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PubMed1998

Solitary fibrous tumour: significance of p53 and CD34 immunoreactivity in its malignant transformation.

AIMS: To clarify the association of p53 and CD34 expression with development of malignant solitary fibrous tumour we have studied 10 cases of solitary fibrous tumour arising in the pleura, retroperitoneum and pelvic cavity with clinicopathological features of malignancy. METHODS AND RESULTS: Tumours were localized solid masses with or without necrosis in eight and they nearly totally occupied the pleural cavity in two. Basic histology of the tumours was the proliferation of spindle cells arranged in 'patternless' pattern or in interlacing bundles with nuclear atypia and mitotic activities of various degree. In two, high-grade foci were present within low or intermediate-grade tumours. Recurrent tumours also showed more atypical features than primary tumours in two. Immunohistochemical studies showed CD34 positivity in seven, but three of them showed marked diminution or complete loss of CD34 expression in high-grade foci or a recurrent tumour. Three high-grade cases showed totally negative staining for CD34. p53 was strongly expressed in cases with fatal outcome, clinical recurrence, nuclear atypia, high mitotic activity or local invasion, whereas almost negative in benign tumours. CONCLUSIONS: Malignant solitary fibrous tumours may occur de novo or by transformation within benign or low-grade tumours and may be associated with p53 mutation. Although CD34 is a useful marker in the diagnosis of solitary fibrous tumour, one should bear in mind that its expression can be lost in high-grade tumours.

Adult↗
PubMed2002

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors.

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.

Adenoma↗
PubMed1995

Beta 2-microglobulin and HLA-DR expression in relation to the presence of Epstein-Barr virus in nasopharyngeal carcinomas.

We have investigated the immunohistochemical expression of beta 2-microglobulin and HLA-DR proteins in nasopharyngeal carcinoma (NPC) in relation to the expression of the Epstein-Barr virus (EBV)-encoded EBER 1-2 mRNAs and LMP-1 protein. beta 2-Microglobulin is expressed in association with MHC-I molecules on most nucleated cells and HLA-DR belongs to the MHC-II molecules which are expressed mostly on antigen-presenting cells. Formalin-fixed paraffin-embedded tissue from 37 NPC cases was stained with immunohistochemistry for beta 2-microglobulin, HLA-DR and LMP-1 proteins and by RNA in situ hybridization for EBER 1-2 mRNAs. beta 2-Microglobulin-positive staining was found in neoplastic cells in all cases. In 19/37 NPC cases the positive staining was found in most neoplastic cells. In the remaining 18 NPC cases more than 25% of the neoplastic cells showed significantly reduced or negative staining in comparison to the normal epithelium and infiltrating small lymphocytes. HLA-DR-positive staining was found in 27/37 NPC cases. EBER 1-2 transcripts were deteced in neoplastic cells in 13/37 NPC cases. LMP-1 expression in tumour cells was found in 4/13 EBER1-2-positive cases. No correlation was found between the presence of EBER1-2 transcripts or LMP-1 protein and the beta 2-microglobulin-reduced expression in NPC. Thus, EBV does not seem to use downregulation of MHC-I to avoid the T cell cytotoxic immune response in NPC. HLA-DR expression was observed in all 13 EBV-positive cases of NPC, suggesting that EBV may participate in the induction of HLA-DR expression in a proportion of NPC.

Adult↗
PubMed1992

Thrombomodulin expression in malignant pleural mesothelioma and pulmonary adenocarcinoma.

Thrombomodulin (TM) is a glycoprotein of molecular weight 75,000 kd that is normally present in restricted numbers of cells, including endothelial and mesothelial cells. In this study, the authors tested the possibility of using anti-TM to facilitate the diagnosis of mesothelioma. All of the 31 mesotheliomas and the two mesothelioma cell lines (MS-1 and MS-2) tested were stained positively with anti-TM. The specificity of anti-TM staining in mesothelioma cells was further confirmed by in situ hybridization of MS-1 cells with a TM-specific probe. The expression of TM in MS-1 cells was increased markedly when these cells were induced by 12-0-tetradecanyl phorbol 13-acetate (TPA) to differentiate. The expression of TM in mesothelioma cells, however, did not correlate with any particular phase of the cell cycle. In an attempt to differentiate pleural mesothelioma from pulmonary adenocarcinoma, the authors compared the expression of TM, carcinoembryonic antigen (CEA), and Leu M1 in these two types of tumors. Only four of 48 (8%) pulmonary adenocarcinomas were stained positively by antibodies to TM. Therefore, immunohistochemical staining with antibodies to TM yielded 100% sensitivity and 92% specificity for diagnosis of mesothelioma. All of the mesotheliomas stained negatively for CEA and Leu M1, except for one, which showed minimal focal positivity for Leu M1. In contrast, 79% and 60% of adenocarcinomas stained positively for CEA and Leu M1, respectively. These findings suggest that immunocytochemical staining with anti-TM should be added to the battery of tests to increase the diagnostic sensitivity and specificity for differentiating mesothelioma from pulmonary adenocarcinoma.

Adenocarcinoma↗
PubMed1988

Immunofluorescent localization of cytokeratin intermediate filaments as a means of defining the presence of recurrent basal cell carcinoma.

Intermediate filament subunits are reliable markers of cytogenetic origin for both normal and neoplastic cells. Immunohistochemical localization of cytokeratin filaments offers a sensitive and specific means of identifying basal or reserve cells when studying histologic sections of skin biopsy specimens. We have applied this technique on eighteen cases in which unequivocal diagnosis or recurrent basal cell carcinoma could not be rendered by conventional histologic techniques. In three of the cases studied, microscopic islands of basal cell carcinoma could be demonstrated by positive staining with cytokeratin antibodies. In the remaining fifteen cases, the possibility of recurrent basal cell carcinoma could be conclusively eliminated on the basis of negative staining with this antibody. Immunolabeling with tissue specific cytokeratin antibodies by indirect immunofluorescent examination may thus constitute a reliable and relatively simple technique that may serve to establish a definitive diagnosis in equivocal cases of suspected recurrent basal cell carcinoma.

Biomarkers, Tumor↗
PubMed2004

Evaluation of P-glycoprotein, HER-2/ErbB-2, p53, and Bcl-2 in primary tumor and metachronous lung metastases in patients with high-grade osteosarcoma.

BACKGROUND: Investigation of the relation between primary tumor and metastatic disease is necessary for the identification of predictive factors for postrecurrence survival (PRS) in patients with recurrent osteosarcoma. METHODS: Cellular levels of P-glycoprotein, ErbB-2, p53, and Bcl-2 expression were evaluated in primary tumor biopsy and metachronous pulmonary metastasis specimens from 19 patients with high-grade osteosarcoma. Results were analyzed for differences between primary tumor and pulmonary metastases and for correlations between expression patterns and survival. RESULTS: Positive staining in lung metastases was noted in 68%, 53%, 32%, and 84% of patients for P-glycoprotein, ErbB-2, p53, and Bcl-2, respectively. These percentages were higher than those observed in primary tumor specimens for all genetic markers evaluated, with a significant difference in the percentage of patients with positive staining for P-glycoprotein (68% vs. 32%; P = 0.05) and a near-significant difference in the percentage of patients with positive staining for Bcl-2 (84% vs. 53%; P = 0.08). Patients with ErbB-2 expression in the primary tumor were more likely to have multiple metastases and shorter recurrence-free intervals compared with patients in whom ErbB-2 expression was not observed, whereas differences in P-glycoprotein, p53, and Bcl-2 expression were not related to differences in metastatic pattern. PRS was influenced by p53 expression levels in pulmonary metastases, with patients who had negative staining for p53 having a significantly better PRS rate relative to patients with positive staining for p53 (3-year PRS rate: p53-negative, 64%; p53-positive, 17%; P = 0.008). CONCLUSIONS: In the current study of patients with high-grade osteosarcoma, most patients exhibited increased cellular expression of P-glycoprotein, ErbB-2, and Bcl-2 in recurrent pulmonary metastases compared with primary tumor. Further studies aimed at investigating the relation between altered p53 expression in lung metastases and postrecurrence survival are recommended.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Immunohistochemical detection of steroid binding in rabbit cornea.

An indirect immunoperoxidase technique was used to demonstrate progesterone binding in rabbit cornea as a measure of steroid absorption into corneal tissues. Tissue preparation prior to immune staining was prepared by both a pre-fixed, paraffin-embedding method and a post-fixed, cryostat sectioning method. Rabbit antiprogesterone antiserum and peroxidase conjugated goat antirabbit antiserum were used as a primary and secondary antibody respectively. The immune staining of the antibody-treated sections was then developed in a solution of 3-3' diaminobenzidine-hydrogen peroxide system. The resulting, brown colored, end-products at the site of reaction indicate the location of bound steroid in the sections. Paraffin-embedded sections showed negative staining in both steroid treated and untreated control corneas while fixed frozen sections showed only positive staining for the treated corneas. The results suggest the preservation of the immunogenicity in the treated frozen sections. The negative result observed in the paraffin-embedded sections may be attributed to the loss of steroid from the tissues during the dehydration and paraffin embedding processes. It was found that the immunoreactive progesterone localized mainly in the epithelial cells and the bottom portion of the stroma. Prior application with an excess of triamcinolone acetonide, to the treated corneas gave negative results indicating the reversible and competitive nature of the binding.

Animals↗
PubMed2001

Correlation of vascular endothelial growth factor expression with fibroblast growth factor-8 expression and clinico-pathologic parameters in human prostate cancer.

Vascular endothelial growth factor (VEGF) mediates neo-angiogenesis during tumour progression and is known to cooperate with the fibroblast growth factor (FGF) system to facilitate angiogenesis in a synergistic manner. In view of this, we have investigated VEGF expression in 67 cases of prostate cancer previously characterized for fibroblast growth factor-8 (FGF-8) expression. Cytoplasmic VEGF staining was detected in malignant cells in 45 out of 67 cases. Cytoplasmic staining was found in adjacent stromal cells in 32 cases, being particularly strong around nests of invasive tumour. Positive VEGF immunoreactivity in benign glands was restricted to basal epithelium. A significant association was observed between tumour VEGF and FGF-8 expression (P = 0.004). We identified increased VEGF immunoreactivity in both malignant epithelium and adjacent stroma and both were found to be significantly associated with high tumour stage (P = 0.0047 and P = 0.0002, respectively). VEGF expression also correlated with increased serum PSA levels (P = 0.01). Among positively stained tumours, VEGF expression showed a significant association with Gleason score (P = 0.04). Cases showing positive VEGF immunoreactivity in the stroma had a significantly reduced survival rate compared to those with negative staining (P = 0.037). Cases with tumours expressing both FGF-8 in the malignant epithelium and VEGF in the adjacent stroma had a significantly worse survival rate than those with tumours negative for both, or only expressing one of the two growth factors (P = 0.029). Cox multivariate regression analysis of survival demonstrated that stromal VEGF and tumour stage were the most significant independent predictors of survival. In conclusion, we report for the first time a correlation of both tumour and stromal VEGF expression in prostate cancer with clinical parameters as well as its correlation to FGF-8 expression.

Aged↗
PubMed1997

Significance of tumor vascularity as a predictor of long-term prognosis in patients with small hepatocellular carcinoma treated by percutaneous ethanol injection therapy.

BACKGROUND/AIMS: We estimated the significance of the vascularity of small hepatocellular carcinoma (HCC) as a predictor of long-term prognosis in patients treated with percutaneous ethanol injection therapy (PEIT/PEI). METHODS: Fifty-four patients who have been followed-up in our hospital and who had HCC less than 20 mm in diameter were observed for 199 to 2074 days. Hepatic angiography (digital subtraction angiography; DSA and ultrasound angiography with intraarterial CO2 microbubbles; USAG) was performed before treatment in all cases, and the vascularity of the tumor was clinically evaluated. The survival rate was analyzed according to this vascularity. RESULTS: Of the 54 tumors, 24 had tumor stain on DSA, while 30 did not, and 38 showed enhancement on USAG, while 16 did not. The 3- and 5-year survival rates were 48.7 and 34.1% and 89.7 and 69.7% of patients with negative staining HCC (p = 0.0723). The rates were 48.6 and 36.7%, respectively, of patients with positive enhancement HCC on USAG, and both rates were 85.7% of patients with negative enhancement HCC (p = 0.0231). CONCLUSIONS: Tumor vascularity will play a role in the long-term prognosis of these patients with small HCC when they are treated with PEIT/PEI.

Administration, Cutaneous↗
PubMed1996

p53 gene alterations and p53 protein in oral epithelial dysplasia and squamous cell carcinoma.

To examine the expression of p53 protein and gene alterations in oral epithelial lesions including epithelial dysplasias and primary squamous cell carcinomas, immunohistochemical and temperature gradient gel electrophoresis (TGGE) methods were applied to formalin-fixed and paraffin-embedded tissues. Morphologically normal mucosal epithelium stained negatively for p53 protein. Three out of 11 (27.3 per cent) epithelial dysplasias and 19 out of 57 (33.3 per cent) primary squamous cell carcinomas stained positively for p53 protein. Although more than half of the cases were positive for p53 protein in stage I, the positive cancer cases were found at other stages with variable frequency. Immunoreactive products were localized in the nucleus, especially in the basal and suprabasal layers. The analysis by TGGE revealed gene alterations in exons 5-8 in 3 out of 3 epithelial dysplasias and 17 out of 19 (89.5 per cent) primary squamous cell carcinomas which were immunohistochemically positive for p53 protein. These results suggest that p53 gene mutation may be involved in carcinogenesis in the oral squamous epithelium even in the early stage of the dysplasia-carcinoma sequence.

Adult↗
PubMed2004

Expression and distribution of c-kit receptor and its ligand in human CNS germ cell tumors: a useful histological marker for the diagnosis of germinoma.

We previously reported the expression of protooncogene c-kit in CNS germ cell tumors and suggested that the soluble form of c-kit (s-kit) may represent a specific clinical marker for germinoma-containing tumors. Here we investigated the expression of stem cell factor (SCF), a specific ligand of c-kit, in CNS germ cell tumor samples from 16 patients, using immunohistochemical methods to assay the expression of c-kit and SCF protein. The immunostaining patterns of c-kit and SCF were almost identical. In all germinoma-containing tumors, c-kit and SCF were diffusely expressed on the surface of germinoma cells; lymphocytes and interstitial cells were negatively stained. In immature teratomas, only some mature components, e.g., cartilage and gland, were immunoreactive for c-kit and SCF. Syncytiotrophoblastic giant cells (STGCs) were negative for both SCF and c-kit, suggesting that germinoma cells primarily coexpress SCF and c-kit. The coexpression of c-kit and SCF may be an important immunohistochemical marker for the diagnosis of CNS germinoma, and the SCF/c-kit pathway may be an alternative molecular target for the treatment of human CNS germinomas.

Biomarkers, Tumor↗
PubMed1993

Localization of cytomegalovirus antigens in liver allografts over time.

Cytomegalovirus (CMV) hepatitis is a common and serious complication of orthotopic liver transplantation. Immunohistochemical studies are the most sensitive methods of diagnosis. We compared immunoperoxidase staining with monoclonal antibodies to CMV immediate early and early antigens with routine hematoxylin-eosin stain. Eleven of 140 liver allograft recipients at our institution had CMV hepatitis identified by hematoxylin-eosin stain on biopsy specimens. We stained serial sections of all previous biopsy specimens and one post-ganciclovir biopsy specimen (when available) from each of these patients. One or both monoclonal antibodies confirmed the original hematoxylin-eosin stain diagnoses. Cytomegalovirus was detected in earlier, hematoxylin-eosin stain-negative biopsy specimens in seven of 11 patients. Detection of immediate early antigen often preceded that of early antigen. Earlier biopsy specimens demonstrated less positive staining, which become more extensive closer in time to the hematoxylin-eosin stain-positive biopsy specimens. Sinusoidal cells became positive earlier than hepatocytes. In one patient occult CMV antigens persisted in biopsy specimens following ganciclovir treatment. We conclude that (1) immunohistochemical staining for CMV antigens can result in earlier detection of viral infection, which may lead to earlier, more effective treatment; (2) CMV infection and antigen expression is focal, requiring extensive examination for diagnosis; (3) extent of occult infection may indicate the extent of active infection in the organ as a whole; (4) most CMV hepatitis begins with infection of sinusoidal lining cells as a result of hematogenous spread from within the allograft or from systemic viremia; and (5) posttreatment biopsy specimens may be more sensitive than resolution of serum liver enzyme abnormalities in evaluating the success of ganciclovir therapy.

Antigens, Viral↗
PubMed1991

Morphological and biochemical evidence that scrapie-associated fibrils are derived from aggregated amyloid-like filaments.

The membrane fraction from scrapie infected mouse brains was dissolved in saturated urea, centrifuged on a 10 to 50% glycerol gradient at 35,000 rpm for 24 h, and fractionated from the bottom of the tube into 11 fractions. PrP was detected throughout the gradient. However, the relative PrP concentrations of fractions 4 and 8 were the highest. The relative PrP concentration versus protein concentration of fractions 1 to 4 was higher than that of the other fractions. Scrapie infectivity also was detected in all fractions. Fractions 2, 3, 4, 7, and 8 produced the shortest incubation periods. Positively stained filamentous aggregates with sizes varying from about 40 x 60 nm to more than 4 microns were observed in fractions 2 and 4 by negative staining. These resembled amyloid filaments. Congo red-stained aggregates showed birefringence under polarized light. Aggregation of the filamentous aggregates was observed by incubation with anti-mouse SAF serum. Fine fibrils 10-18 nm in width were partially dissociated from the aggregates by brief exposure to the detergent Sarkosyl. These facts suggest that SAF are not products of self-assembly from subunit structures liberated from membranes by exposure to detergent, but exist as aggregates of amyloid-like filaments from which SAF are dissociated by detergent extraction.

Animals↗
PubMed1978

Preparation and characterization of liposomes containing the Ca2+-activated photoprotein, obelin.

1. Liposomes bearing different net surface charges have been prepared and their ability to entrap the Ca2+-activated photoprotein, obelin, has been studied 2. Negatively-charged liposomes, composed of egg-yolk lecithin, cholesterol and phosphatidylserine, consistently produced the most homogeneous populations of liposomes after sonication, as shown by electron microscopy after negative staining. These consisted of a large proportion of uni- and bilamellar vesicles within the size range of 20--50 nm, external diameter. 3. Sonicated negatively-charged liposomes had a mean aqueous obelin space of 6.8 +/- 0.8 microliter/mumol of phospholipid compared to a mean space for inulin [14C]carboxylic acid of 5.1 +/- 0.8 microliter/mumol of phospholipid. 4. Sonication reduced the Ca2+ permeability of the negatively-charged liposomes, as measured by the utilization of entrapped obelin. 5. Preparations of uncharged (no phosphatidylserine) and positively-charged (stearylamine instead of phosphatidylserine), sonicated liposomes contained a greater proportion of larger vesicles, which were more permeable to Ca2+ than sonicated, negatively-charged liposomes. 6. Obelin, trapped within sonicated, negatively-charged liposomes, responded to increases in the free Ca2+ concentration within the liposomes caused by the bivalent-cation ionophore A23187 at concentrations as low as 19 nM. 7. The effect of A23187 was inhibited by Mg2+ at a low concentration of Ca2+ (10 muM), but not at 1 mM Ca2+. 8. It was concluded that obelin could be trapped in the aqueous compartment of sonicated liposomes which remained relatively impermeable to Ca2+. Furthermore, trapped obelin could respond to changes in the free Ca2+ concentration within these liposomes.

Calcimycin↗
PubMed1992

Prediction of doxorubicin resistance in gastrointestinal cancer by P-glycoprotein staining.

Feasibility of immunohistochemical staining of P-glycoprotein for the prediction of doxorubicin resistance in gastrointestinal cancers was examined. Among 10 cancer cell lines which consist of two gastric cancer cell lines and eight colon cancer cell lines, seven cell lines were stained positively by the monoclonal antibody to P-glycoprotein, C219. In consequence of the evaluation on the effect of doxorubicin on these tumour cells by means of succinic dehydrogenase inhibition test (SDI test), zero out of seven cell lines stained positively by C219 was sensitive to doxorubicin, but two out of three cell lines stained negatively were sensitive. Among 23 fresh surgical specimens of gastrointestinal cancers which consisted of 15 gastric cancers and eight colon cancers, seven tumour tissues were stained positively by C219. All P-glycoprotein positive tumours were resistant to doxorubicin. On the other hand, four of 16 P-glycoprotein tumours were sensitive to doxorubicin. These data indicate that positively stained cancer cells by C219 are resistant to doxorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

The role of early expression of inducible nitric oxide synthase in human breast cancer.

Nitric oxide synthases are expressed in breast cancer. To elucidate the clinical role of the inducible NOS (i-NOS) in human breast cancer, 161 primary breast cancer tissues were stained immunohistochemically. Staining patterns for i-NOS were correlated with classical prognostic factors such as lymph node status, age, hormonal receptor status, tumour size and tumour differentiation. With classical prognostic factors such as lymph node status, age, hormonal receptor status, tumour size and tumour differentiation. Patients survival was also analysed. Sixty-one percent of the tumours stained positively for i-NOS. Detection of i-NOS was positively correlated with increasing tumour size and decreasing tumour differentiation (P=0.018 and P=0.039, respectively). However, in the 50 year age group, i-NOS staining also correlated with lymph node status. Patients with i-NOS-positive breast carcinomas had a significantly worse overall survival rate versus those with negative stains (5-year survival rate 84.8% versus 67.1%; P=0.049; log-rank test). To date, this is the largest analysis of i-NOS expression in breast cancer patients and the only study to assess survival.

Adult↗
PubMed1982

Aluminum associated bone disease: clinico-pathologic correlation.

A chemical, aurine tricarboxylic acid was used to identify aluminum in bone in a group of chronic renal failure patients with severe osteomalacia. Thirteen out of 49 (26%) patients had a positive stain at the bone-osteoid junction. The presence of aluminum at the staining site was confirmed by electron dispersion x-ray analysis. The patients' biopsies staining negative with this clinical, although having high bone aluminum levels, had more significant features of hyperparathyroidism, both biochemically and histologically than those having a positive stain. The usefulness of this technique to diagnose presymptomatic aluminum related osteomalacia is emphasized.

Adolescent↗
PubMed2000

Immunohistochemical analysis of cytokeratin expression in reactive eccrine syringofibroadenoma-like lesion: a comparative study with eccrine syringofibroadenoma.

In addition to solitary eccrine syringofibroadenoma (ESFA), there is another type of ESFA which is associated with underlying dermatoses (reactive ESFA-like lesion). Five lesions in 4 patients of reactive ESFA-like lesion were analyzed by an immunohistochemical method using 13 kinds of anti-cytokeratin (CK) antibodies. Two cases of solitary ESFA were also studied by the same procedure for comparison. Suprabasal staining pattern of AE1 and MNF116, which stain CKs 6, 16 and 17, markers of hyperproliferative state, was observed diffusely in 5 lesions of reactive ESFA-like lesions except for focal negative staining in one case, and was observed focally in one case of solitary ESFA. Furthermore, differentiation-specific cytokeratin expression was reduced in 3 of 5 lesions of reactive ESFA-like lesions. Both ESFA and reactive ESFA showed basically similar immunoreactivity suggesting differentiation toward the dermal duct. The above slight difference in immunoreactivity between both lesions may be explained due to inflammatory infiltrates associated with underlying dermatoses.

Adenoma, Sweat Gland↗