Kazuhide Nakagaki - Academia.edu (original) (raw)

Papers by Kazuhide Nakagaki

Journal of Neuroimmunology, 1998

Immunology, 1995

Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infecte... more Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infected with the filarial nematode Brugia pahangi were fused with mouse myeloma cell line P3X63.Ag8.653 cells. Of the several canine immunoglobulin-producing clones from this fusion, one was found to produce canine IgE specific for a filarial nematode antigen. The cell line has undergone limiting dilution cloning six times over the past 3 years and continues to produce monoclonal antibody of the IgE subclass at a rate of greater than 3 mg/l. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell culture supernatant protein that bound to protein A beads, showed bands at molecular weights (MW) of approximately 75,000 and 25,000 that were characteristic of epsilon and kappa or lambda chains, respectively. A mouse monoclonal antibody specific for canine IgE bound the 75,000 MW band, as demonstrated by Western blot. Western blots of aqueous extracts of adult filarial nema...

Parasitology Research, Jun 26, 2015

The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have ... more The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have shown that parasite-derived cell-free DNA (cfDNA) can be detected in host blood and may be a promising diagnostic marker for parasitic infections. Thus, our aim was to detect D. immitis-derived cfDNA in host serum by nested PCR. Sera were collected from 12 dogs with natural D. immitis infections; eight were microfilaria (mf)-positive, and the remaining four were mf-negative. Culture fluids derived from single-sex adult D. immitis worms (mf-producing females and males) were also tested for cfDNA. All mf-positive sera were positive by nested PCR, whereas no amplification products were detected in mf-negative sera. The culture fluid of mf-producing females was positive by nested PCR but that of males was negative. All products amplified by nested PCR were sequenced to confirm that the amplicons were those of D. immitis. These results indicate that D. immitis DNA circulates freely in dog serum, except in mf-negative dogs. Additionally, D. immitis cfDNA may primarily be derived from the mf, and adult worms appeared to be minor contributors of cfDNA concentrations in serum; however, the contribution of D. immitis cfDNA derived from larvae of other developmental stages is unclear. An evaluation of the kinetics of D. immitis cfDNA in host serum throughout the parasite life cycle could facilitate the development of early molecular diagnostic techniques. To the best of our knowledge, this is the first report of the detection of mitochondrial DNA from a filarial parasite in host serum.

日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology, Mar 31, 2012

Journal of Immunological Methods, Sep 1, 2018

During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrop... more During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), sargramostim, in patients with autoimmune pulmonary alveolar proteinosis (aPAP), we conducted a pharmacokinetic study of single-dose sargramostim inhalation. Several problems were encountered whereby sargramostim formed an immune-complex with GM-CSF autoantibodies (GMAbs) immediately after entering the body; thus, we could not measure the concentration of sargramostim using a commercial high sensitivity enzyme-linked immunosorbent assay (ELISA). Moreover, the ELISA could not discriminate inhaled sargramostim from intrinsic GM-CSF. To solve these problems, we developed a novel ELISA system with a capture antibody that is specific for sargramostim and a detection antibody capable of binding with GM-CSF. This system quantified the serum sargramostim concentration, but not E. coli-, CHO-, or HEK293Tderived human recombinant GM-CSF. Using this system, serum pharmacokinetics were estimated in five patients after inhalation of 250 μg sargramostim, with a mean Cmax of 9.7 ± 2.85 pg/ml at a Tmax of 2 ± 1.22 h.

PubMed, Aug 1, 1990

Filarial glomerulonephritis was studied using Dirofilaria immitis infected dogs. Of 34 infected d... more Filarial glomerulonephritis was studied using Dirofilaria immitis infected dogs. Of 34 infected dogs examined, 15 dogs (44.1%) had histopathological lesions in the kidney. These lesions included an increased number of mesangial cells and increased thickness of the matrix, the infiltration of the small round and plasma cells into the interstitium and thickening of the basement membrane. Deposits of IgG were demonstrated in the infected dogs, whereas C3 deposits were found in all dogs. Combined immunoglobulin and complement deposits were not always found in the dogs with histopathological lesions. The mean concentration (expressed as absorbance) of circulating immune complexes (CIC) was 0.675 +/- 0.517 in infected dogs, and 0.132 +/- 0.092 in uninfected dogs. Although there was significant difference in the level of CIC between infected and uninfected dogs (P less than 0.001), 11 dogs (32.4%) in infected group were negative. Otherwise, the CIC levels were correlated to the adult worm burden (r = 0.848; P less than 0.001) but not to the number of circulating microfilariae (mf) (r = 0.398; P less than 0.05). Transfer of mf to 7 naive dogs was performed to clarify the role of mf in the pathogenesis of filariasis. Antibodies to crude mf antigen became detectable two weeks after the transfer. Neither pathologic findings nor deposits of IgG and C3 in the kidney were found in dogs examined 20 days or 70 days after transfer. There was no evidence that histopathological lesions were induced by live mf, suggesting that adult worm burdens may be more closely related to filarial nephropathy.

Cytokine, Aug 1, 2014

To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has... more To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli-or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5 h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60 pM). Intermediate-term stimulation (24 h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168 h) was higher at low concentrations of crhGM-CSF (15-30 pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72 h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro.

The Japanese journal of veterinary science, 1981

Journal of pharmacobio-dynamics, 1981

Cytokine & Growth Factor Reviews, Aug 1, 2010

Numerous reports have documented the presence of autoantibodies working against naturally occurri... more Numerous reports have documented the presence of autoantibodies working against naturally occurring cytokines in humans in health and disease. In most instances, their physiological and pathophysiological significance remains unknown. However, recent advances in the methodologies for detecting cytokine autoantibodies and their application in research focused on specific disorders have shown that some cytokine autoantibodies play an important role in the pathogenesis of disease. Additionally, levels of cytokine autoantibodies may also correlate with disease severity and progression in certain infectious and autoimmune diseases but not in others. This suggests that cytokine-specific pathogenic differences exist. While multiple lines of evidence support the notion that high avidity cytokine autoantibodies are present and likely to be ubiquitous in healthy individuals, their potential physiological role, if any, is less clear. It is believed that they may function by scavenging pro-inflammatory cytokines and thereby inhibiting deleterious 'endocrine' effects, or by serving as carrier proteins, providing a 'reservoir' of inactive cytokines and thus modulating cytokine bioactivity. A central hypothesis is that sustained or repeated high-level exposure to cytokines triggers defects in T-cell tolerance, resulting in the expansion of existing cytokine autoantibody-producing B cells.

The Japanese journal of veterinary science, 1988

Journal of Comparative Pathology, May 1, 1994

The lungs of rabbits experimentally infected with Dirofilaria inzmitis were examined histopatholo... more The lungs of rabbits experimentally infected with Dirofilaria inzmitis were examined histopathologically, to compare the changes with those seen in human pulmonary dirofilariasis. Eight rabbits were infected subcutaneously with two to eight immature worms to induce pulmonary dirofilariasis. Obstructive changes, similar to those reported in human pulmonary dirofilariasis, were observed in the blood vessels surrounding the worms. A form of arteritis, similar to occlusive arteritis, and periarteritis were also observed in the lungs of the rabbits. These results suggest that experimentaIly induced dirofilariasis in the rabbit is a useful animal model for human pulmonary dirofilariasis.

Parasitology Research, 1993

For the identification of circulating parasite antigens associated with immune-complex glomerulon... more For the identification of circulating parasite antigens associated with immune-complex glomerulonephritis in dogs infected with Dirofilaria immitis, monoclonal antibodies (mAbs) were generated against adult worms. A total of 11 mAbs were selected for cloning because of their high productivity and their lack of crossreactivity with Toxocara canis in indirect immunofluorescence tests. The ability of mAbs to detect circulating antigens was examined using an antigen-capture enzyme-linked immunosorbent assay. Of the 11 mAbs, only NAK-I, an IgG2a mAb, was capable of detecting circulating antigens in 75% of infected dogs. However, this mAb was highly species-specific in its detection of circulating antigens, since sera from dogs infected with other nematode parasites were negative. Furthermore, the mAb detected antigens at the same glomerular sites in which IgG and/or C3 were deposited. The antigen deposits were observed along capillaries and/or in mesangial cells. The epitope recognized by this mAb is probably a carbohydrate, as it remained stable for 1 h at 100 ~ C and was sensitive to periodate treatment. Two bands of 62 and 26 kDa, respectively, were detected on Western blots by the mAb when sera from dogs infected with D. immitis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted. The pathology of filarial infections has been shown to be partly associated with immune-mediated factors (Zuidema 1971). It has been reported that glomerulonephritis in dogs is caused by infection with Dirofilaria immitis

The Japanese journal of veterinary science, 1988

Journal of the Japan Veterinary Medical Association, 1988

Veterinary Parasitology, Oct 1, 2000

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyze... more Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.

Journal of Neuroimmunology, 1998

Immunology, 1995

Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infecte... more Canine popliteal lymph node cells taken at the onset of clinical disease from a rear limb infected with the filarial nematode Brugia pahangi were fused with mouse myeloma cell line P3X63.Ag8.653 cells. Of the several canine immunoglobulin-producing clones from this fusion, one was found to produce canine IgE specific for a filarial nematode antigen. The cell line has undergone limiting dilution cloning six times over the past 3 years and continues to produce monoclonal antibody of the IgE subclass at a rate of greater than 3 mg/l. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell culture supernatant protein that bound to protein A beads, showed bands at molecular weights (MW) of approximately 75,000 and 25,000 that were characteristic of epsilon and kappa or lambda chains, respectively. A mouse monoclonal antibody specific for canine IgE bound the 75,000 MW band, as demonstrated by Western blot. Western blots of aqueous extracts of adult filarial nema...

Parasitology Research, Jun 26, 2015

The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have ... more The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have shown that parasite-derived cell-free DNA (cfDNA) can be detected in host blood and may be a promising diagnostic marker for parasitic infections. Thus, our aim was to detect D. immitis-derived cfDNA in host serum by nested PCR. Sera were collected from 12 dogs with natural D. immitis infections; eight were microfilaria (mf)-positive, and the remaining four were mf-negative. Culture fluids derived from single-sex adult D. immitis worms (mf-producing females and males) were also tested for cfDNA. All mf-positive sera were positive by nested PCR, whereas no amplification products were detected in mf-negative sera. The culture fluid of mf-producing females was positive by nested PCR but that of males was negative. All products amplified by nested PCR were sequenced to confirm that the amplicons were those of D. immitis. These results indicate that D. immitis DNA circulates freely in dog serum, except in mf-negative dogs. Additionally, D. immitis cfDNA may primarily be derived from the mf, and adult worms appeared to be minor contributors of cfDNA concentrations in serum; however, the contribution of D. immitis cfDNA derived from larvae of other developmental stages is unclear. An evaluation of the kinetics of D. immitis cfDNA in host serum throughout the parasite life cycle could facilitate the development of early molecular diagnostic techniques. To the best of our knowledge, this is the first report of the detection of mitochondrial DNA from a filarial parasite in host serum.

日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology, Mar 31, 2012

Journal of Immunological Methods, Sep 1, 2018

During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrop... more During a clinical trial of a Saccharomyces cerviciae-derived recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), sargramostim, in patients with autoimmune pulmonary alveolar proteinosis (aPAP), we conducted a pharmacokinetic study of single-dose sargramostim inhalation. Several problems were encountered whereby sargramostim formed an immune-complex with GM-CSF autoantibodies (GMAbs) immediately after entering the body; thus, we could not measure the concentration of sargramostim using a commercial high sensitivity enzyme-linked immunosorbent assay (ELISA). Moreover, the ELISA could not discriminate inhaled sargramostim from intrinsic GM-CSF. To solve these problems, we developed a novel ELISA system with a capture antibody that is specific for sargramostim and a detection antibody capable of binding with GM-CSF. This system quantified the serum sargramostim concentration, but not E. coli-, CHO-, or HEK293Tderived human recombinant GM-CSF. Using this system, serum pharmacokinetics were estimated in five patients after inhalation of 250 μg sargramostim, with a mean Cmax of 9.7 ± 2.85 pg/ml at a Tmax of 2 ± 1.22 h.

PubMed, Aug 1, 1990

Filarial glomerulonephritis was studied using Dirofilaria immitis infected dogs. Of 34 infected d... more Filarial glomerulonephritis was studied using Dirofilaria immitis infected dogs. Of 34 infected dogs examined, 15 dogs (44.1%) had histopathological lesions in the kidney. These lesions included an increased number of mesangial cells and increased thickness of the matrix, the infiltration of the small round and plasma cells into the interstitium and thickening of the basement membrane. Deposits of IgG were demonstrated in the infected dogs, whereas C3 deposits were found in all dogs. Combined immunoglobulin and complement deposits were not always found in the dogs with histopathological lesions. The mean concentration (expressed as absorbance) of circulating immune complexes (CIC) was 0.675 +/- 0.517 in infected dogs, and 0.132 +/- 0.092 in uninfected dogs. Although there was significant difference in the level of CIC between infected and uninfected dogs (P less than 0.001), 11 dogs (32.4%) in infected group were negative. Otherwise, the CIC levels were correlated to the adult worm burden (r = 0.848; P less than 0.001) but not to the number of circulating microfilariae (mf) (r = 0.398; P less than 0.05). Transfer of mf to 7 naive dogs was performed to clarify the role of mf in the pathogenesis of filariasis. Antibodies to crude mf antigen became detectable two weeks after the transfer. Neither pathologic findings nor deposits of IgG and C3 in the kidney were found in dogs examined 20 days or 70 days after transfer. There was no evidence that histopathological lesions were induced by live mf, suggesting that adult worm burdens may be more closely related to filarial nephropathy.

Cytokine, Aug 1, 2014

To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has... more To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli-or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5 h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60 pM). Intermediate-term stimulation (24 h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168 h) was higher at low concentrations of crhGM-CSF (15-30 pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72 h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro.

The Japanese journal of veterinary science, 1981

Journal of pharmacobio-dynamics, 1981

Cytokine & Growth Factor Reviews, Aug 1, 2010

Numerous reports have documented the presence of autoantibodies working against naturally occurri... more Numerous reports have documented the presence of autoantibodies working against naturally occurring cytokines in humans in health and disease. In most instances, their physiological and pathophysiological significance remains unknown. However, recent advances in the methodologies for detecting cytokine autoantibodies and their application in research focused on specific disorders have shown that some cytokine autoantibodies play an important role in the pathogenesis of disease. Additionally, levels of cytokine autoantibodies may also correlate with disease severity and progression in certain infectious and autoimmune diseases but not in others. This suggests that cytokine-specific pathogenic differences exist. While multiple lines of evidence support the notion that high avidity cytokine autoantibodies are present and likely to be ubiquitous in healthy individuals, their potential physiological role, if any, is less clear. It is believed that they may function by scavenging pro-inflammatory cytokines and thereby inhibiting deleterious 'endocrine' effects, or by serving as carrier proteins, providing a 'reservoir' of inactive cytokines and thus modulating cytokine bioactivity. A central hypothesis is that sustained or repeated high-level exposure to cytokines triggers defects in T-cell tolerance, resulting in the expansion of existing cytokine autoantibody-producing B cells.

The Japanese journal of veterinary science, 1988

Journal of Comparative Pathology, May 1, 1994

The lungs of rabbits experimentally infected with Dirofilaria inzmitis were examined histopatholo... more The lungs of rabbits experimentally infected with Dirofilaria inzmitis were examined histopathologically, to compare the changes with those seen in human pulmonary dirofilariasis. Eight rabbits were infected subcutaneously with two to eight immature worms to induce pulmonary dirofilariasis. Obstructive changes, similar to those reported in human pulmonary dirofilariasis, were observed in the blood vessels surrounding the worms. A form of arteritis, similar to occlusive arteritis, and periarteritis were also observed in the lungs of the rabbits. These results suggest that experimentaIly induced dirofilariasis in the rabbit is a useful animal model for human pulmonary dirofilariasis.

Parasitology Research, 1993

For the identification of circulating parasite antigens associated with immune-complex glomerulon... more For the identification of circulating parasite antigens associated with immune-complex glomerulonephritis in dogs infected with Dirofilaria immitis, monoclonal antibodies (mAbs) were generated against adult worms. A total of 11 mAbs were selected for cloning because of their high productivity and their lack of crossreactivity with Toxocara canis in indirect immunofluorescence tests. The ability of mAbs to detect circulating antigens was examined using an antigen-capture enzyme-linked immunosorbent assay. Of the 11 mAbs, only NAK-I, an IgG2a mAb, was capable of detecting circulating antigens in 75% of infected dogs. However, this mAb was highly species-specific in its detection of circulating antigens, since sera from dogs infected with other nematode parasites were negative. Furthermore, the mAb detected antigens at the same glomerular sites in which IgG and/or C3 were deposited. The antigen deposits were observed along capillaries and/or in mesangial cells. The epitope recognized by this mAb is probably a carbohydrate, as it remained stable for 1 h at 100 ~ C and was sensitive to periodate treatment. Two bands of 62 and 26 kDa, respectively, were detected on Western blots by the mAb when sera from dogs infected with D. immitis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted. The pathology of filarial infections has been shown to be partly associated with immune-mediated factors (Zuidema 1971). It has been reported that glomerulonephritis in dogs is caused by infection with Dirofilaria immitis

The Japanese journal of veterinary science, 1988

Journal of the Japan Veterinary Medical Association, 1988

Veterinary Parasitology, Oct 1, 2000

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyze... more Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.