation of MIBG in ARI-D rats. There was a heterogeneous decrease of NET expression in STZ-D rats, but not in ARI-D or control rats. CONCLUSION: The gathered data indicate that the enhanced polyol pathway correlates with the decrease in regional cardiac sympathetic nervous function, and this impairment may lead to the reduction of NET protein in cardiac sympathetic nerves of the diabetic inferior wall.

Differential production of Th1- and Th2-type chemokines by mouse Langerhans cells and splenic dendritic cells.

Some chemokines specifically attract T helper 1 (Th1) cells, whereas others attract T helper 2 (Th2) cells. In this study, we investigated the capacity of Langerhans cells (LC) to produce Th1- and Th2-type chemokines in comparison with that of splenic CD11c(+) dendritic cells (DC). We prepared highly purified (>95%) LC from BALB/c mouse skin using the panning method. With regard to Th1-type chemokines, exogenous stimulus, such as interferon-gamma (IFN-gamma), lipopolysaccharide, or polyinosinic-polycytidylic acid, was mandatory for the production of substantial amounts of CXCL10, CXCL9, and CXCL11 both in LC and splenic DC. LC, as a whole, exhibited low ability to produce Th1-type chemokines in comparison with splenic DC. As for Th2-type chemokines, LC, but not splenic DC, produced high levels of CCL22 and CCL17 constitutively during culture even without exogenous stimuli. The production of Th2-type chemokines was regulated in a complicated manner. In particular, interleukin-4 upregulated, and IFN-gamma downregulated both CCL22 and CCL17 production by LC. Of note, LC produced much more amounts of Th2-type chemokines than splenic DC under any conditions tested. Finally, Th1- and Th2-type chemokines produced by LC were shown to be functional using chemokine receptor-transfected-2B4 T cells. The high production of CC chemokine receptor 4 ligands by LC in the absence of IFN-gamma may be an important character discriminating LC from other DC.

The capacity of the natural ligands for CD28 to drive IL-4 expression in naïve and antigen-primed CD4+ and CD8+ T cells.

The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of naïve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Naïve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for naïve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when naïve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.

Anti-HER-2/neu immune responses are induced before the development of clinical tumors but declined following tumorigenesis in HER-2/neu transgenic mice.

HER-2/neu oncogene products have been implicated as a potential target of T cell-mediated immune responses to HER-2/neu-induced tumors. Using HER-2/neu transgenic mice (oncomice), we investigated whether, and if so how, anti-HER-2/neu immune responses are induced and modulated in these oncomice from birth to tumor initiation. Female oncomice carrying the activated HER-2/neu oncogene displayed apparent hyperplasia in mammary glands at 10 weeks of age and developed mammary carcinomas around an average age of 26 weeks. Unfractionated spleen cells from 10- to 15-week-old oncomice that were cultured without any exogenous stimuli exhibited cytotoxicity against the F31 tumor cell line established from an HER-2/neu-induced mammary carcinoma mass. The final antitumor effectors were a macrophage lineage of cells. However, this effector population was activated, depending on the stimulation of oncomouse CD4(+) T cells with oncomouse-derived antigen-presenting cell (APC) alone or with wild-type mouse APC in the presence of F31 membrane fractions, suggesting the presence of HER-2/neu-primed CD4(+) T cells and HER-2/neu-presenting APC in 10- to 15-week-old oncomice. These antitumor cytotoxic responses were detected at approximately 5 weeks of age and peaked at age 10 to 15 weeks. However, the responses then declined at tumor-bearing stages in which the expression of target proteins could progressively increase. This resulted from the dysfunction of CD4(+) T cells but not of APC or effector macrophages. These results indicate that an anti-HER-2/neu CD4(+) T cell-mediated immune response was generated at the pretumorigenic stage but did not prevent tumorigenesis and declined after the development of clinical tumors.

A mechanism underlying STAT4-mediated up-regulation of IFN-gamma induction inTCR-triggered T cells.

IL-12 promotes T(h)1 development/IFN-gamma expression by activating STAT4. However, it is still unclear how STAT4 elicits IFN-gamma promoter activation. Here, we investigated the mechanism by which IL-12-activated STAT4 functions for IFN-gamma induction in TCR-triggered T cells. TCR stimulation induced high levels of IFN-gamma production depending on co-stimulation with IL-12. IL-12 stimulation greatly enhanced the promoter-binding activity of c-Jun/AP-1, a critical transcription factor for IFN-gamma gene expression in wild-type T cells, but not in STAT4-deficient (STAT4(-/-)) T cells. Comparable amounts of c-Jun were induced by TCR stimulation in both wild-type and STAT4(-/-) T cells irrespective of IL-12 co-stimulation. However, c-Jun bound to STAT4 in IL-12-co-stimulated wild-type T cells. c-Jun forming a complex with STAT4 efficiently interacted with the AP-1-related sequence of the IFN-gamma promoter. Such an enhanced c-Jun binding did not occur in STAT4(-/-) T cells. These results show that STAT4 contributes to enhancing IFN-gamma expression by up-regulating the binding of TCR signal-induced AP-1 to the relevant promoter sequence.

Induction of tumor regression by administration of B7-Ig fusion proteins: mediation by type 2 CD8+ T cells and dependence on IL-4 production.

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8(+) effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4(-/-) and STAT6(-/-) mice. However, it was elicited in IFN-gamma(-/-) and STAT4(-/-) mice. By contrast, IL-12-induced tumor regression occurred in IL-4(-/-) and STAT6(-/-) mice, but not in IFN-gamma(-/-) and STAT4(-/-) mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8(+) T cells, although the induction of these effector cells required CD4(+) T cells. Tumor regression induced by CD8(+) T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8(+) T cells (Tc2) leading to Tc2-mediated tumor regression.

Evaluation of interleukin-12-induced interferon-y production in vitro by peripheral blood mononuclear cells in patients with chronic liver disease.

BACKGROUND/AIMS: Interleukin-12 plays an important role in anti-tumor immune response by induction of interferon-gamma production by T cells and NK cells, and by activation of cytotoxic T cells and NK cells. We evaluated interleukin-12-induced interferon-gamma production as one of the immunological markers of patients with chronic liver diseases. METHODOLOGY: Interleukin-12-induced interferon-gamma production was measured in vitro in peripheral blood mononuclear cells from 28 hepatocellular carcinoma patients, 10 liver cirrhosis patients, 14 chronic hepatitis patients and 16 healthy individuals. RESULTS: The hepatocellular carcinoma patients exhibited a reduced interleukin-12 responsiveness for interferon-gamma production compared to the liver cirrhosis patients, the chronic hepatitis patients and the healthy individuals. The reduced interferon-gamma production seemed to roughly reflect clinical stage in the hepatocellular carcinoma patients. The interferon-gamma production correlated with neither alpha-fetoprotein nor protein induced by vitamin K absence II. CONCLUSIONS: The level of interleukin-12-induced interferon-gamma production by peripheral blood mononuclear cells in the patients with hepatocellular carcinoma was significantly lower than that in the patients with liver cirrhosis which is thought to be a premalignant state. The measurement of interferon-gamma production may be useful in evaluating severity of chronic liver disease from an immunological point of view.

A new norepinephrine transporter imaging agent for cardiac sympathetic nervous function imaging: radioiodinated (R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine.

Changes of the cardiac norepinephrine transporter (NET) have been reported in several cardiac failures. (R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine (MIPP), the 3-phenoxy-3-phenylpropylamine analogue iodinated at the 2-position of the phenoxy ring, was synthesized and evaluated as a potential radiopharmaceutical for investigating the cardiac norepinephrine transporter by single photon emission computed tomography (SPECT). (R)-[(125)I]MIPP was synthesized via a halogen exchange reaction under no-carrier-added conditions and purified by high-performance liquid chromatography (HPLC) with high radiochemical yield (60%) and high radiochemical purity (> 98%). The binding affinity of (R)-MIPP for cardiac NET was measured in terms of the displacement of [(3)H]desipramine and (R)-[(125)I]MIPP from binding sites in rat heart membranes. The binding data revealed that the affinity of (R)-MIPP was 5 times that of nisoxetine which is a selective NET inhibitor. In biodistribution studies, (R)-[(125)I]MIPP showed a high uptake followed by rapid clearance in the heart. (R)-[(125)I]MIPP binding sites were saturable and the administration of nisoxetine and desipramine, selective NET inhibitors, decreased the cardiac accumulation of (R)-[(125)I]MIPP. These results suggested that (R)-[(123)I]MIPP may be an useful radiopharmaceutical for imaging cardiac sympathetic nervous functions.