Multinucleated polyploid cardiomyocytes undergo an enhanced adaptability to hypoxia via mitophagy.

AIMS: There is a large subpopulation of multinucleated polyploid cardiomyocytes (M*Pc CMs) in the adult mammalian heart. However, the pathophysiological significance of increased M*Pc CMs in heart disease is poorly understood. We sought to determine the pathophysiological significance of increased M*Pc CMs during hypoxia adaptation. METHODS AND RESULTS: A model of hypoxia-induced cardiomyocyte (CM) multinucleation and polyploidization was established and found to be associated with less apoptosis and less reactive oxygen species (ROS) production. Compared to mononucleated diploid CMs (1*2c CMs), tetraploid CMs (4c CMs) exhibited better mitochondria quality control via increased mitochondrial autophagy (mitophagy). RNA-seq revealed Prkaa2, the gene for AMPKα2, was the most obviously up-regulated autophagy-related gene. Knockdown of AMPKα2 increased apoptosis and ROS production and suppressed mitophagy in 4c CMs compared to 1*2c CMs. Rapamycin, an autophagy activator, alleviated the adverse effect of AMPKα2 knockdown. Furthermore, silencing PINK1 also increased apoptosis and ROS in 4c CMs and weakened the adaptive superiority of 4c CMs. Finally, AMPKα2-/- mutant mice exhibited exacerbation of apoptosis and ROS production via decreases in AMPKα2-mediated mitophagy in 4c CMs compared to 1*2c CMs during hypoxia. CONCLUSIONS: Compared to 1*2c CMs, hypoxia-induced 4c CMs exhibited enhanced mitochondria quality control and less apoptosis via AMPKα2-mediated mitophagy. These results suggest that multinucleation and polyploidization allow CM to better adapt to stress via enhanced mitophagy. In addition, activation of AMPKα2 may be a promising target for myocardial hypoxia-related diseases.

Clinical Features of Isolated Bulbar Palsy of Amyotrophic Lateral Sclerosis in Chinese Population.

BACKGROUND: Progressive bulbar palsy (PBP) is a classic phenotype of bulbar onset amyotrophic lateral sclerosis (ALS) with more rapid progression and worse prognosis. However, as an often under-understood variant of ALS, isolated bulbar palsy (IBP) appears to progress more slowly and has a relatively benign prognosis. This study aimed to investigate the natural course and clinical features of IBP in Chinese population and to compare them with those of PBP. METHODS: The clinical data of patients with bulbar onset ALS were collected from January 2009 to December 2013. Revised ALS Functional Rating Scale (ALSFRS-R), forced vital capacity (FVC), and follow-up evaluation were performed, and the differences in basic clinical features, ALSFRS-R, FVC, and primary outcome measures between IBP and PBP were analyzed. The independent t-test, Chi-square test, Mann-Whitney U-test, and Kaplan-Meier analysis were used. RESULTS: Totally 154 patients with bulbar onset ALS were categorized into two groups, 33 with IBP and 121 with PBP. In the IBP group, the male to female ratio was 0.7 to 1.0, and the mean onset age was 58.5 years. The mean duration from the onset was 16.0 months, and the mean ALSFRS-R score was 43.4 at patients' first visit to our hospital. In 14 IBP patients performing FVC examination, the mean FVC value was 90.5% and there were only two cases with abnormal FVC. In 26 IBP patients completing follow-up, 15 (58%) suffered death or tracheotomy and the mean survival time was 40.5 months. Significant differences were noted in sex ratio, onset age, ALSFRS-R score, upper motor neuron limb signs, pure lower motor neuron (LMN) bulbar signs, FVC, and survival time between IBP and PBP. CONCLUSIONS: IBP was evidently different from PBP, which was characterized with the predominance of female, pure LMN bulbar signs, an older onset age, a relative preservation of respiratory function, and a better prognosis.

Cardiac resident macrophages are involved in hypoxia­induced postnatal cardiomyocyte proliferation.

Induction of cardiomyocyte proliferation, the most promising approach to reverse myocardial attrition, has been gaining importance as a therapy for cardiovascular disease. Hypoxia and macrophages were previously independently reported to promote cardiomyocyte proliferation in mice. However, whether hypoxia promotes cardiomyocyte proliferation in humans, and the association between hypoxia and macrophages in cardiomyocyte proliferation, have not to the best of our knowledge been previously investigated. The present study investigated the cardiomyocyte proliferation in 22 acyanotic and 29 cyanotic patients. Cardiomyocyte proliferation in a hypoxic mouse model (15% O2) was subsequently performed and the macrophage subsets were analyzed. A C­C chemokine receptor type 2 (CCR2) inhibitor was used to increase the number of resident macrophages in order to investigate the effect of macrophages on cardiomyocyte proliferation. The results demonstrated that cardiomyocyte proliferation in the cyanotic infant group was significantly increased compared with the acyanotic infant group and the hypoxia­treated C57BL/6J neonates confirmed the hypoxia­induced cardiomyocyte proliferation. However, hypoxia did not induce the proliferation of isolated cardiomyocytes. Notably, hypoxia treatment increased the number of cardiac resident macrophages in neonate hearts. Furthermore, increasing the number of resident macrophages significantly enhanced cardiomyocyte proliferation. In conclusion, postnatal hypoxia promoted cardiomyocyte proliferation in humans and animals, and cardiac resident macrophages may be involved in this process. Therefore, this novel mechanism may provide a promising strategy for cardiovascular disease treatment.

Association Between Rectus Abdominis Denervation and Ventilation Dysfunction in Patients with Amyotrophic Lateral Sclerosis.

BACKGROUND: Spontaneous potentials in electromyography (EMG) of paraspinal muscles are associated with diaphragm denervation and, therefore, poor respiratory function in amyotrophic lateral sclerosis (ALS) is understandable. EMG changes in the rectus abdominis (RA) display an effect similar to those in paraspinal muscles with respect to the function of lower motor neurons in the thoracic spinal cord. The RA denervation was examined to determine its association with ventilation dysfunction in ALS. METHODS: We collected the clinical data of 128 patients with sporadic ALS in Department of Neurology of Peking University Third Hospital from 2009 to 2013. EMG, Revised ALS Functional Rating Scale (ALSFRS-R) and forced vital capacity (FVC) were performed in all patients and the differences in the EMG changes in RA between those with and without FVC ≥ 80% were analysed. RESULTS: The mean FVC value was 83.4% ± 17.1% (range: 45%-131%) of the predicted value. A total of 79 patients displayed FVC ≥80%, and 49 patients displayed FVC <80%. Compared with the patients displaying a normal FVC (60/79, 75.9%), spontaneous activity in RA was significantly different among those patients displaying an FVC <80% (47/49, 95.9%). In addition, spontaneous potentials in RA were more frequently detected in patients exhibiting dyspnea (32/33, 97.0%) than in patients without dyspnea (75/95, 78.9%). CONCLUSION: Spontaneous potentials in RA are associated with ventilation dysfunction and dyspnea in ALS patients.

[Basic and clinical researches on amyotrophic lateral sclerosis/motor neuron disease].

This study includes three aspects: (1) we have reported some novel or rare mutations of SOD1 (Cu/Zn superoxide dismutase) gene in Chinese families of ALS/MND, and found quite different features from Western patients in polymorphisms with some candidate genes such as vascular endothelial growth factor (VEGF) in sporadic ALS/MND in China. Meanwhile, we have so for established a complete clinical database with more than 1 200 cases; (2) we have established some neurophysiologic techniques of diagnosis and differential diagnosis at early-stage for ALS/MND, which include trigemino-cervical response, sternocleidomastoid and rectus electromyography, contact heat evoked potentials, and motor unit number estimate; (3) we have attempted some experimental and clinical treatments for ALS/MND, which include gene and stem cell therapies in animal models, and a pilot clinical trial of granulocyte colony stimulating factor (G-CSF) for ALS/MND patients (NCT00397423).

The immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4(+) T cells, CD8(+), CD3(-)CD56(+), CD3(+)CD56(+), and gammadeltaT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3(+)CD56(+) cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4(+) cells in the tumor tended to produce more IL-10 but less IFN-gamma, whereas CD8(+) cells showed impaired capacity for the production of both IFN-gamma and perforin. Consistent with previous reports, we observed a significant increase of Foxp3(+) cells in the tumor tissue. Intriguingly, although over 90% of CD4(+)CD25(high) cells were found to be Foxp3(+), the majority of Foxp3(+) cells were identified in the CD4(+)CD25(medium) and CD4(+)CD25(-) subsets. In support of its role as a negative regulator, CD4(+)CD25(high) cells suppressed the proliferation of CD4(+)CD25(-) cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.

Hyperthermia on immune regulation: a temperature's story.

Over the last decade the linkage between hyperthermia, heat shock proteins and fever with the body's immune system has been well investigated. The immunomodulatory function of hyperthermia has been found to be quite sensitively regulated by temperature, as different levels of heating can bring different modulatory effect on different sensitive targets. Understanding these intrinsic mechanisms could bring new inspirations on the design of clinical trials combining local tumor hyperthermia with immunotherapy in cancer patients. This review will attempt to tell the story about the effect of temperature on immune regulation, with special emphasis on the clinical application of hyperthermia and the feasibility of combining it with immunotherapy in the clinic.

The specific immune response to tumor antigen CP1 and its correlation with improved survival in colon cancer patients.

BACKGROUND & AIMS: The present study was undertaken to determine the expression of a newly identified tumor antigen cancer-placenta 1 (CP1) in colorectal carcinoma (CRC) and explore the CP1-specific immune response in CRC patients and its correlation with patient survival. METHODS: CP1 expression was determined by reverse-transcription polymerase chain reaction, immunohistochemistry, and Western blot analysis. Serum antibodies against CP1 were detected by enzyme-linked immunosorbent assay, and T-cell response was measured by interferon-gamma/granzyme-B release enzyme-linked immunospot assays. The HLA-A2-restricted epitopes in CP1 were predicted by bioinformatics and then experimentally validated by enzyme-linked immunospot assay. RESULTS: CP1 expression was detected in a significant number of CRC tissues, reaching 47.6% at the messenger RNA (mRNA) level and 28.6% at the protein level. Of patients with CP1 mRNA(+) tumors, more than 50% had CP1-responsive CD4(+) and CD8(+) T cells and 30% spontaneous-occurring antibodies against CP1. Further studies revealed 2 dominant HLA-A2-restricted epitopes in the CP1 antigen: p31-39 and p58-66. In a follow-up study up to 33 months after surgery, 9 of the 10 patients with CP1-specific CD8 T-cell response survived, whereas 6 of the 8 nonresponders died. Kaplan-Meier analysis indicated a significant correlation between T-cell response and patient survival. CONCLUSIONS: CP1 represents a new class of tumor-specific shared antigen. Its high expression in CRC tissues, prevalence of CP1-specific immune responses in CP1 mRNA(+) CRC patients, and positive correlation with survival suggest that the antigen may be a useful target for cancer immunotherapy.

Specific CD8(+ )T cell responses to HLA-A2 restricted MAGE-A3 p271-279 peptide in hepatocellular carcinoma patients without vaccination.

The MAGE-A3 protein, one of the promising tumor antigens for immunotherapy, is highly expressed in human hepatocellular carcinoma (HCC). In this study, we estimated the specific CD8(+) T cell immune response to MAGE-A3 p271-279 peptide (M3(271)) in the peripheral blood of HCC patients without antigen vaccination in order to evaluate its immunotherapeutic potential in these patients. After expansion in vitro, the functional IFN-gamma producing M3(271) specific CD8(+) T cells were detected in 30.8% (8/26) of HLA-A2(+)MAGE-A3(+) HCC patients. The effector CD8(+ )T cells could release cytotoxic molecules of granzyme B and perforin after restimulation with natural HLA-A2(+)MAGE-A3(+) HCC cell lines in the samples tested. The functional supertype of HLA-A2 in the presentation of HLA-A*0201 restricted M3(271) peptide has been identified in the Chinese HCC patients of Han ethnicity, that widely expanded the applicability of this tumor peptide vaccine in Chinese HCC patients. Thus, the functionally detectable pre-existence of M3(271)-specific CD8(+) T cells in HCC patients makes M3(271) a potential target for immunotherapy in these patients. The responsive CD8(+ )T cells to both NY-ESO-1 and MAGE-A3 antigens provide a rationale for the application of a bivalent vaccine in HCC patients with tumors expressing both antigens.

[Estimation of an NY-ESO-1 expressing HCC cell line by NY-ESO-1b specific CD8+T cells in vitro induced by HLA-A2 restricted NY-ESO-1b peptide].

OBJECTIVE: To analyze the effect of an NY-ESO-1 expressing HCC cell line by NY-ESO-1b specific CD8(+)T cells in vitro induced by HLA-A2 Restricted NY-ESO-1b peptide and to further analyze the possibility of NYjESOj1 as a vaccine in HCC treatment. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from an HLA-A2(+) HCC patient and NY-ESO-1 specific CD8(+)T cells were in vitro induced by HLA-A2 Restricted NY-ESO-1b peptide. Next we estimated the effect that NY-ESO-1 specific CD8(+)T cells recognized and killed NY-ESO-1 expressing HepG2 cells by enzyme-linked immunospot(ELISPOT). RESULTS: These in vitro induced NY-ESO-1b specific CD8(+)T cells could recognize HepG2 cells stably transfected with NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. And the data also showed that NY-ESO-1 specific CD8(+)T cells could efficiently kill NY-ESO-1 expressing HepG2 cells. CONCLUSION: NY-ESO-1 protein can be processed by HCC cells, and that HLA-A2 restricted NY-ESO-1b peptide (157-165) can also be presented on the surfaces of cells and recognized by NY-ESO-1b specific CD8(+)T cells in vitro induced.

Identification of two naturally presented MAGE antigenic peptides from a patient with hepatocellular carcinoma by mass spectrometry.

In the absence of efficient systemic chemotherapy, immunotherapy is considered a hopeful treatment for controlling recurrence of hepatocellular carcinoma (HCC). The identification of proper antigenic peptides presented by MHC class I molecules is a critical step for the development of therapeutic vaccines against tumors. Currently, the "reverse immunology" approach is the most commonly used technique in the identification of the tumor-associated T cell epitopes. However, it is based on T cell dependent approach and cannot fully reflect the actual presentation of epitope in tumor in vivo. In the present study, we managed to identify the naturally presented MAGE epitopes of HCC directly by epitope prediction, HPLC differential analysis and MS detection. We successfully detected a naturally processed peptide FLWGPRALV (MAGE-3(271-279), HLA-A2-restricted) with an estimated number of 38-39 copies/cell in HCC. To our knowledge, this is the first evidence that the naturally processed MAGE-3(271-279) can be isolated and identified from the tumor tissue of HCC patient. Furthermore, specific CD8(+) T cell responses to this epitope were also found after tumor relapse by IFN-gamma release Cytospot and tetramer assay indicating that MAGE-3(271-279) was indeed presented by HCC cells in vivo. In addition, another new antigen peptide was found, which may be derived from MAGE-1. Our findings demonstrate the potential of the direct approach for identification of tumor-associated epitopes. This approach may become a useful tool for the development of vaccine against cancer in the future.

The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients.

PURPOSE: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2-restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. EXPERIMENTAL DESIGN: In HLA-A2(+) patients with NY-ESO-1(+) HCC, we analyzed T-cell antigen-dependent interferon (IFN)-gamma and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-gamma-producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. RESULTS: The frequency of specific CD8(+) T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8(+) T cells was 0.3% (89 +/- 59 per 2.5 x 10(4) CD8(+) cells) and 1.2% as measured by IFN-gamma release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b-specific CD8(+) T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b-specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer(+)CD8(+) T cells were also present. The CD8(+) T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8(+) T-cell responses in HCC patients was observed. CONCLUSIONS: The elevated frequency of specific CD8(+) T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.

T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS.

The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-gamma) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-gamma-secreting T-cell response in HLA-A2(+) donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2(+) healthy donors or in HLA-A2(-) donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2(+) SARS-CoV-infected patients.

[Effect of CD28 costimulator on T cell receptor(TCR)-induced apoptosis of thymocytes].

AIM: To analyze apoptosis and its degree of thymocytes at early and intermediate phases induced by anti-TCRalphabeta mAb or anti-TCRalphabeta mAb+anti-CD28 mAb stimuli, and to analyze the influence of CD28 costimulator on TCR-induced apoptosis of thymocyte subsets. METHODS: Thymocytes were freshly prepared and were cultured for 20 h in the presence of anti-TCRalphabeta mAb+anti-CD28 mAb or anti-TCRalphabeta mAb along, The cultured cells were stained with fluorescein labelled Annexin V, PI, anti-CD4 mAb and anti-CD8 mAb, then color reagents. The apoptotic cells were analyzed by FACS. RESULTS: Compared with the spontaneous apoptosis of thymocytes cultured in medium alone, CD28 costimulator markedly enhanced the number of thymocyte apoptosis at early and intermediate phases; under the action of double signaling stimulators, the apoptosis of CD4(+) CD8(+)(DP) thymocytes were substantially increased, and the expression of CD28 were also upregulated on these apoptotic DP cells. CONCLUSION: Influence of CD28 costimulator on TCR-induced apoptosis of thymocyte subsets might be related to thymocyte's mature degree. Double signaling may induce apoptosis of DP thymocytes.