Yasuhito Abe

Anti-cytokine nature of natural human immunoglobulin : one possible mechanism of the clinical effect of intravenous immunoglobulin therapy

Natural human immunoglobulins are now widely used to treat many clinical diseases. Historically, this therapy began as a supplement for patients with agammaglobulinemia (Barandun et al. 1981/82). Pneumonia, bacterial meningitis and other infectious episodes often seen in patients with this disorder have been improved by intravenous administrations of natural immunoglobulin (Ersoy et al. 1992, Liese et al. 1992, Popescu et al. 1992). Rather than being just a supplement, immunoglobulin is now being used because of its therapeutic effects. One of its most prominent features is its antipyretic effects against infection (Iwata et al. 1987). Some specific antibody fractions against causative microorganisms may be present in natural immunoglobulin. However, it now appears that some unidentified mechanisms play important roles in the outcome of this therapy (Blanchette et al. 1992, Horiuchi et al. 1993, Ronda et al. 1993). Natural human immunoglobulin preparations have been widely used in cases of severe infection such as septic shock and bacterial meningitis {Ruiz et al. 1993, Saladino et al. 1992, Schedel et al. 1991, Wortel et al. 1993). Subsidence of an infection as evidenced by the alleviation of high-grade fever, and the improvement of survival rate have been noticed with the use of natural polyvalent immunoglobulin preparations from simple stock plasma, as well as IgM-enriched specific preparations (Ersoy et al. 1992, Iwata et al. 1987, Shimozato et al. 1990, Taylor et al. 1992). Another example ofthe clinically indicated disorders for this therapy is autoim-

Induction of myasthenia by immunization against muscle-specific kinase

Muscle-specific kinase (MuSK) is critical for the synaptic clustering of nicotinic acetylcholine receptors (AChRs) and plays multiple roles in the organization and maintenance of neuromuscular junctions (NMJs). MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering at the postsynaptic membrane. Although autoantibodies against the ectodomain of MuSK have been found in a proportion of patients with generalized myasthenia gravis (MG), it is unclear whether MuSK autoantibodies are the causative agent of generalized MG. In the present study, rabbits immunized with MuSK ectodomain protein manifested MG-like muscle weakness with a reduction of AChR clustering at the NMJs. The autoantibodies activated MuSK and blocked AChR clustering induced by agrin or by mediators that do not activate MuSK. Thus MuSK autoantibodies rigorously inhibit AChR clustering mediated by multiple pathways, an outcome that broadens our general comprehension of the pathogenesis of MG.

NEDD8 protein is involved in ubiquitinated inclusion bodies.

Proteolysis by the ubiquitin–proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin‐like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin–proteasome system. NEDD8 (neural precursor cell‐expressed and developmentally down‐regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin–proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinsons disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimers disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin–proteasome system. Copyright

Lymphokine-Activated Killer T-Cell-Originated Protein Kinase Phosphorylation of Histone H2AX Prevents Arsenite-Induced Apoptosis in RPMI7951 Melanoma Cells

Purpose: Arsenic is a valuable therapeutic tool in cancer treatment. Lymphokine-activated killer T-cell-originated protein kinase (TOPK) is highly expressed in cancer cells, but its specific function is still unknown. We investigated the role of TOPK in arsenic-induced apoptosis in RPMI7951 human melanoma cells. Experimental Design: Expression of TOPK was evaluated in different melanoma cell lines, and liquid chromatography-tandem mass spectrometry analysis was used to identify proteins binding with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of arsenic on TOPK, histone H2AX, and apoptosis in RPMI7951 cells. Results: Melanoma cell lines expressing high levels of TOPK were more resistant to arsenite (As3+)-induced apoptosis. As3+ treatment induced phosphorylation of TOPK and histone H2AX in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry results indicated that TOPK could bind with histone H2AX, and in vitro and in vivo assays confirmed that TOPK binds with and phosphorylates histone H2AX. As3+ treatment caused phosphorylation of TOPK, which colocalized with phosphorylated histone H2AX in the nucleus. TOPK small interfering RNA cells exhibited a decreased phosphorylation of histone H2AX with As3+ treatment. As3+-induced apoptosis was decreased in H2AX−/− cells but increased in TOPK small interfering RNA cells. Conclusions: TOPK binds with histone H2AX and inhibits As3+-induced apoptosis through phosphorylation of histone H2AX. Melanoma cell lines with high levels of TOPK are more resistant to As3+-induced apoptosis. Therefore, inhibition of TOPK activity combined with As3+ treatment may be helpful in the treatment of melanomas.

MuSK antibodies in AChR Ab-seropositive MG vs AChR Ab-seronegative MG

Hoch et al.1 recently reported a novel specific target antigen, the muscle-specific receptor tyrosine kinase (MuSK), for the autoantibodies in acetylcholine receptor antibody (AChR-Ab)-seronegative myasthenia gravis (MG) patients. MuSK is expressed at the postsynaptic membrane of neuromuscular junctions and has multiple roles in the clustering of AChR by the agrin secreted from motoneuron terminus. Therefore, autoantibodies against MuSK may cause severe inhibition of AChR clustering, leading to reduced numbers, altered distribution of AChRs in mature muscle, or both. MuSK antibodies are found in ∼37.5 to 70% of patients with AChR-Ab-seronegative MG but not in those with AChR-Ab-seropositive MG, a clear differentiation of the two forms of disease.1–5⇓⇓⇓⇓ Furthermore, a lack of detectable MuSK antibodies was reported in MG patients with thymoma. To evaluate MuSK antibodies clinically in patients with MG, we developed a …

Expression and phosphorylation of TOPK during spermatogenesis

Among normal organs and tissues, the MAPKK‐like mitotic protein kinase TOPK is expressed exclusively in the testis. We analyzed the expression and phosphorylation of TOPK to address the functional role of this kinase during spermatogenesis. TOPK protein is expressed mainly in the cytosol of spermatocytes and spermatids, but not in spermatids and spermatogonia in situ. TOPK‐Thr‐9, a cdk1/cyclin B target residue, was specifically phosphorylated during mitotic and meiotic phases, while TOPK‐Thr‐198, a key amino acid for the ATP pocket, was constantly phosphorylated irrespective of the cell cycle. These data indicate that spermatogenic germ cells with vital proliferation activity express TOPK. As TOPK‐Thr‐9 was phosphorylated during both mitosis and meiosis, TOPK was indicted to play a role in cytokinesis and/or chromosomal segregation but not in DNA replication.

Myasthenia gravis experimentally induced with muscle-specific kinase.

Here we present the first evidence that muscle‐specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG‐like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin‐independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin‐independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin‐independent AChR clustering. MuSK plays multiple roles in pre‐patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.

Enzyme-linked immunosorbent assay (ELISA) for human epidermal growth factor (hEGF)

An enzyme-linked immunosorbent assay for human epidermal growth factor (hEGF) by the sandwich method with the use of orthophenylene diamine (OPDA) for the substrate of the enzyme reaction was developed. This assay showed high sensitivity, and there was no problem with the recovery rates even without any pretreatment of samples. The changes of hEGF levels in plasma and serum depend on the condition of blood storage from sampling until separation of the cell content. The levels of hEGF markedly increased with time in serum, especially when stored at room temperature but less so in plasma. Changes of hEGF levels in plasma were negligible for up to 3 h if the blood was stored at 4 degrees C until separation.

Protective Effects of Anti-Neutrophil Antibody against Myocardial Ischemia/Reperfusion Injury in Rats

Neutrophil activation initiates myocardial ischemia/reperfusion (I/R) injuries. The aim of this study is to evaluate the in vitro functions of an anti-neutrophil monoclonal antibody, Urge-8, and its therapeutic efficacy against myocardial ischemia (MI) in rats. We measured in vitro functions of rat neutrophils including chemotactic activity, superoxide production, phagocytic function, and neutrophil degranulation. MI was induced in Wistar rats by clamping the left coronary artery for 1 h. Rats received either isotype-negative control IgG1 (control group, n = 20), 250 µg/kg of Urge-8 before (pre-treatment group, n = 20) or after (post-treatment group, n = 20) MI. The three groups were compared during the first 24 h after reperfusion with respect to changes in mean arterial pressure, heart rate, body temperature, biochemistry, serum cytokines, myocardial neutrophil infiltration, survival rate, and size of MI. Urge-8 effectively suppressed in vitro functions of rat neutrophils including chemotactic activity, superoxide production, phagocytic function, and neutrophil degranulation. The Urge-8 treated groups showed higher levels of arterial pressure and survival rate, lower values of interleukin-6 and interleukin-8, lower grade of myocardial neutrophil infiltration, and smaller MI size as compared to the control group. In conclusion, Urge-8 is effective against myocardial I/R injury by suppressing certain functions and myocardial infiltration of neutrophils in rats.

Enzyme-linked immunosorbent assay (ELISA) for human tumor necrosis factor (hTNF)

Tumor necrosis factor (TNF), a monokine released mainly by macrophages, has the ability to kill cells, especially tumor cells [l]. Since the first report by Carswell et al in 1975 [2], many researchers have given a great deal of attention to this factor because of its potential for use in cancer therapy. Assays for TNF have mainly been by bioassay using L929 cells [3,4], but this approach is not suited to human sera because of assay imprecision. In order to overcome this problem, we developed an ELISA for hTNF.