Autoantibody to transcriptional intermediary factor-1ß as a myositis-specific antibody: clinical correlation with clinically amyopathic dermatomyositis or dermatomyositis with mild myopathy.

BACKGROUND: Myositis-specific autoantibodies (MSAs) are associated with unique clinical subsets in polymyositis/dermatomyositis (PM/DM). Autoantibodies against transcriptional intermediary factor (TIF)-1γ and TIF-1α are known to be MSAs. Previously, we reported that TIF-1ß is also targeted in patients with DM with or without concomitant anti-TIF-1α/γ antibodies. OBJECTIVES: To evaluate the clinical features of seven cases with anti-TIF-1ß antibodies alone. METHODS: Serum autoantibody profiles were determined, and protein and RNA immunoprecipitation studies were conducted. Western blotting was performed to confirm autoantibody reactivity against TIF-1ß. RESULTS: Anti-TIF-1ß antibody was identified by immunoprecipitation assay in 24 cases. Among them, seven patients were positive for anti-TIF-1ß antibody alone. Six of the seven patients were classified as having DM. Among the six cases of DM, two patients had no muscle weakness and normal creatine kinase (CK) levels, and were classified as having clinically amyopathic DM. Four patients had muscle weakness, but three of them had normal serum CK levels that responded well to systemic steroids. Characteristic features of DM included skin rashes, such as Gottron sign, periungual erythema, punctate haemorrhage on the perionychium and facial erythema including heliotrope, which were observed in 86%, 57%, 86% and 71% of our cases, respectively. One of the seven patients had appendiceal cancer. None of the patients had interstitial lung disease. CONCLUSIONS: Seven patients were confirmed to have anti-TIF-1ß antibody without any other MSAs, including TIF-1α/γ antibodies, and six of them were diagnosed with DM. We suggest that anti-TIF-1ß antibody is an MSA, and that it is associated with clinically amyopathic DM or DM with mild myopathy.

Antimelanoma differentiation-associated protein 5 antibody level is a novel tool for monitoring disease activity in rapidly progressive interstitial lung disease with dermatomyositis.

BACKGROUND: Antimelanoma differentiation-associated protein (anti-MDA)5 antibodies are associated with rapidly progressive interstitial lung disease (RP-ILD) in patients with clinically amyopathic dermatomyositis (CADM) or dermatomyositis (DM). OBJECTIVES: We aimed to evaluate the relevance of monitoring anti-MDA5 antibody levels for the management of RP-ILD in patients with CADM or DM. METHODS: Twelve patients with CADM (n = 10) or DM (n = 2) accompanied by RP-ILD were included. Baseline characteristics and outcomes were recorded. Serial measurements of anti-MDA5 antibody levels were measured. All patients were treated with corticosteroids, tacrolimus and intravenous cyclophosphamide. RESULTS: All patients achieved RP-ILD remission after combined immunosuppressive therapy for a mean of 6·8 months, with significant decreases noted in the mean anti-MDA5 antibody levels at remission. Six (50%) patients became anti-MDA5 antibody negative after therapy. After a mean follow-up of 31 months, RP-ILD relapse was observed in four (33%) patients in both the anti-MDA5 antibody sustained positive group and the negative conversion group. However, relapsed patients in the sustained positive group relapsed earlier than those in the negative conversion group. Thus, a decrease in anti-MDA5 antibody levels during remission was associated with longer remission. Relapses were associated with a reincrease of anti-MDA5 antibody levels in four of four (100%) patients. In contrast, none of the patients without reincrease in anti-MDA5 antibody exhibited symptoms of relapse during follow-up. Therefore, reincrease in anti-MDA5 antibody levels was associated with relapse. CONCLUSIONS: The anti-MDA5 antibody level is a novel parameter for monitoring and a good predictor of RP-ILD relapse in patients with CADM or DM.

Incidence and risk factors for herpes zoster in patients undergoing liver transplantation.

BACKGROUND: Herpes zoster (HZ) is the most common manifestation of latent varicella zoster virus reactivation, which occurs naturally as a result of aging or in immunocompromised patients. Solid organ transplant recipients are at increased risk for HZ owing to their chronic immunosuppression. Although several reports investigated risk factors for the development of HZ in heart or renal transplantation, data in liver transplantation (LT) are limited. METHODS: We evaluated clinical data retrospectively in 377 adult patients undergoing LT between January 2005 and December 2012 in our institution. We analyzed the incidence rate of HZ and the standardized incidence ratio (SIR) by comparing with the general Japanese population. We additionally investigated risk factors for HZ after LT. RESULTS: HZ developed in 27 (7.16%) of the 377 patients after LT. The incidence rate of HZ after LT was 17.83 per 1000 person-years, which was significantly higher than in the general Japanese population (SIR = 4.61; 95% confidence interval [CI], 4.13-5.14). Multivariate analysis showed that older age (hazard ratio [HR] = 3.95; P < 0.001) and exposure to mycophenolate mofetil (HR = 3.03; P = 0.007) were independent risk factors for HZ after LT. CONCLUSIONS: This is the first and largest study, to our knowledge, to investigate the incidence rate of HZ and risk factors for development of HZ after LT in the Japanese population. Further investigations to focus on immunosuppressive regimens to reduce the risk for HZ incidence in this high-risk population could establish a new protocol of immunosuppression after LT.

Novel and rapid enumeration method of peripheral blood stem cells using automated hematology analyzer.

INTRODUCTION: The number of infused CD34(+) cells is crucial to the success of peripheral blood stem cell transplantation (PBSCT). Here, we present, for the first time, a new method of enumerating hematopoietic progenitor cells (HPCs) for PBSCT. METHOD: This novel method is based on hemolysis and chemical staining, followed by flow cytometry-based optical detection, conducted using an automated hematology analyzer (XN series, Sysmex). CD34(+) cells and HPCs were compared in 76 granulocyte colony-stimulating factor (G-CSF)-mobilized blood or apheresis samples taken from healthy donors (n = 18) or patients undergoing autologous PBSCT (n = 6). RESULTS: There was a strong correlation between the numbers of HPCs and CD34(+) cells (R(2)  = 0.958). The expected total number of HPCs in the final products, which was estimated from HPCs in pre-apheresis PB or mid-apheresis products, also correlated well with the total number of CD34(+) cells in the final products. The change in HPCs in PB closely resembled that of CD34(+) cells during mobilization. Experiments using immunomagnetic beads suggested that the majority of CD34(+) cells existed in HPCs, and vice versa. CONCLUSION: Hematopoietic progenitor cells may serve as surrogates for CD34(+) cells in PBSCT. However, further investigations are required to verify this.

Altered expression of dermokine in skin disorders.

BACKGROUND: Although dermokine-ß, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl-terminal domain shares a high pI value with many cytokines, suggesting similar functions. OBJECTIVE: To better understand the biology of dermokine, we here determined its localization under pathological conditions and examined factors that regulate its expression. METHODS: We generated an anti-human dermokine-ß/γ monoclonal antibody cross-reacting with the mouse protein. Using this antibody, immunohistological staining and Western blotting of dermokine-ß/γ were performed with various tissue samples. RESULTS: Although human dermokine-ß/γ was expressed in almost all granular layers, upper spinous layers of the skin were also stained with anti-dermokine-ß/γ antibody in inflammatory skin disorders. Dermokine-ß/γ was expressed in keratoacanthoma and a part of well-differentiated squamous cell carcinoma (SCC). However, dermokine-ß/γ was not detected in poorly differentiated SCC or tumours derived from non-keratinocytes. In mice, dermokine-ß/γ-expressed keratinocytes were increased in models of contact hypersensitivity, ultraviolet-irradiated skin injury and wound healing. Consistent with expanded distribution in inflammatory skin diseases, proinflammatory cytokines such as interleukin-1ß, interleukin-12, and tumour necrosis factor-α augmented dermokine-ß/γ expression in cultured human keratinocytes. In contrast, growth factors including epidermal growth factor, insulin-like growth factor-I, keratinocyte growth factor and transforming growth factor-α significantly reduced dermokine expression. CONCLUSION: These results provide novel insights into the physiological and pathological significance of dermokine in the epidermis.

Autoantibody-mediated regulation of B cell responses by functional anti-CD22 autoantibodies in patients with systemic sclerosis.

Studies have demonstrated that B cells play important roles in systemic sclerosis (SSc), especially through the CD19/CD22 autoimmune loop. CD22 is a B cell-specific inhibitory receptor that dampens B cell antigen receptor (BCR) signalling via tyrosine phosphorylation-dependent mechanism. In this study, we examined the presence and functional property of circulating autoantibodies reacting with CD22 in systemic sclerosis. Serum samples from 10 tight skin (TSK/+) mice and 50 SSc patients were assessed for anti-CD22 autoantibodies by enzyme-linked immunosorbent assays using recombinant mouse or human CD22. The association between anti-CD22 antibodies and clinical features was also investigated in SSc patients. Furthermore, the influence of SSc serum including anti-CD22 autoantibodies for CD22 tyrosine phosphorylation was examined by Western blotting using phosphotyrosine-specific antibodies reacting with four major tyrosine motifs of CD22 cytoplasmic domain. Anti-CD22 autoantibodies were positive in 80% of TSK/+ mice and in 22% of SSc patients. Patients positive for anti-CD22 antibodies showed significantly higher modified Rodnan skin thickness score compared with patients negative for anti-CD22 antibodies. Furthermore, anti-CD22 antibodies from patients' sera were capable of reducing phosphorylation of all four CD22 tyrosine motifs, while sera negative for anti-CD22 antibodies did not affect CD22 phosphorylation. Thus, a subset of SSc patients possessed autoantibodies reacting with a major inhibitory B cell response regulator, CD22. Because these antibodies can interfere CD22-mediated suppression onto B cell activation in vitro, SSc B cells produce functional autoantibodies that can enhance their own activation. This unique regulation may contribute to the autoimmune aspect of SSc.

Usefulness of measuring reticulocyte hemoglobin equivalent in the management of haemodialysis patients with iron deficiency.

The evaluation of iron status in dialysis patients provides information essential to the planning of adequate recombinant human erythropoietin treatment. The cellular iron status of the patients can be determined from the recently available measurement of reticulocyte hemoglobin equivalent (RET-He). RET-He is measured on the basis of automated fluorescent flow cytometry which in the reticulocyte channel, using a polymethine dye, also measures the mean value of the forward light scatter intensity of mature red blood cells and reticulocytes. These values equate with reticulocyte hemoglobin content. In this study, to clarify the accuracy of RET-He in diagnosing iron deficiency in dialysis patients, we initially compared RET-He with such iron parameters as serum ferritin levels, transferrin saturation and content of reticulocyte hemoglobin (CHr) which has been established as indicators of functional iron deficiency. Secondly, we investigated the changes in RET-He during iron supplementation for iron-deficient patients to determine whether this marker is a prospective and reliable indicator of iron sufficiency. The participants in this study were 217 haemodialysis patients. Iron deficiency was defined as havsing a transferrin saturation (TSAT) < 20% or serum ferritin < 100 ng/ml. Conventional parameters of red blood cells and RET-He were measured by on a XE-2100 automated blood cell counter (Sysmex). CHr was measured on an ADVIA120 autoanalyser (Siemens). RET-He mean value was 32.4 pg and good correlation (r = 0.858) between RET-He and CHr is obtained in dialysis patients. Receiver operating characteristic curve analysis revealed, values of the area was 0.776 and at a cutoff value of 33.0 pg, a sensitivity of 74.3% and a specificity of 64.9%, were achieved. Iron supplements given to the patients with low TSAT or ferritin, RET-He responded within 2 weeks, and this seemed to be a potential advantage of using RET-He in the estimation of iron status. RET-He is a new parameter, equivalent value to CHr, and is easily measurable on the widely spread and popular blood cell counter and is a sensitive and specific marker of iron status in dialysis patients.

The clinical relevance of serum antinuclear antibodies in Japanese patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disorder with excessive fibrosis of the skin and various internal organs. Although SSc is a heterogeneous disease, it has been reported that the particular antinuclear antibodies (ANA) are often indicative of clinical features, disease course and overall severity. OBJECTIVE: To clarify the association of clinical and prognostic features with serum ANA in Japanese patients with SSc. METHODS: We studied 203 Japanese patients diagnosed with SSc, who visited our hospital during the period 1983-2005. Six SSc-related ANA were identified using indirect immunofluorescence, double immunodiffusion and immunoprecipitation assays. RESULTS: Patients with SSc were classified into six ANA-based subgroups and a group without ANA. As expected, antitopoisomerase I antibody (Ab, n=64), anti-RNA polymerases (RNAP) Ab (n=12) and anti-U3 RNP Ab (n=5) were associated with diffuse cutaneous SSc, whereas anticentromere Ab (ACA, n=75), anti-Th/To Ab (n=7) and anti-U1 RNP Ab (n=10) were frequently detected in patients with limited cutaneous SSc. Clinical features of the ANA-negative group (n=10) were heterogeneous. Consistent with previous findings in Caucasian and/or black African patients, antitopoisomerase I Ab was associated with the involvement of vascular and pulmonary fibrosis, leading to decreased survival rate. However, no patients with anti-RNAP Ab developed renal crisis and the frequency of isolated pulmonary hypertension in patients with ACA, anti-Th/To Ab or anti-U3 RNP Ab was similar to that in other ANA-based subgroups. CONCLUSION: These results indicate that the clinical relevance of SSc-related ANA in Japanese patients differs in some aspects from that in Caucasian and/or black African patients.

Identification of a novel autoantibody reactive with 155 and 140 kDa nuclear proteins in patients with dermatomyositis: an association with malignancy.

OBJECTIVE: Myositis-specific autoantibodies (MSAs) are a useful tool in diagnosis, defining clinical subsets and predicting prognosis of dermatomyositis (DM) and polymyositis (PM). In this study, we identified a novel MSA reactive with 155 and 140 kDa nuclear proteins [anti-155/140 antibody (Ab)] and determined the clinical feature of DM patients positive for this autoantibody (autoAb). METHODS: Sera from 52 Japanese patients with DM, 9 with PM, 48 with systemic lupus erythematosus (SLE), 126 with systemic sclerosis and 18 with idiopathic interstitial pneumonia were examined by immunoprecipitation assays. Positive sera were further characterized by immunodepletion and immunofluorescence staining. RESULTS: Seven of the 52 (13%) Japanese patients with DM immunoprecipitated 155 and 140 kDa proteins from 35S-labelled K562 leukaemia cell extract. No patients with SLE, systemic sclerosis or idiopathic interstitial pneumonia as well as healthy controls were positive for this autoAb. Patients with anti-155/140 Ab developed heliotrope rash, Gottron's papules or sign and flagellate erythema significantly more frequently than those negative. Notably, internal malignancy was found at significantly higher frequency in those positive than those negative (71 vs 11%; P < 0.005). In contrast, none of these patients positive for this autoAb had interstitial lung disease. CONCLUSIONS: This novel MSA is associated with cancer-associated DM and may serve as a diagnostic serological marker for this specific subset.

A clue for telangiectasis in systemic sclerosis: elevated serum soluble endoglin levels in patients with the limited cutaneous form of the disease.

BACKGROUND: The transforming growth factor-beta (TGF-beta) system plays a critical role both in systemic sclerosis (SSc) and hereditary hemorrhagic telangiectasia (HHT). Endoglin, known as a gene responsible for HHT, is a TGF-beta receptor preferentially expressed on endothelial cells. The role of endoglin in SSc is potentially intriguing since limited cutaneous SSc (lcSSc) and HHT share several symptoms, including telangiectasia. OBJECTIVE: To determine serum levels of soluble endoglin (sEndoglin) and clinical associations in patients with SSc. METHODS: Serum sEndoglin levels were examined by ELISA in 70 patients with SSc, 20 patients with systemic lupus erythematosus and 20 healthy individuals. RESULTS: Serum sEndoglin levels were significantly elevated in patients with lcSSc compared with diffuse cutaneous SSc and systemic lupus erythematosus patients as well as normal controls. Patients with elevated sEndoglin levels had telangiectasia more frequently than those with normal sEndoglin levels. Furthermore, pulmonary artery pressure was positively correlated with sEndoglin levels in patients with lcSSc. CONCLUSION: Abnormal expression/function of endoglin may be linked to lcSSc-specific manifestations.

Blue-dye technique complements four-node sampling for early breast cancer.

AIMS: To examine four-node axillary sampling assisted by a blue dye (4NAS/dye) technique as a sentinel node biopsy (SNB) for breast cancer. METHODS: Lymphatic mapping was performed by injection of patent blue for 33 consecutive cases with breast cancer. Axillary sampling was performed until four nodes were obtained. This was followed by back-up axillary lymph node dissection to examine the feasibility of 4NAS/dye. The same study with 30 cases was conducted at an independent hospital to confirm the feasibility of this method. This method was then applied to 101 consecutive clinically node-negative patients to avoid axillary-node dissection, with intraoperative diagnosis made by frozen section examination. RESULTS: The median numbers of blue-stained nodes and nodes excised by 4NAS/dye were 1.7 and 3.4, respectively. The identification rate of sentinel lymph nodes (SNs) was 81.8% using the dye alone and 97.0% when the combination was used. Pathological examination revealed that the nodal status was correctly predicted by the dye alone in 62.5% of cases with metastasis, whereas in 100% by 4NAS/dye. The dye alone was not sufficient to identify SNs, especially in cases with prior excisional biopsy. The identification rate of SNs and the accuracy rate in another feasibility study were 100% and 92.5% in 30 consecutive cases, respectively. 4NAS/dye successfully detected SNs in 100 of 101 cases of the subsequent observational study with an acceptable post-operative axillary morbidity and thus succeeded as an SNB. CONCLUSIONS: The 4NAS/dye method is reliable for the detection of SNs. This method could be applied to observational studies without radio-isotope.

Determination of first cleavage plane: the relationships between the orientation of the mitotic apparatus for first cleavage and the position of meiotic division-related structures in starfish eggs.

In order to understand when the orientation of the first cleavage plane is fixed along the animal-vegetal axis in starfish eggs, the behavior of the sperm aster was examined by indirect immunofluorescence staining. After duplication, the sperm aster organizes the mitotic apparatus for first cleavage perpendicular to the cleavage plane. The sperm aster located in the egg periphery just after fertilization and moved to the site close to the animal pole rather than the egg center by meiosis II. At early metaphase II, duplication of the sperm aster was detected but the axis of the resultant sperm diaster randomly pointed. Subsequently, its axis had already turned perpendicular to the animal-vegetal axis before pronucleus fusion. These results indicate that the orientation processes of the sperm diaster consist of positioning before its duplication and successive determining its azimuth. Furthermore, the azimuth and position of the mitotic apparatus for first cleavage did not change by shifting or eliminating the meiotic division-related structures such as the germinal vesicle, meiotic spindle, and female pronucleus by micromanipulation. These results show that none of them determines the first cleavage plane. Therefore, we discuss the pointing mechanism of the first cleavage plane without the influence of these meiotic division-related structures.

Up regulated expression of fractalkine/CX3CL1 and CX3CR1 in patients with systemic sclerosis.

BACKGROUND: Fractalkine expressed on endothelial cells mediates activation and adhesion of leucocytes expressing its receptor, CX(3)CR1. Soluble fractalkine exhibits chemotactic activity for leucocytes expressing CX(3)CR1. OBJECTIVE: To determine the role of fractalkine and its receptor in systemic sclerosis (SSc) by assessing their expression levels in patients with this disease. METHODS: The expression of fractalkine and CX(3)CR1 in the skin and lung tissues was immunohistochemically examined. Circulating soluble fractalkine levels were examined by enzyme linked immunosorbent assay (ELISA). Blood samples from patients with SSc were stained for CX(3)CR1 with flow cytometric analysis. RESULTS: CX(3)CR1 levels on peripheral monocytes/macrophages and T cells were found to be raised in patients with diffuse cutaneous SSc. The numbers of cells expressing CX(3)CR1, including monocytes/macrophages, were increased in the lesional skin and lung tissues from patients with diffuse cutaneous SSc. Fractalkine was strongly expressed on endothelial cells in the affected skin and lung tissues. Soluble fractalkine levels were significantly raised in sera and were associated with raised erythrocyte sedimentation rates, digital ischaemia, and severity of pulmonary fibrosis. CONCLUSIONS: Up regulated expression of fractalkine and CX(3)CR1 cooperatively augments the recruitment of mononuclear cells expressing CX(3)CR1 into the affected tissue of SSc, leading to inflammation and vascular injury.

Autoantibodies to a collagen-specific molecular chaperone, heat-shock protein 47, in systemic sclerosis.

Heat-shock proteins are highly conserved and immunogenic proteins, which may be involved in the initiation and perpetuation of autoimmune diseases. Heat-shock protein 47 (HSP47) is expressed by collagen-secreting cells such as fibroblasts and serves as a collagen-specific molecular chaperone that plays a crucial role in collagen metabolism. Abnormal collagen accumulation and autoimmunity are characteristics of systemic sclerosis (SSc). We determined the presence and prevalence of autoantibodies to HSP47 in patients with SSc and also in tight-skin (TSK/+) mice, an animal model for SSc. Anti-HSP47 autoantibodies were present in SSc patients with a frequency of 26%, while patients with systemic lupus erythematosus, those with dermatomyositis, those with keloid and healthy subjects did not have anti-HSP47 antibodies. IgG1 and IgG2 were the major Ig isotypes of the autoantibodies. Patients positive for anti-HSP47 had a significantly shorter duration of disease than those who were negative. Anti-HSP47 autoantibodies were also positive in 79% of TSK/+ mice. Thus, autoantibodies to HSP47 were present in the sera from SSc patients as well as those from TSK mice, and may be associated with the pathogenesis of SSc.

Gene expression profile analysis of regenerating liver after portal vein ligation in rats by a cDNA microarray system.

AIMS: We assessed changes in gene expression of hypertrophied liver after portal vein ligation (PL) in a test group of rats compared to a control group, which had the same size liver but no PL. METHODS: The portal veins of the left and median lobes in the test group were ligated in an initial operation. Four days after the PL, the liver volume of the posterior caudate lobe (5%) increased two-fold and comprised 10% of the liver. A 90% hepatectomy was then performed, leaving only the hypertrophied posterior caudate lobe, and leaving the normal anterior and posterior caudate lobes (10%) in the control (sham) group. A comparison of the expression profiles between two groups was performed using cDNA microarrays and the hepatic ATP level was measured. RESULTS: The survival rate for the PL group was significantly higher than for the sham group at 4 days after the hepatectomy (56.3% and 26.7%, P < 0.05). Gene expression of cyclin D1, proliferating cell nuclear antigen, cyclin A and B was upregulated, and the cyclin-dependent kinase inhibitor was downregulated. Increases were observed in: (i) pyruvate dehydrogenase, the tricarboxylic acid cycle cycle regulator, (ii) acyl-CoA dehydrogenase, the oxidation regulator, and (iii) cytochrome oxidases, the oxidative phosphorylation regulator. Hepatic ATP concentration after hepatectomy was better maintained in the PL group than in the sham group (0.48 +/- 0.01 micromol/ml vs. 0.33 +/- 0.01 micromol/ml, P < 0.05). CONCLUSION: The regenerating liver increased tolerance for extended hepatectomy compared to normal liver. It is believed that this is because the induced rapid regeneration of the remaining liver after hepatectomy increases ATP metabolism.

Detecting human CD34+ and CD34- hematopoietic stem and progenitor cells using a Sysmex automated hematology analyzer.

In clinical medicine, particularly in the newly developing stem cell therapies required to support the practice of regenerative medicine, the measurement of both CD34+ and CD34- hematopoietic stem cells (HSC)/hematopoietic progenitor cells (HPC) is important in obtaining more accurate information about the total HSC/HPC content in various stem/progenitor cell sources. We report the results of an investigation into methods of detecting CD34+ and CD34- HSC/HPC using the immature information (IMI) channel incorporated into the Sysmex XE-2100 and SE-9000 automated hematology analyzers. In this study, CD34+ and CD34- HSC/HPC were separated by immunologic methods and quantified by flow cytometry (FACScan) and IMI channel analysis. In addition, CD34-/CD133+ HSC were prepared by a sequential antibody-based positive selection strategy. These cells appeared in the same area as CD34+ cells in the IMI channel of the automated hematology analyzer. These findings confirmed that an automated hematology analyzer can be used to measure both CD34+ and CD34- HSC. These results may explain the difference in HSC/HPC counts sometimes observed between the automated hematology analyzer and flow cytometric methods for CD34+ measurement. The results of this study demonstrated the potential of automated cell counting methods for measuring HSC content in cellular products for both research and clinical applications.