Clinical utility of the basophil activation test for analysis of allergic transfusion reactions: a pilot study.

BACKGROUND AND OBJECTIVES: In previous studies, we demonstrated that the basophil-activating effects of supernatants found in residual-transfused platelet concentrates (PC-SNs) on whole blood basophils in cases of allergic transfusion reactions (ATRs) could be assessed by the basophil activation test (BAT) in terms of allergen/IgE dependency. However, in these studies, the basophils were derived from third-party healthy volunteers. In this study, we performed BAT using patients' own blood basophils to analyse ATRs. MATERIALS AND METHODS: The BAT was performed in two cases of severe ATRs using residual PC-SNs and the patients' own basophils in the presence and absence of dasatinib, an inhibitor of IgE-mediated basophil activation. RESULTS: In both cases, PC-SNs exhibited basophil-activating activity against the patients' basophils, but not against basophils from third-party healthy volunteers. In addition, basophil activation was inhibited in the presence of dasatinib, indicating that the basophils were activated in an allergen/IgE-dependent manner. Of note, the basophils in Case 2, but not in Case 1, were activated by PC-SNs from some unrelated non-haemolytic transfusion reaction cases. CONCLUSION: This pilot study indicates that the BAT may be useful in clarifying the causal relationship between ATRs and transfused blood as well as in elucidating the mechanisms behind ATRs considering the allergen/IgE-dependent pathway.

Sorafenib plus hepatic arterial infusion chemotherapy with cisplatin versus sorafenib for advanced hepatocellular carcinoma: randomized phase II trial.

BACKGROUND: Sorafenib (Sor) is acknowledged as a standard therapy for advanced hepatocellular carcinoma (HCC). This trial was conducted to evaluate the effect of addition of hepatic arterial infusion chemotherapy with cisplatin (SorCDDP) to Sor for the treatment of advanced HCC. PATIENTS AND METHODS: We conducted a multicenter open-labeled randomized phase II trial in chemo-naïve patients with advanced HCC with Child-Pugh scores of 5-7. Eligible patients were randomly assigned 2:1 to receive SorCDDP (sorafenib: 400 mg bid; cisplatin: 65 mg/m2, day 1, every 4-6 weeks) or Sor (400 mg bid). The primary end point was overall survival. RESULTS: A total of 108 patients were randomized (Sor, n = 42; SorCDDP, n = 66). The median survival in the Sor and SorCDDP arms were 8.7 and 10.6 months, respectively [stratified hazard ratio (95% confidence interval), 0.60 (0.38-0.96), P = 0.031]. The median time to progression and the response rate were, respectively, 2.8 months and 7.3% in the Sor arm and 3.1 months and 21.7% in the SorCDDP arm. The adverse events were more frequent in the SorCDDP arm than in the Sor arm, but well-tolerated. CONCLUSION: SorCDDP yielded favorable overall survival when compared with Sor in patients with advanced HCC. CLINICAL TRIAL REGISTRATION: UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number: UMIN000005703.

Combination of Sonodynamic and Photodynamic Therapy against Cancer Would Be Effective through Using a Regulated Size of Nanoparticles.

Nanoparticles have been used for many functional materials in nano-sciences and photo-catalyzing surface chemistry. The titanium oxide nanoparticles will be useful for the treatment of tumor by laser and/or ultrasound as the sensitizers in nano-medicine. We have studied the combination therapy of photo- and sono-dynamic therapies in an animal tumor model. Oral-administration of two sensitizers titanium oxide, 0.2%-TiO2 nanoparticles for sono-dynamic and 1 mM 5-aminolevulinic acid for photodynamic therapies have resulted in the best combination therapeutic effects for the cancer treatment. Our light microscopic and Raman spectroscopic studies revealed that the titanium nanoparticles were distributed inside the blood vessel of the cancer tissue (1-3 µm sizes). Among these nanoparticles with a broad size distribution, only particular-sized particles could penetrate through the blood vessel of the cancer tissue, while other particles may only exhibit the side effects in the model mouse. Therefore, it may be necessary to separate the optimum size particles. For this purpose we have separated TiO2 nanoparticles by countercurrent chromatography with a flat coiled column (1.6 mm ID) immersed in an ultrasonic bath (42 KHz). Separation was performed with a two-phase solvent system composed of 1-butanol-acetic acid-water at a volume ratio of 4:1:5 at a flow rate of 0.1 ml/min. Countercurrent chromatographic separation yielded fractions containing particle aggregates at 31 and 4400 nm in diameter.

Comparison of neutralization profiles for anti-HCV reactive donor samples with or without detectable HCV RNA.

BACKGROUND AND OBJECTIVES: At Japanese Red Cross (JRC) Blood Centers, all donated blood is screened for hepatitis C virus (HCV) by serological and nucleic acid amplification testing. Donor plasma that tested reactive for anti-HCV by serological test is disqualified even if the donor tests negative for HCV RNA. These test results reflect both true-positive results because of past HCV infection and false-positive results because the cross-reactivity of plasma IgG, which current testing methods are unable to distinguish. To characterize these antibody test results, we examined the neutralizing activity of these plasma samples. MATERIAL AND METHODS: Donor plasma samples that tested reactive for anti-HCV by serological test but negative for HCV RNA (n = 43) were analysed for determining their neutralizing activities measured by the inhibition of the cellular entry of pseudoparticles harbouring HCV envelope glycoproteins (HCVpp). RESULTS: Strong and broad neutralizing activities against HCVpp entry similar to the samples that tested reactive for anti-HCV serological test and positive for HCV RNA (considered to be derived from individuals with chronic HCV infection) were observed in three of 43 plasma samples from donors who tested anti-HCV reactive but HCV RNA negative. CONCLUSION: By examining the neutralizing activities of plasma samples, we identified individuals with a past HCV infection from those in whom we were unable to confirm HCV infection according to the current testing algorithms of JRC, which do not perform anti-HCV confirmatory tests.

The association between risk factors and time of onset for thrombocytopenia in Japanese patients receiving linezolid therapy: a retrospective analysis.

WHAT IS KNOWN AND OBJECTIVE: Linezolid (LZD) is an oxazolidinone antibiotic that is active against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The major adverse effect related to its use in humans is reversible myelosuppression, which mostly manifests as thrombocytopenia. This retrospective study was conducted to identify risk factors that might contribute towards the development of thrombocytopenia due to intravenous administration of LZD. METHOD: Patients who were administered LZD between January 2008 and March 2013 were included. Thrombocytopenia was defined as a decrease in platelet count of ≥10 × 10(4) cell/µL from baseline or of ≥30%. RESULTS: A total of 47 patients were included in this study. These patients were divided into two groups: 22 patients (46·8%) were assigned to a non-thrombocytopenia group and 25 patients (53·2%) to a thrombocytopenia group. Multivariate logistic regression analysis revealed significant intergroup differences in duration of LZD treatment [odds ratio (OR) = 1·278; 95% confidence interval (CI) = 1·068-1·529; P = 0·007] and white blood cell (WBC) count (>12000 cells/µL; OR = 10·399; 95% CI = 1·667-64·882; P = 0·012). WHAT IS NEW AND CONCLUSIONS: This finding suggests that duration of LZD treatment and WBC count (>12000 cells/µL) are risk factors associated with thrombocytopenia resulting from LZD administration.

Prospective study of deep vein thrombosis in patients with spinal cord injury not receiving anticoagulant therapy.

STUDY DESIGN: Prospective cross-sectional study. OBJECTIVES: To investigate the timing of deep vein thrombosis (DVT) onset secondary to spinal cord injury without anticoagulant therapies. SETTING: Spinal Cord Injury Center in Hokkaido, Japan. METHODS: Between November 2012 and June 2013, patients with spinal cord injury who were admitted to our hospital within 1 day after the injury and treated surgically within 24 h underwent a neurological examination, leg vein ultrasonography and D-dimer test 1, 3, 7, 14 and 28 days after surgery. All patients received treatment with intermittent pneumatic compression and elastic stockings, but without any anticoagulant. RESULTS: DVT developed in 12 patients (11 men and 1 women), with a mean age of 62.2 years (range, 41-80 years; mean age of total sample, 63.2 years (range, 25-78 years)), all distal to the popliteal vein. DVT occurred more often with a more severe paralysis (66.3%, AIS A and B). The median (± standard error) length of time from the operation to DVT detection was 7.5±2.2 days. The mean D-dimer level upon DVT detection was 14.6±11.8 µg ml(-1), with no significant differences between those who developed DVT and those who did not at any of the time points. CONCLUSION: These results suggest that DVT can develop at the very-acute stage of spinal cord injury and the incidence increases with a more severe paralysis. DVT detection was more reliable with ultrasonography, which should be used with DVT-preventive measures, beginning immediately after the injury, for the management of patients with spinal cord injury.

Studies on expression of aldehyde dehydrogenase in normal and cancerous tissues of thyroids.

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells.

Encephalitis caused by human herpesvirus-6B in pancreas-after-kidney transplantation.

Human herpesvirus-6 (HHV-6) is a common pathogen among children, classically presenting with fever and rash that resolves without specific therapy. HHV-6 can be reactivated in the immunosuppressed patient. After bone marrow and solid organ transplantation, HHV-6 has been linked to various clinical syndromes, including undifferentiated febrile illness, encephalitis, myelitis, hepatitis, pneumonitis, and bone marrow suppression. However, HHV-6 encephalitis after pancreatic transplant has rarely been reported. Early diagnosis and treatment of HHV-6 encephalitis may be important for affected patients. We report the case of a 53-year-old pancreas-after-kidney transplant recipient who initially presented with high fever and confusion 3 weeks after operation. We managed to save the patient's life and preserve the pancreas graft function. We also review previously reported cases of HHV-6B encephalitis in solid organ transplant recipients.

A new transfectant panel cell line-based MoAb-independent antigen capture assay system for detection of CD36 antibody.

BACKGROUND AND OBJECTIVES: CD36 antibody (Ab) causes several disorders: neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and non-haemolytic transfusion reactions. However, there is no gold-standard test for CD36 Ab. MATERIALS AND METHODS: We developed a transfectant panel cell line-based MoAb-independent antigen capture assay system for detection of CD36 Ab and compared it with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) system in terms of sensitivity and specificity. RESULTS: Our new system was characterized by (1) gene-transfected cell lines, but not panel platelets; (2) not being hampered by HLA Abs; and (3) no need to use CD36 MoAbs to ensure the antigen specificity of this detection system. In addition, it showed a much better receiver operating characteristic curve than the MAIPA system. CONCLUSIONS: The present results indicate that our new system permits highly sensitive and specific detection of CD36 Ab.

Kv1.3 blockers ameliorate allergic contact dermatitis by preferentially suppressing effector memory T cells in a rat model.

BACKGROUND: The Kv1.3 voltage-gated potassium channel is selectively upregulated upon activation in effector memory T (TEM ) cells in inflamed tissue, and plays an important role in maintenance of T-cell activation. Although Kv1.3 blockers have been shown to ameliorate allergic contact dermatitis (ACD) in a rat model, it remains unknown whether the effect of Kv1.3 blockers on ACD is mediated by suppressing TEM cell function and/or whether naive T-cells or central memory T (TCM ) cells are influenced. AIM: To analyse the detailed mechanism of Kv1.3 blockers in a rat model of ACD. METHODS: We examined the effects of a Kv1.3 blocker on inflammation and production of the effector cytokine interferon (IFN)-γ in inflamed tissue in rat ACD. Single-cell suspensions were isolated from inflamed rat ears (TEM cells), and regional lymph nodes (naive T/TCM cells), and the effect of Kv1.3 blockers on anti-CD3-stimulated IFN-γ production in vitro was measured. RESULTS: The Kv1.3 blocker significantly suppressed ear inflammation and IFN-γ production at the protein level in vivo. It also suppressed in vitro IFN-γ production from TEM cells from inflamed tissues, but did not suppress the function of naive T/TCM cells from lymph nodes. CONCLUSIONS: We found that the Kv1.3 blocker ameliorated ACD by inhibiting TEM cell functions only, thus Kv1.3 blockers could be a potentially selective therapeutic agent for TEM cell-mediated inflammatory skin diseases without producing harmful side-effects.

A standardized immunofluorescence test method with human neutrophil antigen-expressing cell lines to enhance antibody detection.

There is an international need for a large-scale human neutrophils antigen (HNA) antibody screening platform to minimize the risk of antibody-mediated transfusion-related acute lung injury. However, sourcing a substantial, reliable source of HNA, as well as the scarcity of well-characterized HNA antisera for validating new screening platforms, remain as major obstacles. This short communication presents an improved protocol for the effective use of HNA-expressing KY cells as a screening platform using eight well-characterized HNA antisera of a single defined specificity.

FUS immunoreactivity of neuronal and glial intranuclear inclusions in intranuclear inclusion body disease.

AIMS: Recent studies have shown that fused-in-sarcoma (FUS) protein is a component of 'neuronal' intranuclear inclusion bodies (INIBs) in the brains of patients with intranuclear inclusion body disease (INIBD). However, the extent and frequency of FUS-immunoreactive structures in INIBD are uncertain. METHODS: We immunohistochemically examined the brain, spinal cord and peripheral ganglia from five patients with INIBD and five control subjects, using anti-FUS antibodies. RESULTS: In controls, the nuclei of both neurones and glial cells were intensely immunolabelled with anti-FUS and neuronal cytoplasm was weakly positive for FUS. In INIBD, neuronal and glial INIBs in the brain and spinal cord were positive for FUS. FUS-positive INIBs were also found in the peripheral ganglia. The proportion of FUS-positive neuronal INIBs relative to the total number of inclusion-bearing neurones ranged from 55.6% to 83.3% (average 73.2%) and that of FUS-positive glial INIBs ranged from 45.9% to 85.7% (average 62.7%). The nucleus and cytoplasm of inclusion-bearing neurones and glial cells showed no FUS immunoreactivity. CONCLUSIONS: These findings suggest that FUS is incorporated into INIBs in both neurones and glial cells and that loss of normal FUS immunoreactivity may result from reduced protein expression and/or sequestration within inclusions.

Expression of hepatocyte growth factor in the skin of amyotrophic lateral sclerosis.

OBJECTIVES: Hepatocyte growth factor (HGF) is one of the most potent survival-promoting factors for motor neurons. Overexpression of neuronal HGF has been shown to result in the attenuation of neuronal cell death and progression of disease in a familial amyotrophic lateral sclerosis (ALS) transgenic mouse model. HGF might be beneficial for motor neuron survival and is a good candidate agent for the treatment of ALS. So far, studies of the skin of ALS have shown unique pathological and biochemical abnormalities. However, there has been no study of HGF in ALS skin. MATERIALS AND METHODS: We made a quantitative immunohistochemical study of the expression of HGF in the skin from 19 patients with sporadic ALS and 16 controls. RESULTS: Hepatocyte growth factor immunoreactivity was positive in the epidermis, some dermal blood vessels, and glands in patients with ALS. These findings became more conspicuous as ALS progressed. The optical density for HGF immunoreactivity of the nucleus and the cytoplasm in the epidermis in ALS was significantly higher (P < 0.001 and P < 0.001) than in controls. There was a significant positive relation (r = 0.53, P < 0.02 and r = 0.73, P < 0.001) between HGF immunoreactivity and duration of illness in the nucleus and the cytoplasm in the epidermis in patients with ALS. CONCLUSIONS: These findings suggest that changes in HGF in ALS skin are related to the disease process and that metabolic alterations of HGF may take place in the skin of patients with ALS.

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies.

BACKGROUND: A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. METHODS: Wild-type ß3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and ß3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. RESULTS AND CONCLUSION: Of the 12 sera containing HPA-1a (n = 2), HPA-3a (n = 6), HPA-6b (n = 3) or HPA-7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay.

Increased expression of TDP-43 in the skin of amyotrophic lateral sclerosis.

OBJECTIVES: Transactivation-responsive DNA-binding protein-43 (TDP-43) was identified as a major component of the ubiquitin-positive inclusions in sporadic amyotrophic lateral sclerosis (ALS). However, there has been no study of TDP-43 in ALS skin. The present study investigates TDP-43 in ALS skin. MATERIALS AND METHODS: We made a quantitative immunohistochemical study of the expression of TDP-43 in the skin from 15 patients with ALS and 15 control subjects. RESULTS: The proportion of TDP-43-positive (TDP-43+) cells in the epidermis in ALS patients was significantly higher (P < 0.001) than in controls. There was a significant positive relationship (r = 0.62, P < 0.02) between the proportion and duration of illness in ALS patients. The optical density of TDP-43+ cells in the epidermis in ALS patients is markedly stronger (P < 0.001) than in controls. There was a significant positive relation (r = 0.72, P < 0.01) between the immunoreactivity and duration of illness in ALS patients. CONCLUSIONS: These data suggest that changes of TDP-43 in ALS skin are likely to be related to the disease process and that metabolic alterations of TDP-43 may take place in the skin of patients with ALS.

The effect of ascorbate on minor recurrent aphthous stomatitis.

AIM: Minor recurrent aphthous stomatitis (MRAS) is a common, painful and inflammatory ailment of the oral cavity with juvenile onset and unknown aetiology. The purpose of this study was to evaluate the potential of ascorbate (vitamin C) to reduce the frequency of MRAS and severity of pain. PATIENTS AND METHODS: Sixteen MRAS patients (9 boys and 7 girls: mean age, 12.0 +/- 2.4 years old) were assigned to take an oral dosage of 2000 mg/m(2)/day ascorbate. SUBJECTS: Their baseline frequency of outbreaks and the level of pains were compared during the treatment; in addition, a crossover clinical trial was performed. Polymorphonuclear leucocytes play a role in the pathogenesis, and then superoxide anion production was evaluated in prior to ascorbate treatment. RESULTS: The data indicated a statistically significant 50% reduction in oral ulcer outbreaks and a decline of pain level. Neutrophils were primed for superoxide anion production in the patients with MRAS. CONCLUSION: Ascorbate may modulate the generation of reactive oxygen species and augment neutrophil apoptosis, which could prevent neutrophil-mediated inflammation. Ascorbate seems to be effective, but the findings of our study were preliminary and it should be re-evaluated with a larger randomized controlled clinical trials.

Thalidomide prevents formation of multinucleated giant cells (Langhans-type cells) from cultured monocytes: possible pharmaceutical applications for granulomatous disorders.

Thalidomide is an effective drug for chronic inflammatory diseases, but the mechanism underlying its immunomodulatory action remains uncertain. Thalidomide has been reported to clinically improve chronic inflammatory granulomatous disorders. In such disorders, the granulomas consist of epithelioid cells, scattered lymphocytes and multinucleated giant cells (MNGC; Langhans-type cells). The present experimental approach permitted the reproduction of MNGC formation from peripheral blood monocytes and examination of thalidomides effect on it. MNGC can be effectively generated from monocytes cultured in the presence of interleukin-4 (IL-4) and macrophage colony-stimulating factor(M-CSF) for 14 days. Thalidomide can inhibit the formation of MNGC in a dose-dependent manner. MNGC formation was partly inhibited by the presence of neutralizing TNF-alpha antibody in the responses induced by IL-4 and M-CSF. Autocrinal TNF-alpha production and modulation of cadhelin expression to regulate cell adhesion might be involved in this inhibitory action of thalidomide. Our results support thalidomides clinical efficacy in the treatment of chronic granulomatous disorders (granulomatosis).

Multi-nucleated giant cell formation from human cord blood monocytes in vitro, in comparison with adult peripheral blood monocytes.

Multi-nucleated giant cells (MGCs; Langhans-type cell), formed from macrophage fusion, are recognized as a hallmark histological feature in chronic inflammation. However, their precise pathological role is still poorly understood, especially for microorganism pathogens in the neonatal immune system, which are capable of surviving intracellularly in phagocytes. To conduct a partial evaluation of the monocyte function of neonates, we investigated the ability of human cord blood monocytes to form MGCs in vitro by stimulating various cytokines and comparing them with adult peripheral blood monocytes. Monocytes from cord blood and adult peripheral blood were isolated and cultured for 14 days with cytokines known to induce MGC in vitro. The fusion index in experiments with a combination of interleukin (IL)-4 and macrophage colony-stimulating factor (M-CSF) and a combination of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly lower in cord blood than in adult blood monocytes (P = 0.0018 and P = 0.0141, respectively). The number of nuclei per MGC was significantly lower in cord blood than in adult blood monocytes in experiments with IL-4 alone, the combination of IL-4 and M-CSF, and the combination of IL-4 and GM-CSF (P < 0.0001). These results suggest the possibility that the susceptibility of newborns to mycobacterium infection is due partly to impaired MGC formation.