PNAS-4, an Early DNA Damage Response Gene, Induces S Phase Arrest and Apoptosis by Activating Checkpoint Kinases in Lung Cancer Cells.

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53(-/-)) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21(Waf1/Cip1) and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21(Waf1/Cip1) is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells.

Integration of metabolomics and transcriptomics reveals the regulation mechanism of the phenylpropanoid biosynthesis pathway in insect resistance traits in Solanum habrochaites.

Solanum habrochaites (SH), a wild species closely related to 'Ailsa Craig' (AC), is an important germplasm resource for modern tomato breeding. Trichomes, developed from epidermal cells, have a role in defense against insect attack, and their secretions are of non-negligible value. Here, we found that the glandular heads of type VI trichomes were clearly distinguishable between AC and SH under cryo-scanning electron microscopy, the difference indicating that SH could secrete more anti-insect metabolites than AC. Pest preference experiments showed that aphids and mites preferred to feed near AC compared with SH. Integration analysis of transcriptomics and metabolomics data revealed that the phenylpropanoid biosynthesis pathway was an important secondary metabolic pathway in plants, and SH secreted larger amounts of phenylpropanoids and flavonoids than AC by upregulating the expression of relevant genes in this pathway, and this may contribute to the greater resistance of SH to phytophagous insects. Notably, virus-induced silencing of Sl4CLL6 not only decreased the expression of genes downstream of the phenylpropanoid biosynthesis pathway (SlHCT, SlCAD, and SlCHI), but also reduced resistance to mites in tomato. These findings provided new genetic resources for the synthesis of phenylpropanoid compounds and anti-insect breeding in S. habrochaites and a new theoretical basis for the improvement of important traits in cultivated tomato.

The antinociceptive effects of intracerebroventricular administration of Chicago sky blue 6B, a vesicular glutamate transporter inhibitor.

Accumulating evidence suggests that vesicular glutamate transporters (VGLUTs), which control the storage and release of glutamate, may play a role in pain processing. Chicago sky blue 6B (CSB6B), which is structurally related to glutamate, is a competitive VGLUT inhibitor without affecting plasma membrane transporters. The present study was designed to investigate the antinociceptive effects of CSB6B in a number of pain models. The hot-plate test was used as an acute thermal pain test. Inflammatory pain was evaluated using acetic acid writhing, formalin, and complete Freund's adjuvant tests. Intracerebroventricular administration of CSB6B did not affect acute thermal pain responses in 50 or 55°C hot plate tests. However, CSB6B attenuated acetic acid-induced writhing in a dose-dependent and time-dependent manner. In addition, CSB6B reduced licking/biting behavior during the second phase, but not during the first phase, following an intraplantar injection of formalin. In the complete Freund's adjuvant test, a significant attenuation of thermal hyperalgesia was also observed in CSB6B-treated mice. At antinociceptive doses, CSB6B did not affect mice spontaneous locomotor activity. The present study shows that pharmacological inhibition of VGLUT activity was sufficient to attenuate experimental inflammatory pain and suggests that regulation of VGLUTs might be a novel therapeutic strategy for the treatment of pain.

Inhibitory effects of the attenuated Salmonella typhimurium containing the IL-2 gene on hepatic tumors in mice.

To observe the inhibitory effects of an attenuated S. typhimurium strain carrying IL-2 gene (TPI) on hepatoma cell line (HepG2) and transplanted tumors in mice. TPI, TPG (an attenuated S. typhimurium strain carrying green fluorescent protein gene), and TP (an attenuated S. typhimurium strain) strains were transfected into HepG2 cells. At 48 h after transfecting, the transfection rate was 82.58 ± 1.74%. The expression level of IL-2 was (99.5 ± 12.2) ng/1 × 10(6) cells. Compared with TPG, TP, and normal mouse groups, the proportion of CD4(+) T and CD8(+) T cells in the blood from the TPI group was higher, the levels of IgM and IgG(1) were significantly increased, and the proliferation activity of splenic lymphocyte was significantly stronger. The transplanted tumor weight in the TPI group was significantly smaller than that in the other two groups. The infiltration of lymphocytes increased in the tumor from TPI group mice. TPI was effectively transfected into cancer cells, which expressed the protein of interest. Oral administration of TPI prolonged survival of mice transplanted with hepatoma cell tumours.

Effect of oral hepatocyte growth factor gene mediated by attenuated salmonella on 2-, 4-, 6-trinitro-benzene-sulfonic-acid-induced ulcerative colitis in rat.

BACKGROUND AND AIM: In order to explore a new therapeutic method, we investigated the effects of exogenously expressed hepatocyte growth factor mediated by attenuated salmonella (TPH) on rats with ulcerative colitis (UC) induced by 2-, 4-, 6-trinitro-benzene-sulfonic acid. METHODS: The UC rats were treated with TPH, attenuated salmonella with a eukaryotic expression vector (TP) or sodium bicarbonate (model control [MC]) every other day. Cluster of differentiation (CD)4(+) and CD8(+) T cells and immunoglobulins in the blood were analyzed by flow cytometry. The HGF expression was determined by immunohistochemistry. A macroscopic-scale observation of the colon and a histological assessment were also carried out. RESULTS: The CD4(+) T counts and the CD4(+) /CD8(+) ratio in the TPH group were significantly lower than that in the MC group. The immunoglobulin M and immunoglobulin G(1) levels in the TPH group were significantly lower than that in the MC group and TP group. After treatment with TPH, the symptoms of the ulcerative rats were significantly alleviated. The colonic lesion grades in the TPH group were lower than that in the TP group and MC group. Significant improvement occurred after the TPH treatment, as evidenced by alleviated mucosal inflammation. At 7 days post-treatment, the HGF expression in the colonic tissues that were treated with TPH was stronger than that in the samples treated with TP. CONCLUSIONS: TPH inhibits the proliferation of T lymphocytes and the antibody production of B lymphocytes. Furthermore, it ameliorates mucosal inflammation and promotes the regeneration of mucosa and the healing of the colonic ulceration.

The characteristics of the synonymous codon usage in hepatitis B virus and the effects of host on the virus in codon usage pattern.

BACKGROUND: Hepatitis B virus (HBV) infection is one of the main human health problem and causes a large-scale of patients chronic infection worldwide.. As the replication of HBV depends on its host cell system, codon usage pattern for the viral gene might be susceptible to two main selections, namely mutation pressure and translation selection. In this case, a deeper investigation between HBV evolution and host adaptive response might assist control this disease. RESULT: Relative synonymous codon usage (RSCU) values for the whole HBV coding sequence were studied by Principal component analysis (PCA). The characteristics of the synonymous codon usage patterns, nucleotide contents and the comparison between ENC values of the whole HBV coding sequence indicated that the interaction between virus mutation pressure and host translation selection exists in the processes of HBV evolution. The synonymous codon usage pattern of HBV is a mixture of coincidence and antagonism to that of host cell. But the difference of genetic characteristic of HBV failed to be observed to its different epidemic areas or subtypes, suggesting that geographic factor is limited to influence the evolution of this virus, while genetic characteristic based on HBV genotypes could be divided into three groups, namely (i) genotyps A and E, (ii) genotype B, (iii) genotypes C, D and G. CONCLUSION: Codon usage patterns from PCA for identification of evolutionary trends in HBV provide an alternative approach to understand the evolution of HBV. Further more, a combined selection of mutation pressure with translation selection on codon usage might shed a light on understanding the evolutionary trends of HBV genotypes.

Identification and characterization of circRNAs as competing endogenous RNAs for miRNA-mRNA in colorectal cancer.

BACKGROUND: Recent studies showed that circRNAs are involved in the biological process of some human cancers. However, little is known about their functions in colorectal cancer (CRC). METHODS: Here we first revealed the expression profiles of circRNAs in the CRC tissues and the adjacent non-tumorous tissues using high-throughput sequencing. The sequence feature, chromosome location, alternative splicing and other characteristics of the circRNAs were also explored. The miRNA and mRNA expression profiles were then obtained by analyzing relevant CRC data retrived from the TCGA database. We obtained and analyzed the competing endogenous RNA (ceRNA) network of the top three pairs of the largest up-regulated and down-regulated circRNAs. RESULTS: In this study, we obtained 50,410 circRNAs in the CRC tissue and the adjacent non-tumor tissues, of which 33.7% (16,975) were new, and revealed differential changes in circRNA expression during colorectal carcinogenesis. We have identified six potential key circRNAs (circPIEZO1-3, hsa_circ_0067163, hsa_circ_0140188, hsa_circ_0002632, hsa_circ_0001998 and hsa_circ_0023990) associated with CRC, which play important roles in carcinogenesis as ceRNA for regulation of miRNA-mRNA network. In the subsequent KEGG analysis, several CRC-related pathways were found. CONCLUSIONS: Our findings advance the understanding of the pathogenesis of CRC from the perspective of circRNAs and provide some circRNAs as candidate diagnostic biomarkers or potential therapeutic targets.

Establishment of a highly sensitive sandwich enzyme-linked immunosorbent assay specific for ovomucoid from hen's egg white.

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.

Newly synthesized podophyllotoxin derivative, LJ12, induces apoptosis and mitotic catastrophe in non-small cell lung cancer cells in vitro.

Deoxypodophyllotoxin (DPT), an active compound isolated from a number of herbs and used in traditional medicine, has been reported to exhibit promising anti­tumor activity. A newly synthesized derivative, N-(1-oxyl­4'-demethyl-4-deoxyp odophyllic)-L­methine-4'-piperazine carbamate (LJ12) may have improved antitumor activity and fewer side effects. The present study assessed the effect of LJ12 on cell viability, apoptosis, cell cycle distribution and mitotic catastrophe in A549 human lung cancer cells in vitro. The molecular mechanisms underlying the antitumor activity of LJ12 were also examined. The results demonstrated that LJ12 reduced A549 cell viability in a time­ and dose­dependent manner, with a lower half maximal inhibitory concentration of ~0.1 µM, compared with another known DPT derivative, etoposide (10 µM). Flow cytometric analysis showed that LJ12 induced tumor cell arrest at the G2/M phase of the cell cycle. The present study also observed an expected concomitant decrease in the numbers of cells cells in the G0/G1 and S phases. LJ12 was found to upregulate the protein expression levels of Cdc2 and Cyclin B1. Furthermore, LJ12 induced tumor cell apoptosis and the protein expression of B cell lymphoma­2­associated X protein, caspase­3 and p53. The present study also observed the formation of giant, multinucleated cells, indicating that LJ12 induced mitotic catastrophe in the tumor cells. These results indicated that LJ12 has anti­non­small cell lung cancer activity in vitro. Further investigations aim to develop LJ12 as a therapeutic agent for the treatment of lung cancer.

Gram Scale Syntheses of (-)-Incarvillateine and Its Analogs. Discovery of Potent Analgesics for Neuropathic Pain.

(-)-Incarvillateine (INCA) is the major antinociceptive component of Incarvillea sinensis, which has been used to treat rheumatism and relieve pain in traditional Chinese medicine. We have developed a concise and general synthetic approach for INCA, which enabled gram-scale asymmetric syntheses of (-)-INCA, (-)-incarvilline, (-)-isoincarvilline, and six other INCA analogues. The synthesis of isoincarvilline was reported for the first time. Three structurally simplified analogues of INCA were also synthesized. In vivo screening found that INCA and two structurally optimized analogues were efficacious in preventing the acetic acid-induced writhing response. Moreover, their analgesic efficacy was demonstrated in formalin induced pain model. More importantly, administration of 20 or 40 mg/kg INCA and two structurally optimized analogues showed strong analgesic effects in spared nerve injury (SNI) model, and their effective doses were lower than the current gold standard, gabapentin (100 mg/kg in this model).

Kinetics and Immunodominance of Virus-Specific T Cell Responses During Hantaan Virus Infection.

Immunodominant T cell responses are important for protection against virus challenge. However, studies screening for the immunodominant T cell responses and following their kinetics in acute Hantaan virus (HTNV) infection are very limited. Herein, the HTNV nucleocapsid protein-specific T cell responses were longitudinally screened in 15 patients with acute HTNV infection, eight of whom had a particularly severe hemorrhagic fever with renal syndrome (HFRS). An extremely impaired IFN-γ-producing T cell response was observed in patients with severe HFRS at the early stage of infection, especially to the immunodominant epitopes detected in the mild to moderate group, namely peptides N127-141, N139-153, N241-255, and N355-369. The initially insufficient T cell response to the immunodominant epitopes may play a role in influencing the severity of HTNV infection. These findings provide information that may aid the design of future vaccines against hantaviruses.

Antinociceptive effects of incarvillateine, a monoterpene alkaloid from Incarvillea sinensis, and possible involvement of the adenosine system.

Incarvillea sinensis is a Bignoniaceae plant used to treat rheumatism and relieve pain in traditional Chinese medicine. As a major component of I. sinensis, incarvillateine has shown analgesic activity in mice formalin tests. Using a series of animal models, this study further evaluated the effects of incarvillateine against acute, inflammatory, and neuropathic pain. Incarvillateine (10 or 20 mg/kg, i.p.) dose-dependently attenuated acetic acid-induced writhing, but did not affect thermal threshold in the hot plate test. In a Complete Freund's Adjuvant model, incarvillateine inhibited both thermal hyperalgesia and paw edema, and increased interleukin-1ß levels. Additionally, incarvillateine attenuated mechanical allodynia induced by spared nerve injury or paclitaxel, whereas normal mechanical sensation was not affected. Incarvillateine did not affect locomotor activity and time on the rotarod at analgesic doses, and no tolerance was observed after 7 consecutive daily doses. Moreover, incarvillateine-induced antinociception was attenuated by theophylline, 1,3-dipropyl-8-cyclopentylxanthine, and 3,7-dimethyl-1-propargylxanthine, but not naloxone, indicating that the effects of incarvillateine on chronic pain were related to the adenosine system, but not opioid system. These results indicate that incarvillateine is a novel analgesic compound that is effective against inflammatory and neuropathic pain, and that its effects are associated with activation of the adenosine system.

[Effects of the interleukin-21 expression in patients with immune thrombocytopenia and its regulation by high-dose dexamethasone].

OBJECTIVE: To investigate the correlation of immunologic thrombocytopenia(ITP) pathogenesis with the abnormal expression of IL-21, and to explore the association of high-dose dexamethasone (HD-DEX) treatment with the IL-21 expression. METHODS: 26 newly diagnosed ITP patients and 24 healthy controls were enrolled in this study. The mononuclear cells and serum were obtain from density gradient centrifugation in the newly diagnosed ITP patients before HD-DXM treatment, and the samples of healthy controls were also used for assays. The protein and mRNA expression of IL-21 on peripheral blood mononuclear cells(MNC) were determined by flow cytometry and real-time reverse-transcription polymerase chain reaction. Plasma levels of IL-21, IFN-γ and IL-4 were determined by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: IL-21 expression on mononuclear cells was significantly higher in ITP patients (13.07%) than that in normal controls (8.2%), the ratio of IL-21/GAPDH mRNA expression on MNC was significantly higher in ITP patients (9.524±0.97) than that in normal controls (3.701±0.60, P<0.01). After HD-DXM therapy, the ratio of IL-21/GAPDH mRNA decreased significantly (5.87±1.21) as compared with the level before treatment. Significantly high levels of serum IL-21, IFN-γ and lower IL-4 were found in ITP patients, as compared with healthy controls. Serum IL-21 and IFN-γ levels in ITP patients decreased significantly after HD-DXM administration (P<0.01), while post-treatment levels of IL-4 were increased significantly, compared with the levels before treatment (P<0.01). CONCLUSION: Therapeutic effect of DXM on ITP associates with down-regalation of IL-21 expression. The increased expression of IL-21 involves in the pathogenesis of ITP.

Thrombocytopenia as a predictor of severe acute kidney injury in patients with Hantaan virus infections.

BACKGROUND: Hematological abnormalities often occur several days before kidney injury in patients with hemorrhagic fever with renal syndrome (HFRS). We aimed to investigate the prevalence and prognostic value of the early hematological markers in patients with HFRS caused by Hantaan virus (HTNV) infection. METHODS: In a retrospective cohort study, we analyzed the case records of 112 patients with acute HTNV infection and evaluated the hematological markers for early prediction and risk stratification of HFRS patients with acute kidney injury (AKI). RESULTS: Of 112 patients analyzed, 66 (59%) developed severe AKI, defined as either receipt of acute dialysis or increased serum creatinine ≥ 354 µmol/L. The prognostic accuracy of hematological markers, as quantified by the area under the receiver-operating-characteristic curve (AUC), was highest with the nadir platelet count (AUC, 0.89; 95% CI, 0.83-0.95), as compared with the admission platelet count (AUC, 0.84; 95% CI, 0.77-0.92), and the admission and peak leukocyte counts. The nadir platelet count correlated moderately with the levels of peak blood urea nitrogen (r = -0.616) and serum creatinine (r = -0.589), the length of hospital stay (r = -0.599), and the number of dialysis sessions that each patient received during hospital stay (r = -0.625). By multivariate analysis, decreased nadir platelet count remained independently associated with the development of severe AKI (odds ratio, 27.57; 95% CI, 6.96-109.16; P<0.0001). CONCLUSIONS: Thrombocytopenia, rather than leukocytosis, is independently associated with subsequent severe AKI among patients with acute HTNV infection.

HLA-A2 and B35 restricted hantaan virus nucleoprotein CD8+ T-cell epitope-specific immune response correlates with milder disease in hemorrhagic fever with renal syndrome.

BACKGROUND: Hantaan virus (HTNV) infection in humans is a serious public health concern in Asia. A potent T cell activation peptide vaccine from HTNV structure protein represents a promising immunotherapy for disease control. However, the T cell epitopes of the HTNV restricted by the HLA alleles and the role of epitope-specific T cell response after HTNV infection remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Five well-conserved novel CD8(+) T-cell epitopes of the HTNV nucleoprotein restricted by the most popular HLA alleles in Chinese Han population were defined with interferon-γ enzyme-linked immunospot assay in 37 patients infected with HTNV during hospitalization. Two epitopes aa129-aa137 and aa131-aa139 restricted by HLA-A2 and B35, respectively, were selected to evaluate the epitope-specific CD8(+) T-cell response. HLA-peptide pentamer complex staining showed that the frequency of single epitope-specific CD8(+) T cell could be detected in patients (95% confidence interval for aa129-aa137: 0.080%-0.208%; for aa131-aa139: 0.030%-0.094%). The frequency of epitope-specific pentamer(+) CD8(+) T-cell response was much higher in mild/moderate patients than in severe/critical ones at the acute stage of the disease. Moreover, the frequency of epitope-specific CD8(+) T cells at acute stage was inversely associated with the peak level of serum creatinine and was positively associated with the nadir platelet counts during the hospitalization. The intracellular cytokine staining and the proliferation assay showed that the effective epitope-specific CD8(+) T cells were characterized with the production of interferon-γ, expression of CD69 and the strong capacity of proliferation. CONCLUSION/SIGNIFICANCE: The novel HLA class I restricted HTNV nucleoprotein epitopes-specific CD8(+) T-cell responses would be closely related with the progression and the severity of the disease, which could provide the first step toward effective peptide vaccine development against HTNV infection in humans.

Identification of three novel CTL epitopes within nucleocapsid protein of Hantaan virus.

Hantaan virus (HTNV) is a member of the Hantavirus genus that causes human hemorrhagic fever with renal syndrome (HFRS) in humans. The CTL response seems to play a key role in control of viral infection, but only a few HTNV epitopes recognized by the CTLs have been reported. Herein, we screened a panel of overlapping peptides covering the HTNV nucleocapsid protein by ELISPOT assays for those that can elicit IFN-γ production in vitro. Three novel CD8(+) CTL epitopes, N197-205 (RYRTAVCGL), N245-253 (KLLPDTAAV), and N258-266 (GPATNRDYL), were defined on the nucleocapsid protein and were found to be restricted by various HLA alleles including A11, A24, and B7. The epitopes were highly conserved among the reported HTNV strains and other hantanviruses, including Dobrava-Belgrade virus and Seoul virus, supporting their potential use in vaccine designs.

[Bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation for therapy of patients with multiple myeloma].

This study was aimed to evaluate the therapeutic efficacy of bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation (autoPBSCT) for patients with multiple myeloma (MM). 5 patients underwent autologous hematopoietic stem cell transplantation. Bortezomib treatment was supplied for patients before autoPBSCT and in the conditioning of transplantation, it was also used in maintaining treatment. Patients with transplantation adopted bortezomib plus melphalan conditioning regimen. The number of infused MNC and number of CD34(+) cells were 4.06×10(8) (4.09×10(8) - 4.37×10(8))/kg and 3.98×10(6) (2.49×10(6) - 8.2×10(6))/kg respectively. The results showed that hematopoiesis was reconstituted in 5 patients, with a neutrophil cell count more than 0.5×10(9)/L at day 14 (13 - 25 days) after transplantation and platelet count more than 50×10(9)/L at day 28 (21 - 41 days) after transplantation. Transplantation-associated death was not observed. 5 patients were disease-free survival. In conclusion, treatment of bortezomib combined with autologous peripheral hematopoietic stem cell transplantation is an effective method for patients with multiple myeloma. Use of bortezomib after transplantation might still be favourable to MM patients, for survival prolongation and life quality improvement.

[Application of Epstein-Barr virus-transformed B lymphoblastic cells in identification of CTL epitopes specific for Hantaan virus].

AIM: To establish Epstein-Barr virus (EBV)-transformed B lymphoblastic cell lines (B-LCL) to present peptides as antigen-presenting cells (APC) and stimulate short-cultured T cells secreting IFN-gamma, by which the T cell epitopes are identified. METHODS: PBMCs from patients with hemorrhagic fever with renal syndrome (HFRS) were transformed using EBV from supernatant of B95-8 cells. ELISPOT assay was then employed to evaluate the IFN-gamma production of short-cultured G9L-specific CD8(+) T cells stimulated with peptide-pulsed autologous B-LCL cells. RESULTS: B-LCL pulsed with G9L or G9L-nested V15R can stimulate G9L-specific CD8(+) T cells producing IFN-gamma, but not B-LCL pulsed with non-homologous I15P. CONCLUSION: B-LCL can efficiently and specifically present peptides to peptide-specific T cells as non-professional APC.

Cellular immune response to Hantaan virus nucleocapsid protein in the acute phase of hemorrhagic fever with renal syndrome: correlation with disease severity.

BACKGROUND: The cellular immune response to Hantaan virus (HTNV) is incompletely understood, especially in humans. METHODS: To investigate the cellular immunity during acute HTNV infection, the magnitude of the CD4(+) and CD8(+) T cell responses to HTNV nucleocapsid protein was quantitated by direct ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot analysis, using an array of overlapping peptides. RESULTS: We found that the combined frequencies of HTNV-specific T cells at the earliest available time point (5-8 days after fever onset) were significantly higher in patients who had mild or moderate hemorrhagic fever with renal syndrome (HFRS) than in those who had severe or critical HFRS (P= .006). Moreover, these frequencies were higher in patients with subsequent mild renal failure (maximum serum creatinine level, 707 micromol/L) (P= .006). Kinetic analysis showed that a decrease in the serum creatinine level during the acute phase of illness was often accompanied by an increase in the magnitude of IFN-gamma-producing T cells. CONCLUSION: Taken together with published data on the similar associations with neutralizing antibody, these data suggest that IFN-gamma-producing T cells may help reduce the risk of progression to acute renal failure caused by HTNV infection.

[Identification of HTNV-NP-specific T lymphocyte epitopes and analysis of the epitope-specific T cell response].

AIM: To identify the nucleocapsid protein of Hantaan virus (HTNV-NP)-specific T lymphocyte epitopes and analyze the epitope-specific T cell response during hemorrhagic fever with renal syndrome (HFRS). METHODS: T lymphocyte epitopes and frequencies of epitope-specific T cells were determined by ELISPOT using PBMCs from HFRS patients stimulated by individual or mixture of overlapping 15-mer peptides spanning the amino acid sequence of HTNV-NP. RESULTS: Out of 10 peptide mixtures, 8 elicited strong HTNV-NP-specific responses in 18 of 47 HFRS patients, and the T cell response was found at early stage of HFRS. Moreover, 17 HTNV-NP-specific T lymphocyte epitopes were identified in 11 patients, and most epitopes were clustered near the center of NP in linear structure. Among them, 14 T lymphocyte epitopes were described for the first time. CONCLUSION: HTNV-NP-specific T cell response can be elicited at early stage of HFRS and T lymphocyte epitopes mainly located in the center of NP, suggesting that it may play an important role in immune protection during HTNV infection.