Search PubMed⌕ Search

Biomedical subjects

T Flotte

Publications and source records attributed to T Flotte.

At least 19 recordsLinked to original sources
PubMed2004

Immunity to adeno-associated virus serotype 2 delivered transgenes imparted by genetic predisposition to autoimmunity.

Adeno-associated virus (AAV) is widely considered a promising vector for therapeutic gene delivery. This promise is based on previous studies assessing AAVs safety and toxicity, ability to infect nondividing cells, elicit a limited immune response and provide long-term gene expression. However, we now find that earlier studies underappreciated the degree of AAV immunogenicity as well as the extent to which genetic background, through regulation of immune responsiveness, influences the duration of gene expression and thereby the effectiveness of AAV-mediated gene therapy. We evaluated antibody responses in 12 mouse strains to AAV serotype 2 (AAV2) and AAV2-expressed transgene products including green fluorescent protein (GFP), human alpha1-antitrypsin and murine interleukin-10. As expected, all immunocompetent mice administered AAV2 developed serologic evidence of immune responsiveness to the virus. However, a previously unidentified serologic prozone effect was observed suggesting that the concentrations of anti-AAV2 antibodies may have historically been subject to marked underestimation. Furthermore, strains with genetic predisposition to autoimmunity (eg, NOD, NZW, MRL-lpr) specifically imparted a functionally deleterious immune response to AAV-delivered transgene products. These findings suggest that more thorough studies of anti-AAV immunity should be performed, and that genetic predisposition to autoimmunity should be considered when assessing AAV efficacy and safety in humans.

Animals↗
PubMed2001

Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors.

Gene therapy using recombinant adeno-associated virus vectors (rAAV) is generally considered safe. During the course of a study designed to determine the long-term efficacy of rAAV-mediated gene therapy initiated in newborn mice with the lysosomal storage disease, mucopolysaccharidosis type VII (MPSVII), a significant incidence of hepatocellular carcinomas and angiosarcomas was discovered. A hepatocellular carcinoma was first detected in a 35-week-old mouse and by 72 weeks of age, three out of five rAAV-treated MPSVII mice had similar lesions. These types of tumors had not been seen previously in long-term studies of MPSVII mice using recombinant enzyme or bone marrow transplantation. In an attempt to ascertain whether mouse strain or GUSB expression confers susceptibility to tumor formation, we histopathologically examined untreated normal mice of the same strain, untreated MPSVII mice, and normal mice overexpressing human GUSB for the presence of tumors and increased hepatocyte replication. The results of these studies do not indicate that MPSVII mice or mice overexpressing human GUSB are susceptible to tumor formation; however, the number of animals examined is too small to draw definitive conclusions. Results from quantitative PCR performed on the tumor samples suggest that the tumors are probably not caused by an insertional mutagenesis event followed by the clonal expansion of a transformed cell. In a separate study, a relatively large group of mice injected with varying doses and types of rAAV vectors had no evidence of hepatic or vascular tumors. Although the mechanism of tumor formation is currently unknown, the tumorigenic potential of rAAV vectors must be rigorously determined in long-term in vivo studies.

Animals↗
PubMed2001

Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology.

Precise removal of nonmelanoma cancers with minimum damage to the surrounding normal skin is guided by the histopathologic examination of each excision during Mohs micrographic surgery. The preparation of frozen histopathology sections typically requires 20-45 min per excision. Real-time confocal reflectance microscopy offers an imaging method potentially to avoid frozen histopathology and prepare noninvasive (optical) sections within 5 min. Skin excisions ( approximately 1 mm thick) from Mohs surgeries were washed with 5% acetic acid and imaged with a confocal cross-polarized microscope. The confocal images were compared with the corresponding histopathology. Acetic acid causes compaction of chromatin that increases light back-scatter and makes the nuclei bright and easily detectable. Crossed-polarization strongly enhances the contrast of the nuclei because the compacted chromatin depolarizes the illumination light whereas the surrounding cytoplasm and normal dermis does not. Fast low-resolution examination of cancer lobules in wide fields of view followed by high-resolution inspection of nuclear morphology in small fields of view is possible; this is similar to the procedure for examining histopathology sections. Both the Mohs surgeon and the patient will potentially save several hours per day in the operating room. Fast confocal reflectance microscopic examination of excisions (of any thickness) may improve the management of surgical pathology and guide microsurgery of any human tissue.

Acetic Acid↗
PubMed2001

Adeno-associated virus (AAV) as a vehicle for therapeutic gene delivery: improvements in vector design and viral production enhance potential to prolong graft survival in pancreatic islet cell transplantation for the reversal of type 1 diabetes.

Most viral gene delivery syslems utilized to date have demonstrated significant limitations in practicality and safety due to the level and duration of recombinant transgene expression as well as their induction of host immunogenicity to vector proteins. Recombinant adeno-associated virus (rAAV) vectors appear to offer a vehicle for safe, long-term therapeutic gene transfer; factors afforded through the propensity of rAAV to establish long-term latency without deleterious effects on the host cell and the relative non-immunogenicity of the virus or viral expressed transgenes. The principal historical limitation of this vector system, efficiency of rAAV-mediated transduction, has recently observed a dramatic increase as the titer, purity, and production capacity of rAAV preparations have improved. In terms of systems that could benefit from such improvements, rAAV gene therapy to enhance solid organ transplantation would appear an obvious choice with islet transplantation forming a promising candidate due to the ability to perform viral transductions ex vivo. Currently, islet transplantation can be used to treat type 1 diabetes yet persisting alloimmune and autoimmune responses represent major obstacles to the clinical success for this procedure. The delivery of transgenes capable of interfering with antigenic recognition and/or cell death [e.g., Fas ligand (FasL), Bcl-2, Bcl-XL] as well as imparting tolerance/immunoregulation [e.g., interleukin(IL)-4, IL-10, transforming growth factor (TGF)-beta], or cytoprotection [e.g., heme oxygenase-1 (HO-1), catalase, manganese superoxide dismutase (MnSOD)] may prevent recurrent type 1 diabetes in islet transplantation and offer a promising form of immunotherapy. Research investigations utilizing such systems may also provide information vital to understanding the immunoregulatory mechanisms critical to the development of both alloimmune and autoimmune islet cell rejection mechanisms and recurrent type 1 diabetes.

Animals↗
PubMed2001

Efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV-delivered transgenes did not interfere with islet cell insulin production and were expressed in both beta- and non-beta-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance longterm graft survival.

Adenoviridae↗
PubMed2000

Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Alanine↗
PubMed2000

Pharmacokinetics and pharmacodynamic effects of nicotine nasal spray devices on cardiovascular and pulmonary function.

BACKGROUND: A nasal spray form of nicotine replacement therapy (Nicotrol NS, McNeil Consumer Products Co, Fort Washington, Pa) has been approved and, because of its rapid absorption across the nasal mucosa, may be more effective than nicotine gum or transdermal patches. We tested the hypothesis that the nicotine absorbed into the nasal mucosa would produce significant changes in hemodynamics and pulmonary function in 20 healthy, nonsmoking men and women. METHODS: In this double-blind, randomized study of Nicotrol NS versus placebo, we measured serum nicotine concentrations, blood pressure, heart rate, and indices of pulmonary function at timed intervals before and after nasal spray administration of 3 mg of nicotine. RESULTS: A peak serum nicotine concentration of 4.71 +/- 3.16 ng/mL occurred 10 minutes after drug administration. The maximum change in systolic blood pressure occurred 5 minutes after dosing and was significantly related to nicotine administration (7.1 +/- 9.4% for the nicotine group vs -1.6 +/- 7.3% for the placebo; P = 0.03). In contrast, neither diastolic blood pressure (P = 0.8) nor heart rate (P = 0.07) changed significantly after nicotine administration, when compared with placebo. Pulmonary function was not altered acutely by a single inhalation of nicotine. Pharmacokinetic modeling revealed a classic one-compartment model in which nicotine is absorbed into the systemic circulation by a zero-order process and eliminated by a first-order process. CONCLUSIONS: In this population of nonsmokers, hemodynamic effects of the nicotine nasal spray were observed shortly after administration and before the peak serum nicotine concentration.

Administration, Intranasal↗
PubMed1998

Influence of light delivery on photodynamic synovectomy in an antigen-induced arthritis model for rheumatoid arthritis.

BACKGROUND AND OBJECTIVE: Minimally invasive synovectomy techniques have been unsuccessful due to lack of selectivity. The purpose of this study was to evaluate the potential of photodynamic therapy to destroy diseased synovium in an antigen-induced arthritis model. STUDY DESIGN/MATERIALS AND METHODS: Three sets of experiments evaluated the biodistribution and treatment effects of Photofrin (PF) in rabbits with bilateral knee antigen-induced arthritis. The first set of experiments evaluated the biodistribution of PF in articular tissues of 30 rabbits from 6-72 hours after systemic injection of 2 mg/kg. In the second series of experiments, light was delivered to the knee joint via cleaved optical fibers, whereas for the third, light was delivered via a 600 microm diffusion tip fiber. Tissues were harvested at 2 and 4 weeks posttreatment. RESULTS: The biodistribution experiments demonstrated maximal uptake in inflamed synovium at 48 hours and a lack of uptake in normal tissues. With bare cleaved fibers, necrosis was observed in one specimen at 2 weeks and was absent in all specimens at 4 weeks. In the third experiment, synovial necrosis was observed in 3 of 7 specimens at 2 weeks and 3 of 8 at 4 weeks. No damage to articular cartilage or periarticular tissues was seen with either mode of light delivery. CONCLUSION: These studies indicate that selective destruction of synovium can be achieved with PF and suggest that optimization of light delivery techniques will play an important role in development of this new technique.

Analysis of Variance↗
PubMed1998

Hair growth cycle affects hair follicle destruction by ruby laser pulses.

It has been shown that normal mode ruby laser pulses (694 nm) are effective in selectively destroying brown or black pigmented hair follicles in adult Caucasians. This study investigated how the various stages of the hair follicle growth cycle influence follicle destruction by ruby laser treatment, using a model of predictable synchronous hair growth cycles in the infantile and adolescent mice. A range of ruby laser pulse fluences was delivered during different stages of the hair growth cycle, followed by histologic and gross observations of the injury and regrowth of hair. Actively growing and pigmented anagen stage hair follicles were sensitive to hair removal by normal mode ruby laser exposure, whereas catagen and telogen stage hair follicles were resistant to laser irradiation. Selective thermal injury to follicles was observed histologically, and hair regrowth was fluence dependent. In animals exposed during anagen, intermediate fluences induced nonscarring alopecia, whereas high fluences induced scarring alopecia. The findings of this study suggest treatment strategies for optimal laser hair removal.

Alopecia↗
PubMed1996

Damage to hair follicles by normal-mode ruby laser pulses.

BACKGROUND: Although many temporary treatments exist for hirsutism and hypertrichosis, a practical and permanent hair removal treatment is needed. OBJECTIVE: Our purpose was to study the use of normal-mode ruby laser pulses (694 nm, 270 microseconds, 6 mm beam diameter) for hair follicle destruction by selective photothermolysis. METHODS: Histologically assessed damage in ex vivo black-haired dog skin after the use of different laser fluences was used to design a human study; 13 volunteers with brown or black hair were exposed to normal-mode ruby laser pulses at fluences of 30 to 60 J/cm2, delivered to both shaved and wax-epilated skin sites. An optical delivery device designed to maximize light delivery to the reticular dermis was used. Hair regrowth was assessed at 1, 3, and 6 months after exposure by counting terminal hairs. RESULTS: Fluence-dependent selective thermal injury to follicles was observed histologically. There was a significant delay in hair growth in all subjects at all laser-treated sites compared with the unexposed shaven and epilated control sites. At 6 months, there was significant hair loss only in the areas shaved before treatment at the highest fluence. At 6 months, four subjects had less than 50% regrowth, two of whom showed no change between 3 and 6 months. Transient pigmentary changes were observed; there was no scarring. CONCLUSION: Selective photothermolysis of hair follicles with the normal-mode ruby laser produces a growth delay consistent with induction of prolonged telogen with apparently permanent hair removal in some cases.

Animals↗
PubMed1996

Flow cytometric DNA ploidy and cells phase fractions in recurrent human pituitary adenomas. A correlative study of flow cytometric analysis and the expression of proliferating cell nuclear antigen.

Flow cytometric analysis was applied to embedded tissue to measure the proliferative activity and the DNA ploidy of 16 recurrent and 17 nonrecurrent pituitary adenomas. The results were compared with data from a previous study which demonstrated that proliferating cell nuclear antigen (PCNA) labeling index was higher in recurrent adenomas than in nonrecurrent adenomas. Flow cytometric analysis as a tool for predicting aggressive behavior has been useful in a variety of human tumors; however, its prognostic value in pituitary adenomas is controversial. Therefore, we decided to explore the relationship of the results of flow cytometry and proliferating cell nuclear antigen labeling indices with the prognosis of pituitary adenomas. Three out of 16 recurrent adenomas and five out of 17 nonrecurrent adenomas demonstrated a DNA aneuploid pattern. All the nonfunctional recurrent adenomas had a diploid pattern, while only 40% of the functional recurrent adenomas had a diploid pattern. The GO/G1 phase fraction was higher in the recurrent adenomas, than in the nonrecurrent ones (p = 0.0005). In contrast, the S-phase fraction and the coefficient of variation were higher in the nonrecurrent adenomas (5.9 +/- 1.0%, 7.0 +/- 0.75, respectively) than in the recurrent ones (2.5 +/- 0.6%, 4.0 +/- 0.2%, respectively) (p = 0.003 and p = 0.001, respectively). The proliferating cell nuclear antigen labeling indices were higher in the recurrent adenomas (18.9 +/- 4.5%) than in the nonrecurrent adenomas (2.6 +/- 1.6%) (p = 0.003). The S-phase of flow cytometry correlated weakly with the proliferating cell nuclear antigen labeling indices when the recurrent and the nonrecurrent adenomas were considered as one group. (r = -0.356, p = 0.033). But no significant correlations were observed when the groups of recurrent (r = -0.311, p = 0.195) and nonrecurrent tumors (r = -0.019, p = 0.942) were compared separately. The results of flow cytometric analysis suggest that recurrent adenomas may have a higher proportion of cells in the presynthetic phase than the nonrecurrent adenomas. This study suggests that flow cytometric analysis is of limited value in predicting recurrence of pituitary adenomas.

Adenoma↗
PubMed1995

In vivo uptake of liposomal benzoporphyrin derivative and photothrombosis in experimental corneal neovascularization.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) has been used successfully to occlude neovascularizations experimentally. We evaluated the vasoocclusive potential of benzoporphyrin derivative (BPD), a new photosensitizer currently in clinical trials. Since liposomally formulated BPD strongly binds to endogenous low density lipoproteins (LDL) after i.v. injection, LDL act as carrier to deliver BPD preferentially to proliferating endothelial cells. STUDY DESIGN/MATERIALS AND METHODS: Corneal neovascularizations in rabbits were used as model. Time-dependent uptake and retention of liposomal BPD were measured in vivo by monitoring the laser-induced fluorescence (LIF). Photothrombosis was induced using a dye laser emitting at 692 nm. RESULTS: A maximal BPD concentration was measured at 60-90 minutes postinjection determining the optimal time interval for treatment. Exposures as low as 10 J/cm2 allowed complete and irreversible neovascular occlusion as documented angiographically. Histology revealed selective endothelial damage, adjacent corneal stroma, or iris vessels, remained intact. Identical results were obtained using BPD directly complexed with LDL suggesting use of a LDL-mediated pathway. CONCLUSION: We suggest BPD-PDT for a selective treatment of neovascular diseases.

Animals↗
PubMed1995

Acute and chronic response of articular cartilage to holmium:YAG laser irradiation.

A Ho:YAG (holmium-yttrium-aluminum-garnet) laser system operating at a wavelength of 2.1 microns has been introduced recently for use in arthroscopic surgery. The acceptability of this new tool will be determined not only by its ability to resect tissue, but also by its long-term effects on articular surfaces. Two studies were performed to evaluate the acute and chronic effects of the laser on cartilaginous tissue. The acute in vitro effects of 2.1-micron laser irradiation were evaluated, including measurement of ablation efficiency, ablation threshold, and thermal damage in meniscal and articular cartilage. To document the chronic effects on articular cartilage in vivo, a 10-week healing study was performed. Eight sheep weighing 30 to 40 kg underwent bilateral arthrotomy procedures. Multiple full-thickness and partial-thickness defects were created. Animals were euthanized at 0, 2, 4, and 10 weeks. The results of the healing study showed (1) no healing of full- or partial-thickness defects in hyaline cartilage at 10 weeks; (2) fibrocartilaginous granulation tissue filling full-thickness defects at 2 and 4 weeks that was no longer evident at 10 weeks; (3) chondrocyte necrosis extending to > 900 microns distal to ablation craters at 4 weeks, with no evidence of repair at later dates; (4) chondrocyte hyperplasia at the borders of the damage zone at 2 weeks but not at euthanization occurring at later periods.

Animals↗
PubMed1995

Biodistribution and phototoxicity of 5-aminolevulinic acid-induced PpIX in an orthotopic rat bladder tumor model.

5-Aminolevulinic acid (ALA) is a precursor of heme biosynthesis. In the penultimate step of this biosynthesis, protoporphyrin IX (PpIX), an effective photosensitizer, is generated. In this study, the biodistribution and photodynamic effect of ALA-induced PpIX were investigated in an orthotopic rat bladder tumor model. A quantitative comparison of PpIX biodistribution by extraction and fluorescence spectroscopy following intravenous and intravesical administration of ALA was made. The tumor to normal bladder wall ratio was 2:1 at 4 hours for both delivery modes. Fluorescence microscopy demonstrated predominantly cellular rather than stromal PpIX fluorescence. Phototoxicity, evaluated 4 hours after ALA administration, was light dose-dependent with the most efficient tumor necrosis being observed upon 150 J/cm.2 of 630 nm. irradiation. These data suggest that optimized photodynamic therapy with ALA-induced PpIX may be an alternative treatment for superficial bladder carcinoma.

Aminolevulinic Acid↗
PubMed1994

Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents.

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

[Selective vascular occlusion by repetitive short laser pulse].

The occlusion of arterioles and venules (30 microns in diameter) by means of repeated dye laser pulses (577 nm, 160 microseconds, 0.5 Hz) was studied in the hamster cheek pouch model. Microscopically visible changes during and after irradiation were recorded and monitored by a video system. The energy necessary per pulse for vessel occlusion with a single pulse (arterioles 5 J/cm2, venules 3.5 J/cm2) can be lowered more than 50% with 100 pulses (1.8 J/cm2, 1.2 J/cm2). Rupture of vessels, which regularly occurs with a single pulse, can be totally avoided with 100 repetitive laser pulses. Investigations of the temperature dependence of the occlusion rate of arterioles showed different interaction mechanisms with one pulse and 100 pulses (mechanical and thermal respectively). Light and electron microscopic investigations supported this concept of selective occlusion using repeated short laser pulses. Possible clinical applications are portwine stains of the eyelid, neovascularisation on the cornea and subretinal neovascularisation.

Animals↗