Fexofenadine Suppresses Delayed-Type Hypersensitivity in the Murine Model of Palladium Allergy.

Palladium is frequently used in dental materials, and sometimes causes metal allergy. It has been suggested that the immune response by palladium-specific T cells may be responsible for the pathogenesis of delayed-type hypersensitivity in study of palladium allergic model mice. In the clinical setting, glucocorticoids and antihistamine drugs are commonly used for treatment of contact dermatitis. However, the precise mechanism of immune suppression in palladium allergy remains unknown. We investigated inhibition of the immune response in palladium allergic mice by administration of prednisolone as a glucocorticoid and fexofenadine hydrochloride as an antihistamine. Compared with glucocorticoids, fexofenadine hydrochloride significantly suppressed the number of T cells by interfering with the development of antigen-presenting cells from the sensitization phase. Our results suggest that antihistamine has a beneficial effect on the treatment of palladium allergy compared to glucocorticoids.

Possible Immune Regulation of Natural Killer T Cells in a Murine Model of Metal Ion-Induced Allergic Contact Dermatitis.

Metal often causes delayed-type hypersensitivity reactions, which are possibly mediated by accumulating T cells in the inflamed skin, called irritant or allergic contact dermatitis. However, accumulating T cells during development of a metal allergy are poorly characterized because a suitable animal model is unavailable. We have previously established novel murine models of metal allergy and found accumulation of both metal-specific T cells and natural killer (NK) T cells in the inflamed skin. In our novel models of metal allergy, skin hypersensitivity responses were induced through repeated sensitizations by administration of metal chloride and lipopolysaccharide into the mouse groin followed by metal chloride challenge in the footpad. These models enabled us to investigate the precise mechanisms of the immune responses of metal allergy in the inflamed skin. In this review, we summarize the immune responses in several murine models of metal allergy and describe which antigen-specific responses occur in the inflamed skin during allergic contact dermatitis in terms of the T cell receptor. In addition, we consider the immune regulation of accumulated NK T cells in metal ion-induced allergic contact dermatitis.

Accumulation of invariant NKT cells into inflamed skin in a novel murine model of nickel allergy.

Nickel (Ni) can cause delayed-type hypersensitivity reactions, which are thought to be mediated by the accumulation of T cells into inflamed skin. Accumulated T cells at the developmental stages in metal allergy are poorly characterized because a suitable animal model has not been established. To investigate the accumulated T cells in allergic inflamed skin, we generated a novel murine model of Ni-induced allergy. The murine model of Ni allergy was induced by two sensitizations of Ni plus lipopolysaccharide solution into the groin followed by three challenges with Ni solution into the footpad. Here we show that a specific TCR repertoire bearing Vα14Jα18, called natural killer (NK) T cells, was expanded monoclonally in BALB/c or C57BL/6 mice. Accumulation of NKT cells was characterized as CD4(+) or CD4(-)CD8(-) T cells. These results suggested that NKT cells are major pathogenic T cells at the elicitation phase of Ni allergy.

High clonality of virus-specific T lymphocytes defined by TCR usage in the brains of mice infected with West Nile virus.

It has been reported that brain-infiltrating T lymphocytes play critical roles in the clearance of West Nile virus (WNV) from the brains of mice. We characterized brain-infiltrating T lymphocytes by analyzing the TCR α- and ß-chain repertoires, T cell clonality, and CDR3 sequences. CD3(+)CD8(+) T cells were localized in the WNV-infected brains. The expression of CD3, CD8, CD25, CD69, perforin, and granzymes positively correlated with viral RNA levels, and high levels of expression of IFN-γ, TNF-α, and IL-2 were detected in the brains, suggesting that Th1-like cytotoxic CD8(+) T cells are expanded in the brains in response to WNV infection. The brain-infiltrating T lymphocytes dominantly used TCR genes, VA1-1, VA2-1, VB5-2, and VB8-2, and exhibited a highly oligoclonal TCR repertoire. Interestingly, the brain-infiltrating T lymphocytes had different patterns of TCR repertoire usages among WNV-, Japanese encephalitis virus-, and tick-borne encephalitis virus-infected mice. Moreover, CD8(+) T cells isolated from the brains of WNV-infected mice produced IFN-γ and TNF-α after in vitro stimulation with peritoneal cells infected with WNV, but not with Japanese encephalitis virus. The results suggest that the infiltrating CD8(+) T cells were WNV-specific, but not cross-reactive among flaviviruses. T cells from the WNV-infected brains exhibited identical or similar CDR3 sequences in TCRα among tested mice, but somewhat diverse sequences in TCRß. The results indicate that WNV-specific CD3(+)CD8(+) T cells expanding in the infected brains are highly oligoclonal, and they suggest that TCR α-chains play a dominant and critical role in Ag specificity of WNV-specific T cells.

Increased positive selection pressure within the complementarity determining regions of the T-cell receptor ß gene in New World monkeys.

Because of the long-term co-evolution of TCR and MHC molecules, numerous nucleotide substitutions have accumulated within the domains of TCRß genes. We previously found that nonsynonymous nucleotide substitutions occurred more frequently in complementarity determining region (CDR)ß than in CDRα, even though only a limited number of common marmoset (Callithrix jacchus) and human T-cell receptor ß variable (TRBV) sequences were compared. This interesting finding raised the question of whether the increased selective pressure within CDRß was species-specific. In this study, we identified 21 TRBV region sequences from the common marmoset and performed comparative sequence analyses of the T-cell receptor α variable (TRAV) and TRBV regions from human, chimpanzee, rhesus monkey, cotton-top tamarin, Ma's night monkey, and common marmoset. The ratios of the number of nonsynonymous nucleotide substitutions per site (d(N) ) to the d(S) values (d(N) /d(S) ) were less than 1 within the framework regions (FRs) of TRAV and TRBV region sequences, suggesting that purifying selection is largely dominant within the FRs. In contrast, the d(N) values were statistically significantly greater for CDRß than for CDRα only in New World monkeys. Also, increased d(N) /d(S) ratios (d(N) /d(S) >1) were observed within CDRß between humans and New World monkeys and, interestingly, between New World monkeys, which share a relatively recent common ancestor. Moreover, phylogenetic analysis by maximum likelihood analysis provided firm evidence to support that positive selection occurred within CDRß along New World monkey lineages. These results suggest that increased positive selection pressure within CDRß is common in New World monkeys rather than being species-specific. This study provides an intriguing insight into the co-evolution of TCR and MHC molecules within primates.

Comprehensive analysis and characterization of the TCR alpha chain sequences in the common marmoset.

The common marmoset (Callithrix jacchus) is useful as a nonhuman primate model of human diseases. Although the marmoset model has great potential for studying autoimmune diseases and immune responses against pathogens, little information is available regarding the genes involved in adaptive immunity. Here, we identified one TCR alpha constant (TRAC), 46 TRAJ (joining), and 35 TRAV (variable) segments from marmoset cDNA. Marmoset TRAC, TRAJ, and TRAV shared 80%, 68-100%, and 79-98% identity with their human counterparts at the amino acid level, respectively. The amino acid sequences were less conserved in TRAC than in TCRbeta chain constant (TRBC). Comparative analysis of TRAV between marmosets and humans showed that the rates of synonymous substitutions per site (d(S)) were not significantly different between the framework regions (FRs) and complementarity determining regions (CDRs), whereas the rates of nonsynonymous substitutions per site (d(N)) were significantly lower in the FRs than in CDRs. Interestingly, the d(N) values of the CDRs were greater for TRBV than TRAV. These results suggested that after the divergence of Catarrhini from Platyrrhini, amino acid substitutions were decreased in the FRs by purifying selection and occurred more frequently in CDRbeta than in CDRalpha by positive selection, probably depending on structural and functional constraints. This study provides not only useful information facilitating the investigation of adaptive immunity using the marmoset model but also new insight into the molecular evolution of the TCR heterodimer in primate species.

Percutaneous pancreatic-duct puncture with rendezvous technique can treat stenotic pancreaticojejunostomy.

Stenotic pancreatico-enteric anastomosis is one of the serious late complications after a pancreaticoduodenectomy. We report a case of stenotic pancreaticojejunostomy with a pancreatic juice fistula drained externally, which was treated using percutaneous procedures combined with a rendezvous method. A 77-year-old woman was referred to our hospital for an endoscopic treatment to remove a percutaneous drainage tube from a fluid collection due to pancreatic juice fistula. She had undergone pylorus-preserving pancreatoduodenectomy with Roux-en-Y reconstruction due to duodenal carcinoma of Vater's papilla 1 year before the referral to our hospital. Soon after the operation, she had developed a fluid collection adjacent to the anastomosis due to pancreatic juice fistulas and subsequently had undergone its percutaneous drainage. After admission, we tried to dilate the stenotic anastomosis with an endoscopic procedure from the anastomosed jejunal lumen, using an oblique-viewing endoscope. The endoscope reached a portion of the anastomosis, but did not allow us to visualize the entire anastomosis. We punctured the main pancreatic duct under ultrasonography and fluoroscopy, and advanced the needle into the anastomosed jejunum through the stenotic anastomosis. After putting a guidewire into the anastomosed jejunum through the needle, we introduced an oblique-viewing endoscope into the anastomosed jejunum and caught hold of the guidewire using grasping forceps. Maintaining tension on the guidewire with a slight pulling force, with some effort we were able to place a 5-Fr drainage catheter into the jejunum percutaneously and through the anastomosis via the main pancreatic duct. Three weeks after these procedures, we performed balloon dilation of the anastomosis. One week after balloon dilation, removed the percutaneous catheter.

Presence of the annular ridge and location of the coronary sinus ostium as morphological features ofthe right atrium and interatrial septum in healthy common marmosets (Callithrix jacchus).

To effectively use a common marmoset (Callithrix jacchus) as an experimental animal species, it is critical to establish a normal characteristics and morphology of the organs of the common marmoset. Although gross morphology of the common marmoset heart is reportedly the same as that of humans, little information is available regarding detailed morphology of the right atrium and the interatrial septum. Heart specimens were collected from three male and 10 female marmosets aged 9 to 65 months to determine the morphological features of the right atrium and the interatrial septum. Ten specimens were evaluated morphologically with a stereoscopic microscope in accordance with preparation and investigation methods designed to facilitate evaluation. Three specimens were histologically evaluated after being stained with hematoxylin-eosin, Elastica van Gieson and periodic acid Schiff. An annular ridge that is not present in the human heart was present in the right atrium and the interatrial septum of the common marmoset hearts. Tissue structure of the annular ridge was similar to atrial myocardial fibers. Furthermore, location of the coronary sinus ostium was different to that in humans. Present findings were used to create a schematic view of the annular ridge in the common marmoset heart. In the common marmoset heart, the annular ridge may function as a valve of the superior vena cava ostium, inferior vena cava ostium, and coronary sinus ostium. Present study provides morphological evidence that common marmosets have a valve-like structure in the right atrium.

Characterization of T cell receptors in a novel murine model of nickel-induced intraoral metal contact allergy.

Nickel is a component of several alloy types that are widely used in our environment, including several dental alloy types that cause intraoral metal contact allergy. However, metal-specific immune responses in the oral mucosa have not been elucidated because a suitable animal model has not been established. In this study, we established a novel murine model of nickel-induced intraoral metal contact allergy and aimed to elucidate the immune response in terms of T-cell receptor repertoire and cytokine profiles in inflamed oral mucosa. The intraoral metal contact allergy model was induced by two sensitizations of nickel plus lipopolysaccharide solution into the postauricular skin followed by a single nickel challenge of the buccal mucosa. Cytokine expression profiles and T-cell phenotypes were determined by quantitative polymerase chain reaction. T cells accumulated in the cervical lymph nodes and inflamed oral mucosa were characterized by analyzing their T-cell receptor α- and ß-chain repertoires, and the nucleotide sequences of complementary determining region 3. Significant swelling and pathological features were histologically evident at 1 day after challenge in mice with nickel allergy. At 1 day after the challenge, CD8-positive T cells producing high levels of T helper 1 type cytokines had accumulated in the allergic oral mucosa. At 7 days after the challenge, excessive nickel allergy in the oral mucosa was suppressed by regulatory T cells. Characterization of the T-cell receptor repertoire in nickel allergic mice revealed the presence of natural killer T cells and T cells bearing Trav6-6-Traj57 at 1 day after the challenge. Our murine model of nickel-induced intraoral metal contact allergy showed that natural killer T cells and T cells bearing Trav6-6-Traj57 might be involved in the immune responses of nickel-induced intraoral metal contact allergy.

Unexpected recalcitrant course of drug-induced erythema multiforme-like eruption and interstitial pneumonia sequentially occurring after nivolumab therapy.

Vemurafenib improves survival of melanoma patients. However, cutaneous side-effects commonly occur in them. Nivolumab and ipilimumab are monoclonal antibodies against programmed death 1 and cytotoxic T-lymphocyte-associated antigen 4, both of which regulate excessive T-cell activation. Although these agents induce antitumor immunity against melanoma, the modified immune condition may result in an unexpected adverse reaction which has not been observed previously. Herein, we report a case who manifested severe erythema multiforme-like eruption with mucosal involvement associated with vemurafenib following nivolumab. The patient also subsequently suffered from ipilimumab-induced interstitial pneumonia with refractory course. Such a case has never been reported. This case suggested that dermatologists should pay special attention to unexpected adverse events of these drugs, and carefully observe cutaneous and respiratory status of patients during the treatment of melanoma.

Overexpression of ErbB4 is an independent marker for lymph node metastasis in Japanese patients with oral squamous cell carcinoma.

OBJECTIVE: Overexpression of the epidermal growth factor receptor (EGFR) family is common in oral squamous cell carcinoma (OSCC). Therefore, we analyzed the expression profiles of the four EGFR family members (ErbB1, ErbB2, ErbB3, and ErbB4) in OSCC of Japanese patients. STUDY DESIGN: Sixty-eight primary tumors and 18 normal oral mucosal tissue specimens were evaluated in this study. We analyzed EGFR family members using quantitative polymerase chain reaction and immunohistochemistry, as well as their relationships with clinical factors. RESULTS: The expression level of ErbB1 messenger RNA (mRNA) was markedly increased in OSCC. By comparing the gene expression levels of EGFR family members in OSCC tissues that had lymph node metastasis with those in the absence of lymph node metastasis, we found that ErbB4 mRNA expression was increased significantly. There was also a significant correlation between the mRNA expression level of ErbB4 and those of ErbB2 and ErbB3 in cases with lymph node metastasis. Moreover, we confirmed protein expression of ErbB4 in the cytoplasm and membrane of tumor cells, which was stronger in cases with lymph node metastasis. CONCLUSIONS: ErbB4 is an independent marker for lymph node metastasis in OSCC.

Downregulation of epidermal growth factor receptor family receptors and ligands in a mutant K-ras group of patients with colorectal cancer.

The present study investigated the expression profiles of the epidermal growth factor receptor (EGFR) family, which consists of four transmembrane tyrosine kinase receptors and their eight ligands, in 122 patients with colorectal cancer (CRC) using reverse transcription-quantitative polymerase chain reaction analysis. On comparison of the CRC primary tumor and matched adjacent normal mucosa (ANM) tissue samples, the mRNA expression levels of ErbB3, but not ErbB1, were significantly increased in CRC tissue samples, compared with those in the ANM tissues. The expression levels of the ligands exhibited opposing trends to their corresponding receptors, including EGF, BTC, AREG, EREG and HB­EGF, which were increased in the CRC tissues, whereas NRG1 and NGR2 were decreased in thee CRC tissues, compared with those in the AMN tissues. Subsequently, the present study investigated the frequency of K-ras mutations in the patients with CRC. The K­ras mutations were found to be present in 36.8% (45/122) of the cases, however, no correlation was observed between K­ras mutations and clinicopathological characteristics. In the CRC tissues, the expression levels of the EGFR family receptors and their ligands were determined in wild-type and mutant K-ras CRC cases. The expression levels of ErbB1, ErbB2, ErbB3, BTC, AREG, EREG, NRG1 and NRG2 were significantly decreased in the mutant K­ras cases, compared with those in the wild­type K­ras cases. These results suggested that the tumorigenesis of CRC with wild­type K­ras was mediated through, not only ErbB1, but also through the ErbB2 and ErbB3 pathways. Notably, although ErbB2 does not bind any ErbB ligands, ErbB2 may activate tumorigenesis via a heterodimer, rather than a homodimer. Therefore, the results of the present study suggest that the most effective strategy to target not only ErbB1, but also ErbB2 and ErbB3, is the use of monoclonal antibody treatment.

Spontaneously developed osteoarthritis in the temporomandibular joint in STR/ort mice.

Temporomandibular joint (TMJ) osteoarthritis is typically a slowly progressive asymmetric disease. Little is known regarding the natural destruction of TMJ articular tissues. The aim of the present study was to investigate morphological changes in the TMJ of STR/ort mice, known to be the model for spontaneous osteoarthritis in the knee joint, and to evaluate STR/ort mice as a suitable animal model for TMJ osteoarthritis. TMJs from 32 STR/ort mice euthanized at 30, 40, 50 or 60 weeks of age, and from 6 CBA mice euthanized at 30, 40 or 60 weeks of age were examined. Toluidine blue and tartrate-resistant acid phosphatase staining were used to assess histological changes in the articular cartilage. Morphological changes in the articular cartilage of the TMJ were evaluated using microcomputed tomography. At the age of 40-50 weeks, 17 (68%) of the 25 STR/ort mice had loss of articular cartilage on histology, with cavitation and erosion of the exposed bone and gradual changes in condylar shape. Furthermore, osteoarthritic morphological changes, and structural alterations were observed by microcomputed tomography. The STR/ort mouse strain appears to develop spontaneous osteoarthritis-like lesions in the TMJ with age, and would be a useful model to study the pathogenesis of TMJ osteoarthritis.

Clinicopathological significance and prognostic value of Wilms' tumor gene expression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, and novel effective treatments and diagnostic tools are urgently required. OBJECTIVE: The selection of appropriate targeting tumor-associated antigens (TAAs) is critical for immunotherapy. Therefore, we analyzed TAA expression levels and investigated their relationship with clinical factors in adjacent normal mucosa (ANM) and CRC tissue. METHODS: We obtained specimens of CRC primary tumors and matched ANM from 137 patients with CRC who underwent surgical resection. The mRNA levels of seven TAAs, Wilms' tumor gene (WT1), kinetochore associated-2 (KNTC2), cell division cycle associated-1 (CDCA1), M phase phosphoprotein-1 (MPHOSPH1), DEP domain-containing 1 (DEPDC1), 34-kDa translocase of the outer mitochondrial membrane (TOMM34) and ring finger protein-43 (RNF43), were analyzed using quantitative real-time reverse transcription-polymerase chain reaction, and their relationships with clinicopathological factors and the cell cycle were analyzed. RESULTS: The expression levels of all seven TAAs were significantly higher in CRC tissues than in ANM. Expression levels of WT1 in CRC tissues did not correlate with the cell cycle. Furthermore, WT1 expression in CRC tissues was significantly related to tumor progression, lymph node metastasis, distant metastasis and clinical stage. CONCLUSIONS: WT1 is a potential marker for prognosis and tumor progression in CRC.

Accumulation of metal-specific T cells in inflamed skin in a novel murine model of chromium-induced allergic contact dermatitis.

Chromium (Cr) causes delayed-type hypersensitivity reactions possibly mediated by accumulating T cells into allergic inflamed skin, which are called irritants or allergic contact dermatitis. However, accumulating T cells during development of metal allergy are poorly characterized because a suitable animal model is not available. This study aimed to elucidate the skewing of T-cell receptor (TCR) repertoire and cytokine profiles in accumulated T cells in inflamed skin during elucidation of Cr allergy. A novel model of Cr allergy was induced by two sensitizations of Cr plus lipopolysaccharide solution into mouse groin followed by single Cr challenge into the footpad. TCR repertoires and nucleotide sequences of complementary determining region 3 were assessed in accumulated T cells from inflamed skin. Cytokine expression profiles and T-cell phenotypes were determined by qPCR. CD3+CD4+ T cells accumulated in allergic footpads and produced increased T helper 1 (Th1) type cytokines, Fas, and Fas ligand in the footpads after challenge, suggesting CD4+ Th1 cells locally expanded in response to Cr. Accumulated T cells included natural killer (NK) T cells and Cr-specific T cells with VA11-1/VB14-1 usage, suggesting metal-specific T cells driven by invariant NKT cells might contribute to the pathogenesis of Cr allergy.

Upregulation of epidermal growth factor receptor 4 in oral leukoplakia.

In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.

Characterization of T cell receptors of Th1 cells infiltrating inflamed skin of a novel murine model of palladium-induced metal allergy.

Metal allergy is categorized as a delayed-type hypersensitivity reaction, and is characterized by the recruitment of lymphocytes into sites of allergic inflammation. Because of the unavailability of suitable animal models for metal allergy, the role of T cells in the pathogenesis of metal allergy has not been explored. Thus, we developed a novel mouse model for metal allergy associated with infiltration of T cells by multiple injections of palladium (Pd) plus lipopolysaccharide into the footpad. Using this model, we characterized footpad-infiltrating T cells in terms of phenotypic markers, T cell receptor (TCR) repertoires and cytokine expression. CD3+ CD4+ T cells accumulated in the allergic footpads 7 days after Pd challenge. The expression levels of CD25, interleukin-2, interferon-γ and tumor necrosis factor, but not interleukin-4 and interleukin-5, increased in the footpads after challenge, suggesting CD4+ T helper 1 (Th1) cells locally expanded in response to Pd. Infiltrated T cells in the footpads frequently expressed AV18-1 and BV8-2 T cell receptor (TCR) chains compared with T cells in the lymph nodes and exhibited oligoclonality. T-cell clones identified from Pd-allergic mouse footpads shared identical CDR3 sequences containing AV18-1 and BV8-2. These results suggest that TCR AV18-1 and BV8-2 play dominant and critical parts in the antigen specificity of Pd-specific Th1 cells.

Immune-related gene expression profile in laboratory common marmosets assessed by an accurate quantitative real-time PCR using selected reference genes.

The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12ß, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12ß and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.

A new method for quantitative analysis of the T cell receptor V region repertoires in healthy common marmosets by microplate hybridization assay.

The common marmoset, Callithrix jacchus, is one of the smallest primates and is increasingly used for an experimental nonhuman primate model in many research fields. Analysis of T cell receptor (TCR) repertoires is a powerful tool to investigate T cell immunity in terms of antigen specificity and variability of TCR expression. However, monoclonal antibodies specific for many TCR Vα or Vß chains have not been created. We have recently identified a large number of TCRα chain variable (TRAV) and TCRß chain variable (TRBV) sequences from a cDNA library of common marmosets. The purpose of this study is to develop a new method for analysis of TCR repertoires in the common marmoset using this sequence information. This method is based on a microplate hybridization technique using 32 TRAV-specific and 32 TRBV-specific oligoprobes following an adaptor-ligation PCR. This enables the easy quantitation of the respective TRAV and TRBV expression levels. No cross-hybridization among specific-oligoprobes and very low variances in repeated measures of the same samples was found, demonstrating high specificity and reproducibility. Furthermore, this method was validated by an antihuman Vß23 antibody which specifically bound to marmoset Vß23. Using this method, we analyzed TCR repertoires from various tissue samples (PBMCs, spleen, lymph node and thymus) and isolated T cell subpopulations (CD4+CD8+, CD4+CD8− and CD4−CD8+) from the thymus of 10 common marmosets. Neither tissue-specific nor T cell subpopulation-specific differences was found in TRAV and TRBV repertoires. These results suggest that, unlike mice, TCR repertoires in the common marmoset are not affected by endogenous superantigens and are conserved among individuals, among tissues, and among T cell subpopulations. Thus, TCR repertoire analysis with high specificity and reproducibility is a very useful technique, with the potential to replace flow cytometric analysis using a panel of TRV-specific antibodies, many of which remain unavailable.

Up-regulation of EGF receptor and its ligands, AREG, EREG, and HB-EGF in oral lichen planus.

OBJECTIVE: This study aimed to investigate the roles of the epidermal growth factor receptor (EGFR) family members and their ligands in oral lichen planus (OLP). STUDY DESIGN: The expressions of 4 EGFR-like receptors and 6 EGF-like ligands were measured in OLP tissues from 10 patients and compared with the levels in normal oral mucosa (NOM) from 10 healthy donors. RESULTS: Of the receptors, only EGFR mRNA and protein were more highly expressed in OLP compared with NOM tissues. Regarding the ligands, the mRNAs of amphiregulin (AREG), epiregulin (EREG), and heparin-binding EGF-like growth factor (HB-EGF) were more highly expressed in OLP compared with NOM tissues. These ligands were strongly expressed by infiltrating lamina propria lymphocytes as well as epithelial keratinocytes in OLP lesions, as shown by immunohistochemistry. CONCLUSIONS: The enhanced EGFR expression on the keratinocytes in OLP lesions and the up-regulation of EGF-like ligands in keratinocytes and infiltrating mononuclear cells could contribute to the carcinogenesis and pathogenesis of OLP.