Evaluation of the Growth-Inhibitory Spectrum of Three Types of Cyanoacrylate Nanoparticles on Gram-Positive and Gram-Negative Bacteria.

The development of novel effective antibacterial agents is crucial due to increasing antibiotic resistance in various bacteria. Poly (alkyl cyanoacrylate) nanoparticles (PACA-NPs) are promising novel antibacterial agents as they have shown antibacterial activity against several Gram-positive and Gram-negative bacteria. However, the antibacterial mechanism remains unclear. Here, we compared the antibacterial efficacy of ethyl cyanoacrylate nanoparticles (ECA-NPs), isobutyl cyanoacrylate NPs (iBCA-NPs), and ethoxyethyl cyanoacrylate NPs (EECA-NPs) using five Gram-positive and five Gram-negative bacteria. Among these resin nanoparticles, ECA-NPs showed the highest growth inhibitory effect against all the examined bacterial species, and this effect was higher against Gram-positive bacteria than Gram-negative. While iBCA-NP could inhibit the cell growth only in two Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, it had negligible inhibitory effect against all five Gram-negative bacteria examined. Irrespective of the differences in growth inhibition induced by these three NPs, N-acetyl-L-cysteine (NAC), a well-known reactive oxygen species (ROS) scavenger, efficiently restored growth in all the bacterial strains to that similar to untreated cells. This strongly suggests that the exposure to NPs generates ROS, which mainly induces cell growth inhibition irrespective of the difference in bacterial species and cyanoacrylate NPs used.

The Deficiency of Indoleamine 2,3-Dioxygenase Aggravates the CCl4-Induced Liver Fibrosis in Mice.

In the present study, we examined the role of indoleamine 2,3-dioxygenase (IDO) in the development of CCl4-induced hepatic fibrosis. The liver fibrosis induced by repetitive administration with CCl4 was aggravated in IDO-KO mice compared to WT mice. In IDO-KO mice treated with CCl4, the number of several inflammatory cells and the expression of pro-inflammatory cytokines increased in the liver. In the results, activated hepatic stellate cells (HSCs) and fibrogenic factors on HSCs increased after repetitive CCl4 administration in IDO-KO mice compared to WT mice. Moreover, the treatment with l-tryptophan aggravated the CCl4-induced hepatic fibrosis in WT mice. Our findings demonstrated that the IDO deficiency enhanced the inflammation in the liver and aggravated liver fibrosis in repetitive CCl4-treated mice.

Kynurenine production mediated by indoleamine 2,3-dioxygenase aggravates liver injury in HBV-specific CTL-induced fulminant hepatitis.

UNLABELLED: Indoleamine 2,3-dioxygenase (IDO), an enzyme that is ubiquitously distributed in mammalian tissues and cells, converts tryptophan to kynurenine, and is also known as a key molecule that promotes apoptosis in lymphocytes and neurons. In this study, we established hepatitis B virus (HBV)-transgenic (Tg)/IDO-knockout (KO) mice and examined the influence of IDO in a murine fulminant hepatitis model induced by HBV-specific cytotoxic T lymphocytes (CTL). An increase of IDO expression in the livers of HBV-Tg/IDO-wild-type (WT) mice administered HBV-specific CTL was confirmed by real-time polymerase chain reaction, western blotting, and evaluating IDO activity. Plasma alanine aminotransferase (ALT) levels in HBV-Tg/IDO-KO mice after HBV-specific CTL injection significantly decreased compared with those in HBV-Tg/IDO-WT mice. An inhibitor of IDO, 1-methyl-d-tryptophan (1-MT), could also attenuated the observed liver injury induced by this HBV-specific CTL. The expression levels of cytokine and chemokine mRNAs in the livers of HBV-Tg/IDO-WT mice were higher than those in the livers of HBV-Tg/IDO-KO mice. The administration of kynurenine aggravated the liver injury in HBV-Tg/IDO-KO mice injected with HBV-specific CTL. Simultaneous injection of recombinant murine interferon (IFN-γ) and kynurenine also increased the ALT levels in HBV-Tg/IDO-KO mice. The liver injury induced by IFN-γ and kynurenine was improved in HBV-Tg/tumor necrosis factor-α-KO mice. CONCLUSION: Kynurenine and IFN-γ induced by the administration with HBV-specific CTL are cooperatively involved in the progression of liver injury in acute hepatitis model. Our results may lead to a new therapy for the acute liver injury caused by HBV infection.

Induction of hepatitis B virus surface antigen-specific cytotoxic T lymphocytes can be up-regulated by the inhibition of indoleamine 2, 3-dioxygenase activity.

Cytotoxic T lymphocytes (CTLs) are thought to be major effectors involved in viral clearance during acute infections, including hepatitis B virus (HBV) infection. A persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which may be reflective of tolerance to HBV. Efficient induction of HBV-specific CTLs leads to the clearance of HBV in patients with a chronic HBV infection. Previously, we reported that α-galactosylceramide (α-GalCer), a specific natural killer T (NKT) cell agonist, enhanced the induction of HBV surface antigen (HBsAg)-specific CTLs. In the present study, we found that inhibition of indoleamine 2,3-dioxygenase (IDO) activity enhanced the induction of HBsAg-specific CTLs after immunization with HBsAg and α-GalCer. The administration of HBsAg and α-GalCer increased the production of interleukin-2 and interleukin-12b, which are crucial for the induction of HBsAg-specific CTLs. The production of these cytokines was more strongly enhanced in IDO knockout mice compared with wild-type mice. In addition, α-GalCer induced the production of IDO in CD11b(+) cells, and these cells inhibited proliferation of HBsAg-specific CTLs. Our results lead to strategies for improving the induction of HBsAg-specific CTLs.

Design and synthesis of photolabile caged cytokinin.

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.

Ability of IDO to attenuate liver injury in alpha-galactosylceramide-induced hepatitis model.

IDO converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, we examined the effect of IDO in α-galactosylceramide (α-GalCer)-induced hepatitis. The increase in IDO expression in the liver of wild-type (WT) mice administered α-GalCer was confirmed by real-time PCR, Western blotting, and IDO immunohistochemical analysis. The serum alanine aminotransferase levels in IDO-knockout (KO) mice after α-GalCer injection significantly increased compared with those in WT mice. 1-Methyl-D-tryptophan also exacerbated liver injury in this murine hepatitis model. In α-GalCer-induced hepatitis models, TNF-α is critical in the development of liver injury. The mRNA expression and protein level of TNF-α in the liver from IDO-KO mice were more enhanced compared with those in WT mice. The phenotypes of intrahepatic lymphocytes from WT mice and IDO-KO mice treated with α-GalCer were analyzed by flow cytometry, and the numbers of CD49b(+) and CD11b(+) cells were found to have increased in IDO-KO mice. Moreover, as a result of the increase in the number of NK cells and macrophages in the liver of IDO-KO mice injected with α-GalCer, TNF-α secretion in these mice was greater than that in WT mice. Deficiency of IDO exacerbated liver injury in α-GalCer-induced hepatitis. IDO induced by proinflammatory cytokines may decrease the number of TNF-α-producing immune cells in the liver. Thus, IDO may suppress overactive immune response in the α-GalCer-induced hepatitis model.

Advantage of IFN-beta/alpha2b same-day administration for ribavirin-intolerant patients with chronic hepatitis C.

AIM: Although interferon (IFN)/ribavirin is the mainstream combination treatment for chronic hepatitis C in patients with a high viral load, ribavirin is problematic for women of childbearing age and patients with anemia. Therefore we needed to establish a new regimen without ribavirin. METHODS: We devised a new regimen (same-day beta/alpha2b) to administer IFN-beta and alpha2b on the same-day, and compared it with IFN-alpha2b alone and IFN-alpha2b plus ribavirin. The cases were 36 patients (26.1%) in whom ribavirin could not be used (young women, anemia, etc.) among 138 patients who underwent IFN treatment after ribavirin release. RESULTS: The percentages of patients withdrawing due to side-effects were 6.8%, 18.8% and 17.0% in the treatment with same-day beta/alpha2b, IFN-alpha2b alone and IFN-alpha2b plus ribavirin groups, respectively. In genotype 1b, the sustained viral response (SVR) was 28.6% (4/14), 13.6% (3/22) and 25.0% (8/32) with a high viral load, and 91.7% (11/12), 27.3% (3/11) and 57.1% (4/7) with a low viral load for the respective groups. According to a study on viral half-life during the early phase of IFN therapy, there was no difference among the regimens of same-day IFN-beta/alpha2b, beta alone, alpha2b alone and twice-daily treatment with IFN-beta. Same-day beta/alpha2b treatment showed a significantly higher SVR rate in moving type patients with a genotype 1b/high viral load. CONCLUSIONS: Same-day beta/alpha2b treatment resulted in few cases where therapy was discontinued and showed a high SVR rate. This regimen is especially appropriate in cases where ribavirin has been deemed unsuitable.

Interaction between LPS-induced NO production and IDO activity in mouse peritoneal cells in the presence of activated Valpha14 NKT cells.

In this study, we demonstrated that lipopolysaccharide (LPS) markedly increased nitric oxide (NO) production and indoleamine 2,3-dioxygenase (IDO) activity in mouse peritoneal cells in the presence of activated Valpha14 natural killer T cells. Moreover, LPS-induced NO production in peritoneal cells from IDO-knockout (KO) mice was more increased than that from wild-type mice. However, there was no significant difference in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein between the wild-type and IDO-KO mice. No significant difference was also observed in the ratio of CD3- and DX5-positive cells and F4/80- and TLR4-positive cells in peritoneal cells between the wild-type and IDO-KO mice. Since the IDO activity was enhanced by an NO inhibitor, NO may be post-translationally consumed by inhibiting the IDO activity. IDO is well known to play an important role in immunosuppression during inflammatory disease. Therefore, the inhibition of IDO by NO may exacerbate inflammation in the peritoneal cavity.

Role of TNF-alpha produced by nonantigen-specific cells in a fulminant hepatitis mouse model.

In previous studies, the mechanisms of acute liver injury and virus exclusion have been examined using a model wherein HBsAg-specific CTL are injected into HBsAg transgenic (Tg) mice. The importance of the role of TNF-alpha in virus exclusion was shown, but its role in liver injury was unclear. We crossed the TNF-alpha knockout mouse and HBsAg-Tg mouse to establish the HBsAg-Tg/TNF-alpha KO mouse, and examined the influence of TNF-alpha on liver injury. The severity of liver damage, as determined by serum alanine aminotransferase activity, was approximately 100 times greater in HBsAg-Tg/TNF-alpha(+/+) than in HBsAg-Tg/TNF-alpha(-/-) mice after i.v. administration of 5 x 10(6) CTLs. This liver damage reached the peak of its severity within 24-48 h, and was restored 7 days later. Histopathological examination showed hepatocellular necrosis and inflammatory cell infiltrate 24 h after the CTL injection in HBsAg-Tg/TNF-alpha(+/+) mice but not in HBsAg-Tg/TNF-alpha(-/-) mice. The liver damage was fatal for all HBsAg-Tg/TNF-alpha(+/+) mice that received 1.5 x 10(7) CTLs. In contrast, 1.5 x 10(7) CTLs could not kill the HBsAg-Tg/TNF-alpha(-/-) mice. The TNF-alpha production level was enhanced after the CTL injection in not only intrahepatic macrophages but also other types of mononuclear cells from non-HBsAg-Tg/TNF-alpha(+/+) mice. An adoptive transfer examination revealed that severe liver damage occurred in HBsAg-Tg/TNF-alpha(-/-) mice that had received mononuclear cells from TNF-alpha(+/+) mice. In conclusion, the present study provides evidence that TNF-alpha produced by intrahepatic non-Ag-specific inflammatory cells is critical in the development of lethal necroinflammatory liver disease.

Upregulation of indoleamine 2,3-dioxygenase in hepatocyte during acute hepatitis caused by hepatitis B virus-specific cytotoxic T lymphocytes in vivo.

BACKGROUND/AIMS: Indoleamine-2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme inducing suppression of T-cell function and immune tolerance. In hepatitis B virus (HBV) transgenic (Tg) mice, the adoptive transfer of HBV-specific cytotoxic T lymphocytes (CTL) causes a necroinflammatory liver disease that is histologically similar to acute viral hepatitis in man. The present study aimed to determine IDO expression in the liver and hepatocytes during an acute hepatitis model. METHODS: Serum l-kynurenine (l-Kyn) concentration in HBV Tg mice administered with HBV-specific CTL was measured over time, together with serum levels of alanine aminotransferase (ALT). Furthermore, we examined the expression of IDO in the total liver and isolated hepatocytes of HBV Tg mice after CTL injection using immunohistochemical analysis and reverse-transcription polymerase chain reaction (PCR). RESULTS: In HBV Tg mice, HBV-specific CTL induced, over the course of several days, a chronic increase in serum l-Kyn levels, which was associated with a sustained enhancement of liver IDO activity. In particular, IDO expression was enhanced in the liver parenchymal cells (hepatocytes) after HBV-specific CTL injection both in immunohistochemical analysis and in reverse-transcription PCR. Moreover, murine recombinant interferon-gamma (IFN-gamma) directly increased the IDO expression in primary hepatocytes in vitro. CONCLUSIONS: Cytotoxic T lymphocytes transduction results in the upregulation of IDO, which might downregulate T-cell responsiveness. Our findings provide evidence that hepatocyte itself expresses IDO and increases levels of l-Kyn in the blood in acute lethal hepatitis of mice. These data indicate that HBV infection facilitates the induction of IDO in response to proinflammatory cytokines, particularly IFN-gamma.

Valpha14 NKT cells activated by alpha-galactosylceramide augment lipopolysaccharide-induced nitric oxide production in mouse intra-hepatic lymphocytes.

Valpha14 natural killer T (Valpha14 NKT) cells activated by alpha-galactosylceramide (alpha-GalCer) secrete a large amount of Th1 and Th2 cytokines. IFN-gamma plays a crucial role in the inflammation response, and is also known as an activator of nitric oxide (NO) production. We previously reported that lipopolysaccharide (LPS)-induced NO production is augmented by alpha-GalCer in mouse peritoneal cells. Since the liver is susceptible to LPS stimulation via the portal vein, we examined the effect of alpha-GalCer on LPS-induced NO production in murine intra-hepatic lymphocytes (IHLs). Although IHLs augmented LPS-induced NO production by alpha-GalCer administration, such an augmentation was not observed in non-treated mice. Furthermore, alpha-GalCer did not augment LPS-induced NO production in IHLs from IFN-gamma knockout mice. In flow cytometry analysis of IHLs from alpha-GalCer-treated mice, the ratio and number of F4/80- and TLR4-positive cells rose as compared with non-treated mice. The liver injury may be induced by LPS and NO under the condition where Valpha14 NKT cells were activated.

Role of Valpha14+ NKT cells in the development of Hepatitis B virus-specific CTL: activation of Valpha14+ NKT cells promotes the breakage of CTL tolerance.

CTLs are thought to be major effectors for clearing viruses in acute infections including hepatitis B virus (HBV). Persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which is thought to reflect tolerance to HBV antigens. In the present study, we found that alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14-positive NKT cells, strongly enhanced the induction and proliferation of HBV-specific CTLs by HBsAg. In HBsAg transgenic mice, which are thought to be tolerant to HBV-encoded antigens, administration of HBsAg or alpha-GalCer alone failed to induce HBsAg-specific CTLs, but they were induced by co-administration of both compounds. Furthermore, by limiting dilution analysis, we confirmed the existence of HBsAg-specific CTL precursors in the HBsAg transgenic mice immunized with HBsAg and alpha-GalCer. A blocking experiment using antibodies to cytokines and CD40 ligand showed that IL-2 and CD40-CD40L interaction mediate the enhancement of CTL induction caused by alpha-GalCer through NKT cell activation. Our results may open up a new method for clearing the virus from patients with persistent HBV infection.

Interferon-beta reduces the mouse liver fibrosis induced by repeated administration of concanavalin A via the direct and indirect effects.

Type I interferons (IFNs), IFN-alpha and IFN-beta, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0.3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN-beta pretreatment. IFN-beta suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN-beta reduced the expressions of transforming growth factor-beta, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN-beta reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

Prevalence of hepatitis B and C virus infection in haematological malignancies and liver injury following chemotherapy.

The aim of this study was to determine the carrier rate of hepatitis virus in patients with haematological malignancies and the incidence of liver injury in these patients following chemotherapy. From January 1996 to September 2002, we studied 601 consecutive, unselected series of patients with haematological malignancies admitted in our hospital unit (Japan). They consisted of 246 cases of acute leukaemia, 218 non-Hodgkin's lymphoma (NHL), 13 adult T-cell leukaemia, and 124 multiple myeloma. Of these 601 patients, 373 were men and 228 were women; their mean age was 61 yr, with a range from 18 to 89 yr. The prevalences of hepatitis B virus (HBV) and hepatitis C virus (HCV) were 7.3% and 10.1%, respectively, in NHL, both higher than those in acute leukaemia (1.7% and 2.9%, P < 0.005) and in general Japanese population (1.2% and 2.6%). The incidence of post-chemotherapy liver injury in 25 HBV carriers (36.0%) was significantly higher than that in 539 non-hepatitis virus carriers (12.6%, P = 0.003) and 37 HCV carriers (10.8%, P = 0.026). Liver injury in HBV carriers was more often present in patients who had been treated with steroids than in those without steroids (72.7% and 0%, P = 0.013). After lamivudine became available in our institution, the incidence of liver injury in HBV carriers was reduced from 53.3% to 10.0% (P = 0.041). The therapeutic strategy for haematological malignancies in hepatitis virus carriers should be further investigated.

Excessive production of tumor necrosis factor-alpha by bone marrow T lymphocytes is essential in causing bone marrow failure in patients with aplastic anemia.

Aplastic anemia (AA) is regarded as an immunological disorder because of the clinical effect of immunosuppressive therapy. Recent studies have reported that cytokines play an important role in the development of AA. In the present study, we measured levels of T-cell derived intracellular cytokine production in peripheral blood and bone marrow (BM) of patients with AA. We demonstrated that BM lymphocytes, particularly CD4(+) and CD8(+) T cells, in patients with AA produced significantly higher amounts of tumor necrosis factor-alpha (TNF-alpha), compared with lymphocytes in normal controls. We have previously reported that expression of TNF receptor (R)1 and TNFR2 in the CD34(+) CD38(-) and CD34(+) CD38(+) fractions of patients with AA is significantly higher than those in normal control. These results indicate that BM stem cells in patients with AA may possess high sensitivity to TNF-alpha. This in turn suggests that TNF-alpha affects hematopoiesis at an earlier stage in AA patients than in normal controls. We strongly support the hypothesis that a simultaneous increase in TNF-alpha production by BM lymphocytes and sensitivity of stem cells to TNF-alpha leads to BM failure in AA.

A definitive role of RhoC in metastasis of orthotopic lung cancer in mice.

PURPOSE: Lung cancer is a major cause of cancer death, and its incidence is increasing in the world. Conventional therapies remain less effective for metastases of lung cancer, leading to poor prognosis of this disorder. The present study investigates pathological roles of RhoC in metastasis of lung cancer using a clinically relevant mouse model of lung cancer. EXPERIMENTAL DESIGN: RhoA, RhoC, dominant-negative Rho (dnRho) or green fluorescent protein gene was retrovirally transduced to murine lung cancer cells. For in vivo study, these transduced cells were intrapulmonary inoculated in syngeneic mice, and subsequently, growth and metastasis were analyzed. Migration and invasion activities were further investigated by in vitro chemotaxic chamber assays. Expression levels and activities of certain matrix metalloproteinases (MMPs) were explored by reverse transcription-PCR and gelatin zymography. RESULTS: Metastasis of lung cancer in the animal model, as well as in vitro migration and invasion, were significantly enhanced or inhibited by overexpression of RhoC or dnRho, respectively, without affecting the growth of primary tumors. Expression levels of certain MMPs and the activity of MMP-2 were significantly enhanced or suppressed by overexpression of RhoC or dnRho, respectively. CONCLUSION: RhoC plays a crucial role in metastasis of lung cancer. RhoC does not affect tumor growth but enhances the metastatic nature of lung cancer by not only stimulating cell motility but also up-regulating certain MMPs. Attenuation of RhoC activity may be a potential target in the development of a novel strategy for treating metastasis of lung cancer.

Role of Valpha 14 NKT cells in the development of impaired liver regeneration in vivo.

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.

Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes.

Recent studies have shown that tumor necrosis factor alpha (TNF-alpha) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-alpha knockout (TNF-alpha(-/-)) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-alpha(-/-) mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-alpha(-/-) mice after two sessions of immunization with the HBV plasmid; however, TNF-alpha was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-alpha(-/-) mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-alpha enhanced the proliferation of a TNF-alpha(-/-) clone but suppressed that of a TNF-alpha(+/+) clone in vitro, TNF-alpha also has a direct effect on the proliferation of CTLs. In conclusion, TNF-alpha is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.

Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anaemia by bone marrow stem cell expression of the tumour necrosis factor receptor.

It is often difficult to distinguish hypoplastic myelodysplastic syndrome (h-MDS) from acquired aplastic anaemia (AA), because of the considerable clinical, cytological and histological similarities between these two disorders. The distinction between AA and h-MDS is important because there is a higher risk of progression to acute leukaemia in patients with h-MDS compared with AA. Recent studies suggest that tumour necrosis factor-alpha (TNF-alpha) plays an important role in the development of AA. In order to determine the potential importance of TNF-alpha in the differential diagnosis of hypoplastic bone marrow (BM) disorders, we examined whether analysis ofTNF-receptor expression could be used as a tool to differentiate AA from h-MDS. Flow cytometric analysis revealed that BM stem cells (CD34+) from AA patients have markedly greater TNF receptor (R) 1 and TNFR2 expression than those from patients with MDS and h-MDS. We suggest that the BM stem cells with a high expression of TNFR in patients with AA may be potently sensitive to TNF-alpha stimulation of differentiation. Thus, we propose that quantification of TNFR expression in BM stem cellsmay be a useful method to distinguish AA from h-MDS.