Makoto Bonkobara

Molecular Cloning of a Dendritic Cell-associated Transmembrane Protein, DC-HIL, That Promotes RGD-dependent Adhesion of Endothelial Cells through Recognition of Heparan Sulfate Proteoglycans

We isolated a novel molecule (DC-HIL) expressed abundantly by the XS52 dendritic cell (DC) line and epidermal Langerhans cells, but minimally by other cell lines. DC-HIL is a type I transmembrane protein that contains a heparin-binding motif and an integrin-recognition motif, RGD, in its extracellular domain (ECD). A soluble fusion protein (DC-HIL-Fc) of the ECD and an immunoglobulin Fc bound to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated polysaccharides (e.g. heparin and fucoidan) inhibited binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an integrin inhibitor (RGDS tetramer) had no effect on binding to SVEC, but prevented adhesion of SVEC. This differential RGD requirement was confirmed by the finding that DC-HIL-Fc mutant lacking the RGD motif can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore, DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing heparan sulfate proteoglycans on endothelial cells, thereby inducing adhesion of SVEC in an RGD-dependent manner. We propose that DC-HIL serves as a DC-associated, heparan sulfate proteoglycan-dependent integrin ligand, which may be involved in transendothelial migration of DC.

Effect of Tyrosine Kinase Inhibition by Imatinib Mesylate on Mast Cell Tumors in Dogs

BACKGROUND Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT. HYPOTHESIS Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit. ANIMALS Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment. METHODS Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks. RESULTS Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission). CONCLUSIONS AND CLINICAL IMPORTANCE Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Expression of NF-κB in epidermis and the relationship between NF-κB activation and inhibition of keratinocyte growth

Summary Background Nuclear factor‐κB (NF‐κB) is a transcription factor involved in a number of signalling pathways in many cell types. NF‐κB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation.

Comparison of expression of glucokinase gene and activities of enzymes related to glucose metabolism in livers between dog and cat.

Plasma glucose and immunoreactive insulin (IRI) concentrations and activities of enzymes related to glucose metabolism in livers were measured in dogs and cats. Nucleotide sequences of the conserved region of glucokinase (GK) cDNA that contained ATP- and glucose-binding domains were determined in canine liver and feline pancreas for design of the species-specific oligonucleotide primers for reverse transcription–polymerase chain reaction (RT-PCR) analysis. There were no significant differences in plasma glucose and IRI concentrations between dogs and cats. In feline liver, although GK activities were not detected, activities of hexokinase, fructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, fructose-1,6-bisphosphatase and glucose-6-phosphatase were significantly higher than those in canine liver. The partial sequences of canine liver GK and feline pancreas GK cDNA were respectively 88% and 89% identical with the rat liver GK cDNA. Expression of GK gene was observed in canine liver and pancreas and feline pancreas with RT-PCR using species specific primers based on the cDNA sequences.

The DC-HIL/syndecan-4 pathway inhibits human allogeneic T cell responses

T‐cell activation is regulated by binding of ligands on APC to corresponding receptors on T cells. In mice, we discovered that binding of DC‐HIL on APC to syndecan‐4 (SD‐4) on activated T cells potently inhibits T‐cell activation. In humans, we now show that DC‐HIL also binds to SD‐4 on activated T cells through recognition of its heparinase‐sensitive saccharide moiety. DC‐HIL blocks anti‐CD3‐induced T‐cell responses, reducing secretion of pro‐inflammatory cytokines and blocking entry into the S phase of the cell cycle. Binding of DC‐HIL phosphorylates SD‐4s intracellular tyrosine and serine residues. Anti‐SD‐4 Ab mimics the ability of DC‐HIL to attenuate anti‐CD3 response more potently than Ab directed against other inhibitory receptors (CTLA‐4 or programmed cell death‐1). Among leukocytes, DC‐HIL is expressed highest by CD14+ monocytes and this expression can be upregulated markedly by TGF‐β. Among APC, DC‐HIL is expressed highest by epidermal Langerhans cells, an immature type of dendritic cells. Finally, the level of DC‐HIL expression on CD14+ monocytes correlates inversely with allostimulatory capacity, such that treatment with TGF‐β reduced this capacity, whereas knocking down the DC‐HIL gene augmented it. Our findings indicate that the DC‐HIL/SD‐4 pathway can be manipulated to treat T‐cell‐driven disorders in humans.

Epidermal Langerhans cell-targeted gene expression by a dectin-2 promoter

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5′-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

Mutations in the fifth immunoglobulin‐like domain of kit are common and potentially sensitive to imatinib mesylate in feline mast cell tumours

The purpose of the current study was to investigate the mutation status of KIT in feline mast cell tumours (MCTs) and to examine the effects of tyrosine kinase inhibition on the phosphorylation of mutant kit in vitro and in clinical cases of cats. Sequence analysis of KIT identified mutations in 42/62 MCTs (67·7%). The vast majority of the mutations were distributed in exons 8 and 9, both of which encode the fifth immunoglobulin‐like domain (IgD) of kit. All five types of kit with a mutation in the fifth IgD were then expressed in 293 cells and examined for phosphorylation status. The mutant kit proteins showed ligand‐independent phosphorylation. The tyrosine kinase inhibitor imatinib mesylate suppressed the phosphorylation of these mutant kit proteins in transfectant cells. In a clinical study of 10 cats with MCTs, beneficial response to imatinib mesylate was observed in 7/8 cats that had a mutation in the fifth IgD of kit in tumour cells. Mutations in the fifth IgD of kit thus appear to be common and potentially sensitive to imatinib mesylate in feline MCTs. These data provide an in vivo model for paediatric mastocytosis where mutations in the fifth IgD of kit also occur.

Validation of the prognostic value of histopathological grading or c-kit mutation in canine cutaneous mast cell tumours: a retrospective cohort study.

The objective of this retrospective cohort study was to validate the prognostic value of histological grading of canine cutaneous mast cell tumours (MCTs) according to the Patnaik (grades I-III) and Kiupel (low, high) grading systems, and to confirm the prognostic significance of internal tandem duplications (ITDs) within exon 11 of the c-kit gene (ITD-Exon11). The baseline characteristics and outcome data from 47 dogs diagnosed with cutaneous MCTs were collected and reviewed. Tumours were graded according to both grading systems and the nucleotide sequence of c-kit was evaluated. Results were analyzed to evaluate predictive factors for overall survival (OS) and progression-free survival (PFS). Log-rank tests indicated that dogs with Patnaik grade III MCTs had significantly reduced OS and PFS compared to those with either grade I or II tumours. However, no significant difference in OS or PFS was observed between grade I and II tumours. The dogs with Kiupel high-grade MCTs had significantly shorter OS and PFS than dogs with low-grade MCTs. The presence of ITD-Exon11 was significantly associated with shorter PFS. The result of Cox regression analysis showed that the Kiupel grading system for OS and PFS, and lymph node metastasis for OS, independently predicted prognosis. Kappa statistics confirmed a significantly higher inter-observer consistency for the Kiupel compared to the Patnaik grading system. These findings demonstrate that the Kiupel grading system is a useful prognostic tool for canine cutaneous MCTs in predicting OS and PFS, while the occurrence of ITD-Exon11 appeared to be a useful predictor for PFS.

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFκB blocking agents

Abstract To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6–10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFκB) in UVB-inducible HIV activation, two types of blockers, NFκB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFκB.

Imatinib elicited a favorable response in a dog with a mast cell tumor carrying a c-kit c.1523A>T mutation via suppression of constitutive KIT activation.

Target therapy using the tyrosine kinase inhibitor imatinib is one of the new therapeutic approaches for canine mast cell tumors (MCTs). In the present report, we demonstrate a clinical response to imatinib in a dog with MCT carrying a c-kit c.1523A>T mutation. Moreover, the effect of this mutation on the phosphorylation status of KIT and the inhibitory potency of imatinib on the phosphorylation of the mutant KIT were examined in vitro. A dog with a MCT tumor mass on the right forelimb sole with lymph node metastasis and mastocytemia was treated with imatinib. The MCT mass markedly shrank and mastocytemia became undetectable with 2 weeks of treatment. The lymph node enlarged by metastasis became normal in size with 5 weeks of treatment. From the sequencing analysis of c-kit in tumor cells, a substitution mutation c.1523A>T that alters the amino acid composition (p.Asn508Ile) within the extracellular domain of KIT was identified. The mutant KIT expressed on 293 cells showed ligand-independent phosphorylation and imatinib suppressed this phosphorylation in a dose-dependent manner. From these findings, imatinib was considered to elicit a clinical response in a canine case of MCT via inhibition of the constitutively activated KIT caused by a c-kit c.1523A>T mutation.