Recent advances regarding the potential roles of invariant natural killer T cells in cardiovascular diseases with immunological and inflammatory backgrounds.

Invariant natural killer T (iNKT) cells, which bear αß-type T-cell antigen-receptors (TCRs), recognize glycolipid antigens in a cluster of differentiation 1d (CD1d)-restricted manner. Regarding these cells, the unique modes of thymic selection and maturation elucidate innateness, irrespective of them also being members of the adaptive immune system as a T-cell. iNKT cells develop and differentiate into NKT1 [interferon γ (IFN-γ)-producing], NKT2 [interleukin 4 (IL-4)/IL-13-producing], or NKT17 (IL-17-producing) subsets in the thymus. After egress, NKT10 (IL-10-producing), follicular helper NKT (NKTfh; IL-21-producing), and regulatory NKT (NKTreg) subsets emerge following stimulation in the periphery. Moreover, iNKT cells have been shown to possess several physiological or pathological roles. iNKT cells exhibit dual alleviating or aggravating roles in experimentally induced immune and/or inflammatory diseases in mice. These findings indicate that the modulation of iNKT cells can be employed for therapeutic use or prevention of human diseases. In this review, we discuss the potential roles of iNKT cells in the development of immune/inflammatory diseases of the cardiovascular system, with emphasis on atherosclerosis, aortic aneurysms, and cardiac remodeling.

MR1 deficiency enhances IL-17-mediated allergic contact dermatitis.

Major histocompatibility complex (MHC) class Ib molecules present antigens to subsets of T cells primarily involved in host defense against pathogenic microbes and influence the development of immune-mediated diseases. The MHC class Ib molecule MHC-related protein 1 (MR1) functions as a platform to select MR1-restricted T cells, including mucosal-associated invariant T (MAIT) cells in the thymus, and presents ligands to them in the periphery. MAIT cells constitute an innate-like T-cell subset that recognizes microbial vitamin B2 metabolites and plays a defensive role against microbes. In this study, we investigated the function of MR1 in allergic contact dermatitis (ACD) by examining wild-type (WT) and MR1-deficient (MR1-/-) mice in which ACD was induced with 2,4-dinitrofluorobenzene (DNFB). MR1-/- mice exhibited exaggerated ACD lesions compared with WT mice. More neutrophils were recruited in the lesions in MR1-/- mice than in WT mice. WT mice contained fewer MAIT cells in their skin lesions following elicitation with DNFB, and MR1-/- mice lacking MAIT cells exhibited a significant increase in IL-17-producing αß and γδ T cells in the skin. Collectively, MR1-/- mice displayed exacerbated ACD from an early phase with an enhanced type 3 immune response, although the precise mechanism of this enhancement remains elusive.

A low-frequency IL4R locus variant in Japanese patients with intravenous immunoglobulin therapy-unresponsive Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis which may be associated with coronary artery aneurysms. A notable risk factor for the development of coronary artery aneurysms is resistance to intravenous immunoglobulin (IVIG) therapy, which comprises standard treatment for the acute phase of KD. The cause of IVIG resistance in KD is largely unknown; however, the contribution of genetic factors, especially variants in immune-related genes, has been suspected. METHODS: To explore genetic variants related to IVIG-unresponsiveness, we designated KD patients who did not respond to both first and second courses of IVIG therapy as IVIG-unresponsive patients. Using genomic DNA from 30 IVIG-unresponsive KD patients, we performed pooled genome sequencing targeting 39 immune-related cytokine receptor genes. RESULTS: The single nucleotide variant (SNV), rs563535954 (located in the IL4R locus), was concentrated in IVIG-unresponsive KD patients. Individual genotyping showed that the minor allele of rs563535954 was present in 4/33 patients with IVIG-unresponsive KD, compared with 20/1063 individuals in the Japanese genome variation database (odds ratio = 7.19, 95% confidence interval 2.43-21.47). Furthermore, the minor allele of rs563535954 was absent in 42 KD patients who responded to IVIG treatment (P = 0.0337), indicating that a low-frequency variant, rs563535954, is associated with IVIG-unresponsiveness in KD patients. Although rs563535954 is located in the 3'-untranslated region of IL4R, there was no alternation in IL4R expression associated with the mior allele of rs563535954. However, IVIG-unresponsive patients that exhibited the minor allele of rs563535954 tended to be classified into the low-risk group (based on previously reported risk scores) for prediction of IVIG-resistance. Therefore, IVIG-unresponsiveness associated with the minor allele of rs563535954 might differ from IVIG-unresponsiveness associated with previous risk factors used to evaluate IVIG-unresponsiveness in KD. CONCLUSION: These findings suggest that the SNV rs563535954 could serve as a predictive indicator of IVIG-unresponsiveness, thereby improving the sensitivity of risk scoring systems, and may aid in prevention of coronary artery lesions in KD patients.

Wavelet transforms on Gelfand-Shilov spaces and concrete examples.

In this paper, we study the continuity properties of wavelet transforms in the Gelfand-Shilov spaces with the use of a vanishing moment condition. Moreover, we also compute the Fourier transforms and the wavelet transforms of concrete functions in the Gelfand-Shilov spaces.

ESCRT-0 protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is targeted to endosomes independently of signal-transducing adaptor molecule (STAM) and the complex formation with STAM promotes its endosomal dissociation.

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.

[Anesthetic management of posterior lumbar spinal fusion in a patient suspected of having acute exacerbation of chronic interstitial pneumonia].

A patient complicated with interstitial pneumonia required emergency posterior lumbar spinal fusion. The blood gas analysis showed relatively benign values (PaO2 81 torr, PaCO2 44 torr, under room air), but the honeycombing lungs were noted in the bilateral lung fields on CT, and the KL-6 level was high (1,000 U x ml(-1)), for which the acute exacerbation of interstitial pneumonia was suspected. Sivelestat sodium administration was initiated during the surgery and continued postoperatively. During surgery, setting the FIO2 at 0.34, the P/F ratio and intra-airway pressure could be maintained at 500 and 25 mmHg, respectively. To reduce postoperative respiratory complication, anesthesia was maintained with desflurane, which is dissipated easily, and 0.5% ropivacaine 15 ml was subcutaneously injected to the surgical field at the time of wound closure to reduce the total doses of intraoperative fentanyl and postoperative analgesics. After the completion of surgery, the endotracheal tube was removed with head elevated position, and the patient was transported back to the ward. No acute exacerbation occurred thereafter, and the patient was discharged 67 days after surgery. The prediction of acute exacerbation of interstitial pneumonia is difficult. Moreover, there is no established preventive method, although the mortality is high. Therefore, physicians should be thoroughly informed about the currently available evidence, including developmental factors.

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal.

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor ß (IL-2Rß) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rß and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rß and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rß together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rß. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor ß (IL-2Rß). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor α (IL-4Rα), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2Rß. Examinations of the Hrs binding regions of the receptors reveal that a hydrophobic amino acid cluster in both IL-2Rß and IL-4Rα is essential for the binding. Whereas the wild-type receptors are delivered to LAMP1-positive late endosomes, mutant receptors lacking the hydrophobic amino acid cluster are sorted to lysobisphosphatidic acid-positive late endosomes rather than LAMP1-positive late endosomes. We also show that the degradation of these mutant receptors is attenuated. Accordingly, Hrs functions during ubiquitin-independent endosomal sorting of the receptors by recognizing the hydrophobic amino acid cluster. These findings suggest the existence of a group of cargo proteins that have this hydrophobic amino acid cluster as a ubiquitin-independent sorting signal.

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus.

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 x 10(4) cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.

Haeme-regulated degradation of delta-aminolevulinate synthase 1 in rat liver mitochondria.

Protein turnover, which occurs at various rates, is critical for the homeostasis of cellular protein levels. However, the proteolysis systems that determine the turnover rate of mitochondrial proteins are largely unknown. Delta-aminolevulinic acid synthase (ALAS) 1, a rate-limiting enzyme in the haeme biosynthesis, is one of the mitochondrial proteins that have a very short lifetime. In this study, to reveal the regulatory mechanisms for ALAS1 degradation, we examined the turnover rates of ALAS1 in rat liver under several conditions. In primary rat hepatocytes, the degradation of ALAS1 was stimulated by haeme, and suppressed by inhibition of haeme biosynthesis. Furthermore, the haeme-stimulated degradation of ALAS1 was observed in the isolated mitochondria. These results suggested that, in mitochondria, there exists an ALAS1 degradation system that is regulated by cellular haeme level and plays a crucial role in the regulation of haeme biosynthesis.