IL-17 eliminates therapeutic effects of oral tolerance in murine airway allergic inflammation.

BACKGROUND: Oral tolerance is a classically used strategy for antigen-specific systemic immunotherapy. However, the roles of IL-17 in modification of oral tolerance are not yet understood. OBJECTIVE: To define the effects of IL-17 on the modification of oral tolerance, the effects of transfer of Th17 cells, administration of IL-17 or anti-IL-17 antibody (αIL-17Ab) to a murine allergic airway inflammation model were investigated. METHODS: Mice sensitized to and challenged with OVA, received OVA feeding, followed by OVA challenges. Transfer of Th17 cells, administration of IL-17 or αIL-17Ab were executed during OVA feeding. Airway hyperresponsiveness (AHR), airway inflammation, Th2 cytokine response and lung pathology were assessed. RESULTS: Administration of IL-17 as well as transfer of Th17 cells aggravated AHR and airway allergic inflammation as compared with the findings in mice subjected to OVA feeding alone, whereas administration of αIL-17Ab ameliorated AHR and airway eosinophilia. The effects of Th17 transfer were presumably attributable to augmentation of endogenous IL-6 production in gut. The number of Foxp3-positive regulatory T (Treg) cells in lungs and Payer's patches was increased in the OVA fed mice, whereas the number of these cells was decreased in the mice subjected to OVA feeding + Th17 cell transfer. Neutralization of IL-6 by monoclonal antibody in the mice subjected to OVA feeding + transfer of Th17 cells restored the effects of oral tolerance. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that IL-17 may inhibit the induction of tolerance to antigen through, at least in part augmenting IL-6 production, thereby suppressing the expansion of Treg cells.

A prostacyclin agonist with thromboxane inhibitory activity for airway allergic inflammation in mice.

BACKGROUND: ONO-1301 is a novel drug that acts as a prostacyclin agonist with thromboxane A(2) (TxA(2)) synthase inhibitory activity. We investigated the effect of ONO-1301 on development of airway allergic inflammation. METHODS: Mice sensitized and challenged to ovalbumin (OVA) received ONO-1301, OKY-046 (TxA(2) synthase inhibitor), beraprost, a prostacyclin receptor (IP) agonist, ONO-1301 plus CAY10449 (selective IP antagonist) or vehicle during the challenge period. Twenty-four hours after the OVA challenge, airway hyperresponsiveness (AHR) to methacholine was assessed and bronchoalveolar lavage was performed. Lung specimens were excised for goblet cell staining and analysis of lung dendritic cells (DCs). Bone marrow-derived dendritic cells (BMDCs) were generated, in the presence or absence of drugs, for analysis of DC function. RESULTS: Mice that received ONO-1301 showed significantly lower AHR, airway eosinophilia, T-helper type 2 cytokine levels, mucus production and lung DCs numbers than vehicle-treated mice. These effects of ONO-1301 were mostly reversed by CAY10449. BMDCs treated with ONO-1301 alone showed lower DC functions, such as expression of costimulatory factors or stimulation to spleen T cells. CONCLUSIONS: These data suggest that ONO-1301 may suppress AHR and airway allergic inflammation through modulation of DCs, mainly mediated through the IP receptor. This agent may be effective as an anti-inflammatory drug in the treatment of asthma.

Appropriate timing of CD40 ligation for RNA-Pulsed DCs to induce antitumor immunity.

Dendritic cell (DC) based anti-cancer immunotherapy is believed to be a promising treatment. However, appropriate conditioning including the method of antigen loading and stimulation for DC maturation is still unclear. Total RNA pulsing is one of the most attractive methods to load antigen, since RNA is easily replicated by the PCR technique and is absolutely free of tumor cell contamination. On the other hand, CD40 ligation is capable of producing one of the most potent signals for immature DCs to start functional maturation, which is required to induce adaptive immunity, resulting in altered migration ability to secondary lymphoid organs and augmented antigen presenting activity. Here, we demonstrate that DCs pulsed with total RNA extracted from tumor cells required CD40 stimulation with an appropriate sequence to present tumor-associated antigens. RNA derived antigens were presented for both CD4+ and CD8+ T cells in an antigen-specific manner. Dendritic cells that were pulsed with RNA followed by the stimulation through CD40 successfully primed antitumor effector T cells in draining LNs and subsequently induced antitumor protective immunity.

Generation of anti-tumour effector T cells from naïve T cells by stimulation with dendritic/tumour fusion cells.

Tumour-draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour-associated antigens in vivo. However, such tumour-specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour-specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti-tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)-based anti-cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30-40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L-positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL-2. After 9 days of culture, the antigen-specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN-gamma upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3-day established pulmonary metastases, these cells with coadministration of IL-2 exhibited anti-tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti-tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.

Phase I/II study of daily carboplatin, 5-fluorouracil and concurrent radiation therapy for locally advanced non-small-cell lung cancer.

A study was undertaken to determine the maximum tolerated dose, the dose-limiting toxicities and the response rate of carboplatin and 5-fluorouracil administered daily with concurrent thoracic radiation therapy in patients with locally advanced non-small-cell lung cancer. In a phase I/II clinical trial, patients with histologically documented, unresectable stage IIIA or IIIB non-small-cell lung cancer (NSCLC) were enrolled. Carboplatin (20-40 mg m(-2) 2-h infusion, daily) and 5-fluorouracil (200 mg m(-2) 24-h continuous infusion, daily) were administered concurrently with radiotherapy on days 1-33. Radiotherapy, with a total dose of 60 Gy, was delivered in 30 fractions on days 1-40. In the phase I portion, the daily dose of carboplatin was escalated from 20 to 40 mg m(-2). Once the maximum tolerated dose (MTD) and recommended dose (RD) of carboplatin was determined, the study entered the phase II portion. In the phase I portion, the daily MTD and RD of carboplatin were 40 and 35 mg m(-2), respectively. The dose-limiting toxicity was neutropenia. In the following phase II study, 30 patients were entered and the objective response rate was 76.7% (95% CI, 62-92%) and the local control rate was 85.7%. The median survival time was 19.8 months, with a survival rate of 70% at 1 year, 36.7% at 2 years. The major toxicities of treatment were neutropenia (>or=grade 3, 87.9%) and thrombocytopenia (>or=grade 3, 23.3%). This combined therapy for locally advanced non-small-cell lung cancer is promising and shows acceptable toxicity.

Successful liquid storage of peripheral blood stem cells at subzero non-freezing temperature.

Although non-frozen storage of peripheral blood stem cells (PBSC) has been extensively studied and utilized clinically, the optimal storage conditions have not been determined. In order to improve the maintenance of clonogenic capacity during storage, we evaluated the feasibility of subzero non-freezing preservation of PBSC and attempted to determine the optimal conditions. Human PBSC were stored in different non-cryopreserved conditions. University of Wisconsin (UW) solution was used as the storage medium for PBSC. The stem cell integrity was optimally maintained when PBSC were preserved in a supercooled state at -2 degrees C in UW solution without any cryoprotectants, and the highest values for nucleated cell survival (91.6%), CFU-GM survival (67.3%) and trypan blue viability (92%) were achieved at 72 h. CFU-GM survival in our storage conditions was significantly better than the survival achieved with hypothermic preservation in autologous serum and ACD-A solution at 4 degrees C (67.3 +/- 9.2% vs 42.9 +/- 15.3%; P < 0.01) or cryopreservation at -80 degrees C (67.3 +/- 9.2% vs 52.7 +/- 10.7%; P < 0.01). Thus, the combination of supercooling and UW solution was the optimal non-freezing method of preserving transplantable PBSC tested here. This method is of clinical utility in peripheral blood stem cell transplantation (PBSCT) for its simplicity and storage efficiency, and has value as a short-term storage method for PBSC to support dose-intensive multicyclic chemotherapy.

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.

Cancer immunogene therapy.

The establishment of cancer in a host involves at least two major events: the escape of tumor cells from normal growth control and their escape from immunological recognition. Because of this nature of their development, cancer cells seem to be predominatly poorly immunogenic. In contrast to the previous idea that cancer cells express no recognizable antigens, recent progress in the identification and characterization of tumor antigens, as well as the expansion of knowledge on the cellular and molecular mechanisms of antigen recognition by the immune system, have raised the possibility of using immunotherapy to treat certain tumors. Information on these mechanisms has been obtained in three crucial areas: 1) the role of cytokines in the regulation of the immune response, 2) the molecular characterization of tumor antigens in both mouse and human tumors, and 3) the molecular mechanisms of T cell activation and antigen presentation. Such information has provided new insight into tumor immunology and immunotherapy. Furthermore, recombinant DNA technology allows for modification of the genome of mammalian cells for therapeutic purposes in several diseases. Several novel strategies have been developed to derive genetically modified tumor cells and use them as cellular vaccines to induce antitumor immunity in animal tumor models. This combined modality of genetically modified tumor cells and immunotherapy has been termed immunogene therapy of tumors. Crucial to this approach has been the ability to transfer into normal or neoplastic cells genes known to increase the immunogenicity of cells, which subsequently can be used to augment immune reactions in tumor-bearing mice or cancer patients. While there has been success in inducing antitumor immunity in some tumor models, there are difficulties and limitations in the application of these gene-modified tumor cells for the treatment of preexisting tumors. In this review, recent progress in cancer immunogene therapy is discussed.

A phase I dose escalation study of multicyclic, dose-intensive chemotherapy with peripheral blood stem cell support for small cell lung cancer.

A phase I dose-escalation study of multicyclic, ifosfamide, carboplatin, and etoposide (ICE) with sequential reinfusion of peripheral blood stem cells (PBSCs) was conducted to determine the maximum-tolerated dose (MTD) of ICE. Twenty-four patients with SCLC (LD: 6, ED: 18) were treated with ifosfamide (3000-9000 mg/m2, 24-h infusion), carboplatin (300-400 mg/m2), and etoposide (300 mg/m2) followed by subcutaneous filgrastim (75 microg/day) from day 4 to the day of PBSC collection. PBSC were harvested when the WBC count reached >/=5 x 109/l. The leukapheresis product was cryopreserved and reinfused on day 4 of the next cycle, which was started 48 h after the last PBSC collection. The ifosfamide dose was escalated as follows: 3000 mg/m2 (level 1), 5000 mg/m2 (level 2), 7000 mg/m2 (level 3), 9000 mg/m2 (level 4). Patients with LD were treated with concurrent radiotherapy at 1.5 Gy twice daily for the initial 3 weeks to a total dose of 45 Gy and MTD, defined separately. Patients were evaluated for hematologic and non-hematologic toxicity, actual dose intensities, as well as response to therapy. The maximum-tolerated dose (MTD) was defined as the dose level at which more than 5 days of grade 4 myelo- suppression or non-hematologic toxicity greater than grade 3 developed in two thirds of the patients. For ED cases, MTD was level 4 and the recommended dose of ifosfamide was 7000 mg/m2. For LD cases, the recommended dose of ifosfamide was 5000 mg/m2. The dose limiting toxicity of multicyclic ICE was hemato- logic toxicity and CNS toxicity which manifested as ataxia. Tumor responses were seen in all patients, with 14 patients showing a complete response. The actual total dose-intensity at the recommended dose level was 2.2 and 1.74, for ED and LD, respectively, compared with previously reported ICE regimens. PBSC support for dose-intensive ICE regimen permitted dose escalation of ifosfamide with a mean interval of 16-17 days. We conclude that this regimen is well tolerated, with acceptable hematological and non-hematological toxicity. Bone Marrow Transplantation (2000) 25, 5-11.

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells.

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.

Critical role of CD11a (LFA-1) in therapeutic efficacy of systemically transferred antitumor effector T cells.

The systemic adoptive transfer of activated T cells, derived from tumor-draining lymph nodes (LNs), mediates the regression of established tumors. In this study, the requirement of cell adhesion molecules, CD11a/CD18 (LFA-1), CD54 (ICAM-1), CD49d/CD29 (VLA-4), and CD106 (VCAM-1), for T cell infiltration into tumors and antitumor function was investigated. Administration of anti-CD11a mAb completely abrogated the efficacy of adoptive immunotherapy for both intracranial and pulmonary metastatic MCA 205 fibrosarcomas. In contrast, adoptive immunotherapy was effective in animals treated with anti-CD49d mAb, anti-CD106 mAb, anti-CD54 mAb, or in CD54 knockout recipients. Trafficking of transferred cells to the intracranial tumor was not affected by any of the mAb. However, the tumor-specific secretion of IFN-gamma by activated LN T cells was suppressed by anti-CD11a mAb or anti-CD54 mAb. To account for the different effects of CD11a and CD54 blockade in vivo, an additional CD11a/CD18 ligand, CD102 (ICAM-2), was demonstrated on tumor-associated macrophages but not on tumor cells. These results show that CD11a mediates a critical function in interactions between effector T cells, tumor cells, and host accessory cells in situ leading to tumor regression.

Clinical features of polymyositis/dermatomyositis with steroid-resistant interstitial lung disease.

Polymyositis and dermatomyositis (PM/DM) without creatine kinase (CK) elevation shows a poor prognosis. PM/DM is complicated with interstitial lung disease (ILD), some of which progress rapidly. To clarify the clinical features of PM/DM from the viewpoint of ILD progression, the clinical data of 25 PM/DM patients with ILD were reviewed. They were classified as responders or non-responders. The patients whose ILD responded to steroid therapy and elicited good clinical courses were termed as responders. On the other hand, the patients who had rapidly progressive ILD resistant to steroid therapy were considered as non-responders. The patients diagnosed to have DM were likely to be steroid-resistant. The non-responder group revealed significantly high aspartate aminotransferase (AST), low CK, low white blood cell (WBC), and low absolute lymphocyte counts in their peripheral blood. High CK/AST may be a favorable predictor of the disease. The percentages of lymphocytes in bronchoalveolar lavage fluid were increased in both groups. However, the percentages of two responders with low CK/AST were lower than those of three non-responders. A steroid-resistant ILD group with PM/DM may be clinically different from a steroid-responsive ILD group.

Purification of L-selectin(low) cells promotes the generation of highly potent CD4 antitumor effector T lymphocytes.

Successful adoptive immunotherapy of cancer requires the identification, isolation, and expansion of tumor-specific immune effector cells. A reliable source of tumor-immune lymphocytes is lymph nodes draining a growing tumor. After in vitro stimulation with anti-CD3 and expansion in IL-2, these cells are capable of mediating the regression of established tumors. In the absence of further Ag stimulation, we recently found that the down-regulation of the homing molecule L-selectin could serve as a surrogate marker for isolation of specific tumor-sensitized T cells. The L-selectin(low) (L-selectin-) T cells proliferated more vigorously than unfractionated or L-selectin(high) cells. In adoptive immunotherapy of established intracranial MCA 205 tumors, L-selectin- cells displayed at least 30-fold greater therapeutic efficacy than unfractionated cells. L-selectin(high) cells did not demonstrate any antitumor effects. Activated L-selectin- cells secreted a number of cytokines, including IFN-gamma, IL-2, IL-4, and IL-10, specifically when stimulated with cognate tumor cells. Further analysis revealed that CD4 T cells alone mediated tumor regression and secreted cytokines. Our results thus demonstrate that the purification of L-selectin- cells led to the generation of CD4 immune effector cells with unusually high therapeutic efficacy against chemically induced tumors. The lack of cytotoxicity and the ability to secrete cytokines suggest that these effector CD4 cells mediate antitumor effects through an indirect mechanism similar to the delayed hypersensitivity reaction.

[Bronchiolitis obliterans and no radiographic abnormalities in a patient with rheumatoid arthritis].

A 53-year-old woman was given a diagnosis of rheumatoid arthritis in 1988, and begun treatment with D-penicillamine in September 1992. She noticed dry coughing and exertional dyspnea that began in April 1993. Chest X-ray and CT films revealed no abnormal opacities. However, bronchiolitis obliterans was suspected because of a low FEV1% (23%). Examination of specimens obtained by thoracoscopic lung biopsy revealed constrictive obliteration by granulation tissue in proximal bronchioles and follicular bronchiolitis. Alveoli and respiratory bronchioles were intact. After corticosteroid and cyclophosphamide pulse therapy, FEV1% increased to 35%. At the time of this writing she was alive 2.5 years after hospitalization.

Contraction and intracellular calcium-ion elevation of cultured human aortic smooth muscle cells by endothelin-1, vasoactive intestinal contractor (VIC) and the derivatives.

Effects of endothelin (ET) family peptides and their derivatives on cellular contraction and calcium-ion level were examined by using cultured human vascular smooth muscle cells (VSM). Contraction of cultured human VSM, isolated from human fetal aortic segments, was induced within 1 min after the treatment with ET-1 (100 nM) as seen in the changes of cytosolic calcium-ion localization. In parallel with the cell contraction, cytosolic calcium-ion level in the human VSM increased very rapidly and then dropped with some oscillation as determined by Anchorage Cell Analyzing System. It was noted that transient calcium-ion mobilization rather than sustained calcium-ion influx was significant in the contraction of cultured human VSM. Vasoactive intestinal contractor (VIC), three amino acids different from ET-1, had less activity in increase of intracellular calcium-ion level and in percent of response cells than ET-1, ET-2, and VIC-S4L6 (one amino acid different from ET-1). EC50 of ET-1, VIC-S4L6, ET-2, and VIC were 0.5 nM, 0.6 nM, 2.0 nM, and 20 nM, respectively. VIC-like peptide (VIC-LP), 16 amino acids fragment of VIC precursor protein, had no effect with a single administration of up to 10 microM. However, the increase in calcium-ion level by VIC was suppressed with a prior treatment of cells with high concentration (10 microM) of VIC-LP. The establishment of cultured human VSM for the simultaneous examination of the contraction and calcium-ion level will provide a new system to study signal transduction of vasocontractor peptides.

Isolation based on L-selectin expression of immune effector T cells derived from tumor-draining lymph nodes.

The ability to generate a large number of tumor-reactive T lymphocytes is the most critical requirement for adoptive immunotherapy. Our laboratory has previously demonstrated that cells from tumor-draining lymph nodes (LNs) are an excellent source of tumor-reactive T lymphocytes. After activation with anti-CD3, these cells readily proliferate in low concentrations of interleukin 2 and acquire effector functions. The adoptive transfer of these cells is capable of mediating the regression of tumors established in the lung as well as in the brain. Here, we analyzed several adhesion molecules on the tumor-draining LN T cells and separated them based on L-selectin expression. The homing receptor L-selectin mediates adhesion to the luminal surface of specialized high endothelial venules, thus regulating lymphocyte recirculation through peripheral LNs. In response to progressive tumor growth, a small population of draining LN T cells down-regulated L-selectin and increased the expression of CD44 and lymphocyte function-associated antigen 1. In adoptive immunotherapy, purified T cells with low L-selectin (L-selectin-) expression constituted all the in vivo antitumor reactivity, whereas isolated high L-selectin (L-selectin+) cells were ineffective. Furthermore, reverse transcription-PCR analysis revealed that L-selectin- cells expressed interleukin 2, IFN-gamma and tumor necrosis factor alpha mRNA upon in vitro stimulation with specific tumor cells. These results suggest that highly potent immune T cells can be isolated based on their pattern of adhesion molecule expression. The ability of the immune effector cells to transcribe cytokine genes when stimulated with tumor cells provides a basis for identifying similar cells for adoptive immunotherapy of cancer in humans.

Low oxygen enhances endothelin-1 (ET-1) production and responsiveness to ET-1 in cultured cardiac myocytes.

To understand the pathophysiological role of ET-1 in heart, the ET-1 production ability and the responsiveness to ET-1 in cultured neonatal rat cardiac myocytes were compared between high (50%) and low (21%) oxygen atmosphere culture conditions. The amount of immunoreactive ET-1 secreted by the cardiac myocytes into the culture medium was much higher at the low oxygen condition. An analysis of ET-1 binding capacity to the cardiac myocytes revealed that the receptor number of ET-1 was about two-fold at low oxygen culture condition. In fact, an addition of ET-1 (1nM) increased in protein synthesis at the low but not the high oxygen culture condition. Thus, the increase in ET-1 production and responsiveness to ET-1 in the cardiac myocytes at the low oxygen culture condition suggests that autocrine ET-1 might be involved in the maintenance of beating ability in heart at hypoxia.