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Biomedical subjects

N Nishimura

Publications and source records attributed to N Nishimura.

At least 19 recordsLinked to original sources

Genetic control of the spontaneous activation of CD4+ Th cells in systemic lupus erythematosus-prone (NZB x NZW) F1 mice.

The F(1) hybrid of autoimmune hemolytic anemia-prone NZB and nonautoimmune NZW strains of mice has been studied as a murine model of systemic lupus erythematosus. Both NZB and F(1) hybrid mice show age-dependent spontaneous activation of peripheral CD4(+) T cells as reflected by the elevated frequencies of CD4(+) T cells positive for CD69 early activation marker. Both strains also show age-dependent abnormal decrease of the frequencies of CD62L(+) naive CD4(+) T cells and/or NTA260(+) memory CD4(+) T cells in the spleen. We studied the multigenic control of these abnormal features of peripheral CD4(+) T cells in (NZB x NZW) F(1) x NZW backcross mice by quantitative trait loci mapping and by association rule analysis. The abnormally elevated frequencies of CD69(+)CD4(+) T cells and decreased frequencies of CD62L(+) naive and/or NTA260(+) memory CD4(+) T cells were under the common genetic control, in which the interaction between MHC and a hitherto unknown locus, designated Sta-1 (spontaneous T-cell activation) on chromosome 12, plays a major role. The allelic effects of these loci likely predispose CD4(+) T cells to the loss of self-tolerance, and are responsible for the accelerated autoimmune phenotypes of (NZB x NZW) F(1) hybrid mice.

Age Factors↗

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells.

Taurine has been reported to enhance cholesterol 7alpha-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine - a structural analogue of taurine - did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.

Animals↗

In vitro and in vivo analysis of human herpesvirus-6 U90 protein expression.

In order to establish a reliable method for the detection of human herpesvirus-6 (HHV-6) B antigens in peripheral blood mononuclear cells (PBMCs) collected from HHV-6 infected patients, we created a polyclonal antibody against the HHV-6 B U90 protein (IEA/ex3) and used it to examine the expression of this protein in virus-infected cells and patients' PBMCs. This antibody reacted with 170 and 195 kDa proteins in HHV-6 B-infected cord blood mononuclear cells. The IEA/ex3 antigen was detected in cord blood mononuclear cells at 6 hr post-infection, and the number of infected cells reached its maximum at 48 hr post-infection. The antigen stained in a punctate pattern and partially localized to the promyelocytic leukemia (PML) protein body. We also examined 60 PBMC samples from 60 febrile children (3-19 months old) and detected IEA/ex3 antigen in the PBMCs by laser-scanning microscopy. HHV-6 was isolated from 31 of the 60 samples. The sensitivity and specificity of the antigen detection were 84% (26/31) and 97% (28/29), respectively, in the samples with virus detected. The mean number of antigen-positive PBMCs was 409/10(6) cells in 20 samples with viral isolation. A significant correlation (r = 0.566; P = 0.008) was observed between the viral load and number of antigen-positive cells. Although IEA/ex3 antigen was detected by laser-scanning microscopy in PBMCs (without cultivation) collected from six patients with isolated virus, it was detected in only one sample by conventional fluorescence microscopy. Increasing the intensity by cultivation (24 hr) resulted in a higher detection rate (5/6) even by conventional fluorescence microscopy, which is available in most hospital laboratories.

Antibodies, Viral↗

Up regulated expression of tumour necrosis factor {alpha} converting enzyme in peripheral monocytes of patients with early systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is accompanied by abnormalities in humoral and cellular immune systems. OBJECTIVE: To determine the genes specifically expressed in the immune system in SSc by analysis of the gene expression profile of peripheral blood mononuclear cells (PBMC) from patients with SSc, including those treated with haematopoietic stem cell transplantation (HSCT). Additionally, to investigate the clinical significance of the up regulation of tumour necrosis factor alpha (TNFalpha) converting enzyme (TACE). METHODS: PBMC from patients with SSc (n = 23) and other autoimmune diseases (systemic lupus erythematosus (SLE, n = 16), rheumatoid arthritis (RA, n = 29)), and from disease-free controls (n = 36) were examined. Complementary DNA arrays were used to evaluate gene expression of PBMC, in combination with real time quantitative polymerase chain reactions. TACE protein expression in PBMC was examined by fluorescence activated cell sorter (FACS). RESULTS: In patients with SSc 118 genes were down regulated after HSCT. Subsequent comparative analysis of SSc without HSCT and healthy controls indicated SSc-specific up regulation for three genes: monocyte chemoattractant protein-3 (p = 0.0015), macrophage inflammatory protein 3alpha (p = 0.0339), and TACE (p = 0.0251). In the FACS analysis, TACE protein was mainly expressed on CD14(+) monocytes both in patients with SSc and controls. TACE expression on CD14(+) cells was significantly increased in patients with early SSc (p = 0.0096), but not in those with chronic SSc, SLE, or RA. TACE protein levels in SSc monocytes correlated with the intracellular CD68 levels (p = 0.0016). CONCLUSIONS: Up regulation of TACE expression was a unique profile in early SSc, and may affect the function of TNFalpha and other immunoregulatory molecules.

ADAM Proteins↗

Granulocyte and monocyte adsorptive apheresis in the treatment of active distal ulcerative colitis: a prospective, pilot study.

AIM: To assess safety and clinical efficacy of granulocyte and monocyte adsorptive apheresis for distal ulcerative colitis. METHODS: Granulocyte and monocyte adsorptive apheresis therapy (five aphereses for 5 consecutive weeks) was performed for 30 consecutive patients with active distal ulcerative colitis. Patient compliance, adverse effects and clinical symptoms were regularly assessed. RESULTS: Adverse effects were noted during nine (6%) apheresis sessions in eight patients; slight headache five, transient abdominal pain with tenesmus two, fever (38 degrees C) one and mild liver dysfunction one. None of these adverse effects was serious and all patients could complete five aphereses. Clinical symptoms (stool frequency and consistency, rectal bleeding, tenesmus and mucus in stools) significantly improved after the third apheresis. Clinical remission (normal stool frequency and no rectal bleeding) was achieved in 21 patients (70%) after five aphereses. The median Disease Activity Index score significantly decreased; from 6 [interquartile range (IQR): 4-7] to 2 (IQR: 1-3) (P < 0.0001). CONCLUSION: In the treatment of active distal ulcerative colitis, granulocyte and monocyte adsorptive apheresis is safe and well-tolerated. Granulocyte and monocyte adsorptive apheresis had a beneficial effect on clinical remission and symptoms. However, randomized-controlled trials would be necessary to assess a definite efficacy of granulocyte and monocyte adsorptive apheresis.

Adolescent↗

Hemichrome formation observed in human haemoglobin A under various buffer conditions.

AIM: To observe hemichrome formation in human haemoglobin A under various buffer conditions. METHOD: Hemichrome formation of human oxyhaemoglobin A (HbO2) was studied spectrophotometrically in 0.1 m buffer at various temperatures and pH values. RESULTS: Following autoxidation in ferrous HbO2, it was evident that formation of hemichrome, which tends to precipitate, occurred at various stages during the course of the autoxidation reaction namely at initial, intermediate or final stages, depending on temperature and pH of the solution. By varying temperature of the solution from 35 to 55 degrees C and pH from 4.5 to 10.5, it is shown here that HbO2 exhibits high susceptibility for hemichrome formation and its occurrence is a function of pH, temperature and progress of autoxidation of HbO2. Unlike HbO2 and its separated haemoglobin chains, monomeric bovine heart myoglobin (MbO2) did not easily form hemichrome. CONCLUSION: These findings provide a clue on the crucial role of haemoglobin molecule for senescent cell recognition or homeostasis in the blood circulation.

Animals↗

Adenoviral interleukin-12 gene transduction into human bronchial epithelial cells: up-regulation of pro-inflammatory cytokines and its prevention by corticosteroids.

BACKGROUND: One of the potential effects of IL-12 is to restore Th1/Th2 balance. Therefore, we investigated the possibility of developing a system for local delivery of IL-12 into the airways by examining protein expression in a human bronchial epithelial cell line (BEAS-2B) after adenoviral IL-12 gene transduction. The effects of dexamethasone on the gene-modified cells were also examined. METHODS: Adenoviral vectors AxCAegfp and Ax1CIhp40ip35 were used to transduce enhanced green fluorescence protein and IL-12 genes, respectively, into BEAS-2B cells. Wild-type and IL-12 gene-transduced BEAS-2B cells were then incubated with or without dexamethasone, and concentrations of IL-12, IFN-gamma, IL-6, IL-8, granulocyte macrophage-colony stimulating factor and chemokines (TARC and RANTES) in the supernatant were measured by ELISA. IL-12 receptor expression was analysed by flow cytometry and RT-PCR. RESULTS: The efficiency of transgene expression in BEAS-2B cells at a multiplicity of infection of 30 was approximately 80%. Gene-modified BEAS-2B cells produced biologically active IL-12, regardless of dexamethasone treatment. While IL-12 gene transduction led to increased production of IL-6 and IL-8 by BEAS-2B cells, expressions of these proteins were suppressed by dexamethasone. Addition of exogenous IL-12 failed to augment BEAS-2B cell IL-6 and IL-8 production, and IL-12 receptor expression by BEAS-2B cells was not detected. CONCLUSIONS: Our findings suggest that adenoviral IL-12 gene transduction may be effective in inducing IL-12 expression in the airways, and could be a potential approach in the management of bronchial asthma.

Adenoviridae↗

Measurement of soluble Fcgamma receptor type IIIa derived from macrophages in plasma: increase in patients with rheumatoid arthritis.

FcgammaRIII (CD16) is found in two alternative forms, a transmembrane FcgammaRIIIa expressed on NK cells and macrophages, and a glycosylphosphatidylinositol-linked FcgammaRIIIb present on neutrophils. Previously, we measured soluble FcgammaRIIIa (sFcgammaRIIIa) in plasma of NA(1 +, 2-) phenotyped donors with the anti-FcgammaRIII monoclonal antibody (MoAb) GRM1, which recognizes NA2-FcgammaRIIIb and FcgammaRIIIa. The level of sFcgammaRIIIa, as well as the total sFcgammaRIII (sFcgammaRIIIa plus sFcgammaRIIIb) in patients with rheumatoid arthritis (RA) was significantly higher than that in healthy controls. In this study, we measured sFcgammaRIIIa(M)(phi) in plasma with a newly developed anti-FcgammaRIII MoAb, MKGR14 (mIgM), which recognizes FcgammaRIIIa(M)(phi) specifically. From the recovery of purified sFcgammaRIIIa(M)(phi), the amount of sFcgammaRIIIa(M)(phi) present was about half that of sFcgammaRIIIa(NK), and that of sFcgammaRIIIa was about 50 times lower than that of sFcgammaRIIIb in pooled plasma from healthy NA(1 +, 2-) phenotyped donors. The level of sFcgammaRIIIa(M)(phi) in RA patients was about four times higher than that in healthy controls. In RA patients, both the sFcgammaRIIIa(M)(phi) and sFcgammaRIIIa levels were increased as proportionally as the Lansbury Index. The sFcgammaRIIIa, but not sFcgammaRIIIa(M)(phi) levels, were increased directly proportional to C-reactive protein. sFcgammaRIIIa(M)(phi) may be a novel marker of disease activity in RA.

Adult↗

Clonal diversity and genetic differentiation in Ilex leucoclada M. patches in an old-growth beech forest.

We investigated clonal diversity within patches of Ilex leucoclada and genetic variation within and among patches using random amplified polymorphic DNA (RAPD) markers in a 1-ha plot within an old-growth beech forest. We found 38 patches that exhibited a clumped distribution in the middle of the plot. We identified a total of 166 RAPD phenotypes among the 215 stems sampled from 27 patches that were completely within the plot. The population showed high clonal diversity within patches (mean number of genets relative to number of stems = 0.79; mean Simpson's D = 0.89). Variation in RAPD phenotypes among patches was highly significant (PhiST in the molecular variance analysis = 0.316, P < 0.001), indicating genetic differentiation among patches. Pairwise genetic distances, PhiST, among patches did not correlate with geographical distances among patches. The cluster analysis based on the genetic distances showed few clear clusters of patches, indicating no spatial genetic structure among patches. High levels of clonal diversity both within patches and within the population may be explained by multiple founders, seedling recruitment during patch-formation, and somatic mutation. The significant genetic differentiation among patches may be caused by separate founding events and/or kin structuring within patches.

Cluster Analysis↗

Rat thyroid hyperplasia induced by gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Effects of gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thyroid function of offspring were investigated in the rat. Pregnant Holtzman rats, TCDD-sensitive strain, were given a single oral dose of 200 ng or 800 ng TCDD/kg on gestational day 15. Parameters related to the thyroid functions were examined on postnatal days (PNDs) 21 and 49. Serum T(4) levels in offspring decreased significantly on PND21 in the two TCDD-exposed groups but increased on PND 49 only in the high-dose group. A dose of 800 ng TCDD/kg exerted a more than 2-fold increase in serum TSH level in male offspring on PNDs 21 and 49. A significant induction of uridine diphosphate-glucuronosyltransferase-1 gene by TCDD was observed on PND 21 but returned to basal levels on PND 49. Gene expression of cytochrome P4501A1 was markedly induced in the liver treated with TCDD. Even a single oral perinatal exposure to 800 ng TCDD/kg resulted in hyperplasia of the thyroid gland of offspring on PND 49. Proliferating cell nuclear antigen immunocytochemistry also supported this finding. Thus, gestational and lactational exposure to TCDD was found to disrupt thyroid hormone homeostasis, which results in a sustained excessive secretion of TSH, followed by the hyperplasia of thyroid follicular cells.

Administration, Oral↗

High-throughput array production using precision glass syringes.

The advantages of using 1, 96, or 384 precision glass syringes in automated high-throughput microdispensers in creating highly uniform and reproducible DNA, protein, and organic compound array filters and slides are described. Using the Hydra Microdispenser and Tango Liquid Handling system, 0.1-5 ng (in 50-300 nL) PCR-amplified, human cancer-related genes and housekeeping genes were spotted onto nylon membranes and coated slides. Protein solutions of 50 microg/mL to 1 mg/mL were spotted onto coated slides or onto MaxiSorp 96-well plates. Up to 6144 spots/membrane and up to 1000 spots/slide were printed. The size of the spots created by glass syringes was uniform and reproducible (precision variation of less than 5%) from spot to spot and membrane to membrane. Using a Tango 384 system, a total of ten 6144-spot filters can be produced in approximately 25 min, translating into a spotting speed of 2.5 min/membrane.

Oligonucleotide Array Sequence Analysis↗

Does temperature affect the outcome of larval competition between Aedes aegypti and Aedes albopictus?

The superior larval competitive ability of Aedes albopictus has been proposed to explain the recent displacement of Aedes aegyptiby the former species inparts of the southeastern U.S. Ae. aegypti persists, however, in sympatry with Ae. albopictus in urban areas of southern Louisiana, Florida, and Texas, and the impact of larval competition between these species has not been investigated at higher temperatures that may be characteristic of these urban environments. We compared growth and survivorship of the two species at controlled temperatures of 24 degrees and 30 degrees C in water-containing tires under conditions of intra- and interspecific competition and with or without leaf litter. When other variables were controlled statistically, the estimated finite rate of increase (lambda') was significantly higher for both species at the higher temperature, and the proportional increases in lambda' did not differ between species. Therefore, our experiment predicts that by itself, temperatures between 24 degrees and 30 degrees C would not alter the outcome of larval competition. Overall, response measures of Ae. albopictus were more sensitive than those of Ae. aegypti to the litter and species/density variables, although the development ofAe. aegypti females was uniquely retarded by a high density of its own species.

Adaptation, Physiological↗

Transduction of KAI1/CD82 cDNA promotes hematogenous spread of human lung-cancer cells in natural killer cell-depleted SCID mice.

KAI1, which is identical to CD82, was initially identified as a metastasis-suppressor gene for human prostate cancer, and its expression is reported to be a favorable prognostic factor for operable human lung cancer. In this study, we examined the functional role of KAI1/CD82 in the late phase of metastatic spread of human lung-cancer cells. For this, KAI1/CD82 cDNA was introduced into KAI1/CD82 low-expressing human lung-cancer cell lines, SBC-3 and PC-14, and then the metastatic potential of the transformants was analyzed by i.v. inoculation of KAI1/CD82-transduced cells, SBC-3/KAI1 and PC-14/KAI1, into NK cell-depleted SCID mice. Contrary to our expectations, KAI1/CD82 gene transfer promoted multiorgan metastasis of i.v.-inoculated human lung-cancer cells, while s.c. tumor growth was unaffected. Cancer cells from metastatic tumors of NK cell-depleted SCID mice injected i.v. with SBC-3/KAI1 expressed appreciable cell-surface KAI1/CD82, and cells not expressing KAI1/CD82 (revertants) were not detected in the tumors. Our findings indicate that under conditions where the host's natural cytotoxicity is suppressed, KAI1/CD82 may enhance the formation of tumors by circulating lung-cancer cells at metastatic sites.

Animals↗

Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust.

Exposure to diesel exhaust (DE) induces lesions in lung epithelium by generation of reactive oxygen species. Glycosaminoglycans (GAG), components of extracellar matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. We investigated how GAG are related to the recovery of lung tissue from injury. Using high-performance liquid chromatography analysis, we determined the amounts of GAG, such as chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) in the lungs of rats exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m(3) as suspended particulate matter, or to filtered air. The contents of CS and HA in the surroundings of the bronchi were significantly increased after exposure to DE. In addition, immunohistochemical staining showed that the number of 8-hydroxydeoxyguanosine-positive cells as a marker of cell damage, and proliferating cell nuclear antigen-positive cells also increased in the same areas in which the levels of GAG were elevated in the lungs of rats exposed to 3 mg/m(3) DE. These results suggest that CS and HA in the lung contribute to cell proliferation and remodeling in the process of recovery from injury caused by exposure to DE.

8-Hydroxy-2'-Deoxyguanosine↗

Induction of metallothionein in the livers of female Sprague-Dawley rats treated with 2,3,7 ,8-tetrachlorodibenzo-p-dioxin.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects against metal toxicity and external stimuli including ionizing or ultraviolet B irradiation. Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to cause an exaggerated oxidative stress response in animals and in different organs, we have studied possible involvement of MT in the oxidative responses induced by TCDD. Female Sprague-Dawley (SD) rats (6-week old) were administered a single oral dose of TCDD that varied from 1.0 to 4.0 microg/kg body weight. The serum and tissues were collected 7 days after dosing. Indicators of oxidative damage were assessed. Significant increases in serum 8-hydroxydeoxyguanosine (8-OHdG) levels were observed in the rats dosed with 2.0 and 4.0 microg TCDD/kg bw. Only 4.0 microg TCDD/kg bw produced a decrease in reduced glutathione concentration in the liver. Immunohistochemical staining revealed a TCDD-induced increase in heme oxygenase-1 (HO-1) expression in the hepatic macrophages (Kupffer cells). Under these conditions, MT protein as well as the mRNAs of MT-I and MT-II, were dose-dependently induced in the liver by TCDD doses from 1.0 microg/kg bw. TCDD-induced MT was found to localize in the parenchymal cells of the liver. Serum concentrations of cytokines (TNF-alpha, IL-1beta and IL-6) were not affected by TCDD. The hepatic concentrations of Cu, Zn and Fe were all increased significantly by TCDD administration. Our results suggest that MT levels are increased in the liver upon exposure to TCDD, perhaps by TCDD-generated reactive oxygen species, and that it may play a protective role in TCDD-induced oxidative stress responses as an antioxidant.

8-Hydroxy-2'-Deoxyguanosine↗

Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-derived dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells in the immune system. Gene transduction of DC with tumor-associated antigen (TAA) or other genes that enhance the immune reaction has been considered theoretically useful for DC-based immunotherapy. However, gene transduction of DC generated from human peripheral blood monocytes has been difficult due to its low efficiency, even when adenoviral vector was used at high multiplicity of infection (MOI). In the present study, we examined the effect of centrifugal force to enhance efficiency of adenovirus-mediated gene transduction into human monocyte-derived DC at various rotor speeds at various temperatures for various times. We judged the transduction efficiency using enhanced green fluorescence protein (EGFP)-expressing adenoviral vector, and the best condition for centrifugal transduction was determined as 2000 x g at 37 degrees C for 2 h at an MOI of 10 or greater. At an MOI of 50 without centrifugation, the gene transduction efficiency was about 66% and mean fluorescence intensity (MFI) of EGFP expression was about 150 (at 37 degrees C for 2 h). With centrifugal transduction (2000 x g at an MOI of 50 at 37 degrees C for 2 h), 86% or more DC were gene-modified, and especially, MFI of EGFP expression was highly enhanced (MFI: about 3100 or greater). Centrifugally gene-transduced DC were not damaged and were thoroughly functional as measured by mixed lymphocyte reaction (MLR). The centrifugal method was also applicable to human monocytes and K562 cells. The centrifugal transduction method with adenoviral vector might be helpful for the generation of gene-modified DC.

Adenoviridae↗

Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust.

Diesel exhaust (DE) has been recognized as a noxious mutagen and/or carcinogen, because its components can form DNA adducts. Mechanisms governing the susceptibility to DE and the efficiency of such DNA adduct formation require clarification. The transcription factor Nrf2 is essential for inducible and/or constitutive expression of a group of detoxification and antioxidant enzymes, and we hypothesized that the nrf2 gene knockout mouse might serve as an excellent model system for analyzing DE toxicity. To address this hypothesis, lungs from nrf2(-/-) and nrf2(+/-) mice were examined for the production of xenobiotic-DNA adducts after exposure to DE (3 mg/m(3) suspended particulate matter) for 4 weeks. Whereas the relative adduct levels (RAL) were significantly increased in the lungs of both nrf2(+/-) and nrf2(-/-) mice upon exposure to DE, the increase of RAL in the lungs from nrf2(-/-) mice exposed to DE were approximately 2.3-fold higher than that of nrf2(+/-) mice exposed to DE. In contrast, cytochrome P4501A1 mRNA levels in the nrf2(-/-) mouse lungs were similar to those in the nrf2(+/-) mouse lungs even after exposure to DE, suggesting that suppressed activity of phase II drug-metabolizing enzymes is important in giving rise to the increased level of DNA adducts in the Nrf2-null mutant mouse subjected to DE. Importantly, severe hyperplasia and accumulation of the oxidative DNA adduct 8-hydroxydeoxyguanosine were observed in the bronchial epidermis of nrf2(-/-) mice following DE exposure. These results demonstrate the increased susceptibility of the nrf2 germ line mutant mouse to DE exposure and indicate the nrf2 gene knockout mouse may represent a valuable model for the assessment of respiratory DE toxicity.

Animals↗