Kazuhide Nakagaki

Anti-cytokine autoantibodies are ubiquitous in healthy individuals

Anti‐cytokine autoantibodies in healthy individuals have been widely reported but the occurrence is variable and inconstant. We hypothesized that cytokine‐binding in vivo may explain their variable and infrequent detection. Therefore, we focused on the detection of the cytokine‐autoantibody complexes and found that anti‐cytokine autoantibody to IL‐2, IL‐8, tumor necrosis factor‐α, vascular endothelial growth factor and granulocyte‐colony stimulating factor were present in all 15 individuals evaluated, while those to IL‐3, osteopontin and macrophage‐colony stimulating factor were not detected in anyone. Autoantibodies against IL‐4, IL‐6, IL‐10, and interferon‐gamma were variously detected. Thus, we discovered that anti‐cytokine autoantibodies to multiple cytokines are ubiquitous in healthy individuals.

Novel aspects on the pathogenesis of Mycoplasma pneumoniae pneumonia and therapeutic implications

Mycoplasma pneumoniae (Mp) is a leading cause of community acquired pneumonia. Knowledge regarding Mp pneumonia obtained from animal models or human subjects has been discussed in many different reports. Accumulated expertise concerning this critical issue has been hard to apply clinically, and potential problems may remain undiscovered. Therefore, our multidisciplinary team extensively reviewed the literature regarding Mp pneumonia, and compared findings from animal models with those from human subjects. In human beings, the characteristic pathological features of Mp pneumonia have been reported as alveolar infiltration with neutrophils and lymphocytes and lymphocyte/plasma cell infiltrates in the peri-bronchovascular area. Herein, we demonstrated the novel aspects of Mp pneumonia that the severity of the Mp pneumonia seemed to depend on the host innate immunity to the Mp, which might be accelerated by antecedent Mp exposure (re-exposure or latent respiratory infection) through up-regulation of Toll-like receptor 2 expression on bronchial epithelial cells and alveolar macrophages. The macrolides therapy might be beneficial for the patients with macrolide-resistant Mp pneumonia via not bacteriological but immunomodulative effects. This exhaustive review focuses on pathogenesis and extends to some therapeutic implications such as clarithromycin, and discusses the various diverse aspects of Mp pneumonia. It is our hope that this might lead to new insights into this common respiratory disease.

Inflammation provoked by Mycoplasma pneumoniae extract: implications for combination treatment with clarithromycin and dexamethasone

Recently, combination treatment with a macrolide and a steroid for Mycoplasma pneumoniae (Mp) pneumonia has been reported to be effective. Thus, the effect of this combination on a mouse model of lung inflammation associated with Mp extract (the LIMEX mouse) was studied. Interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) were induced in Mp extract-treated RAW264.7 cells, and this induction was inhibited by dexamethasone, parthenolide, SB203580 or LY294002. This suggested that Mp extract activates nuclear factor κB-, p38- and PI-3K-linked pro-inflammatory signals. The LIMEX mice were then either treated with or without clarithromycin and/or dexamethasone. Clarithromycin administration enhanced the production of IL-6, TNF-α, macrophage inflammatory protein-1α, monocyte chemotactic protein-1 and RANTES, while their production was perfectly suppressed by the combination of clarithromycin and dexamethasone. IL-17, IL-23, keratinocyte-derived chemokine (KC) and interferon-γ levels were not affected by clarithromycin treatment, but they were significantly suppressed by the combination of dexamethasone and clarithromycin. Collectively, some components of Mp extract provoked an inflammatory reaction in the RAW 264.7 cell line and LIMEX mice. Whereas the lung reaction in LIMEX mice was further exacerbated by clarithromycin treatment, it was resolved by the combinational treatment with clarithromycin and dexamethasone.

IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.

Identification of a mechanism for lung inflammation caused by Mycoplasma pneumoniae using a novel mouse model

Human Mycoplasma pneumoniae (MP) pneumonia is characterized by alveolar infiltration with neutrophils and lymphocytes and lymphocyte/plasma cell infiltrates in the peri-bronchovascular area (PBVA). No mouse model has been able to mimic the pathological features seen in human MP pneumonia, such as plasma cell-rich lymphocytic infiltration in PBVA. To figure out the mechanism for inflammation by MP infection using a novel mouse model that mimics human MP pneumonia, mice were pre-immunized intraperitoneally with Th2 stimulating adjuvant, alum, alone or MP extracts with an alum, followed by intratracheal challenge with MP extracts. The toll-like receptor-2, which is the major receptor for mycoplasma cell wall lipoproteins, was strongly up-regulated in alveolar macrophages in a latter group after the pre-immunization but prior to the intratracheal challenge. Those findings demonstrated that acceleration of innate immunity by antecedent antigenic stimulation can be an important positive-feedback mechanism in lung inflammation during MP pneumonia.

Prevalence of Dirofilaria immitis among shelter dogs in Tokyo, Japan, after a decade: comparison of 1999–2001 and 2009–2011

Changes in the seroprevalence of Dirofilaria immitis infection among shelter dogs between a decade ago and the present were evaluated. Serum samples were collected from 200 adult dogs in urban and suburban areas in Tokyo, Japan, during two 2-year periods (April 1999 to March 2001 and April 2009 to March 2011). Sera were tested for the presence of D. immitis antigen using a specific commercialized kit. The seroprevalence of D. immitis infection was 46% in 1999–2001 and 23% in 2009–2011. A decrease was observed in the prevalence of infection between 1999–2001 and 2009–2011; in particular, the prevalence in urban areas decreased significantly compared with that in suburban areas (P < 0.01). There was no significant difference in prevalence between the sexes in each period, but there was a significant difference between mixed-breed and purebred dogs (P < 0.01). The decrease in prevalence of canine heartworm disease in urban areas could be related to better veterinary care.

Detection of Dirofilaria immitis DNA in host serum by nested PCR

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.

Light chain (κ/λ) ratio of GM-CSF autoantibodies is associated with disease severity in autoimmune pulmonary alveolar proteinosis☆☆

Previous studies demonstrated that antigranulocyte colony-stimulating factor autoantibody (GMAb) was consistently present in patients with autoimmune pulmonary alveolar proteinosis (aPAP), and, thus, represented candidature as a reliable diagnostic marker. However, our large cohort study suggested that the concentration of this antibody was not correlated with disease severity in patients. We found that the κ/λ ratio of GMAb was significantly correlated with the degree of hypoxemia. The proportion of λ-type GMAb per total λ-type IgG was significantly higher in severely affected patients than those in mildly affected patients, but the proportion of κ-type was unchanged. The κ/λ ratio was significantly correlated with both KL-6 and SP-D, which have been previously reported as disease severity markers. Thus, the light chain isotype usage of GMAb may not only be associated with the severity of aPAP, but may also represent a useful disease severity marker.

Expression of canine Kdap in normal, hyperplastic and neoplastic epidermis.

Keratinocyte differentiation-associated protein, Kdap, is a recently identified small secretory protein that may act as a soluble regulator for the cornification and/or desquamation of keratinocytes. To clarify the role of Kdap in the terminal differentiation of keratinocytes, detailed in situ localisation of Kdap was studied using canine skin with normal, hyperplastic and neoplastic epidermis. In normal canine trunk skin, Kdap was expressed by granular keratinocytes, with polarity to the apical side of the cells, suggesting that canine Kdap is present in lamellar granules, as in humans. Expression of Kdap was widespread in the spinous layers in hyperplastic epidermis, but was undetectable in squamous cell carcinomas. These findings suggest that Kdap is closely related to the delay of terminal differentiation and/or release of cells in hyperplastic epidermis.

Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro

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 (0.5h) 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 60pM). Intermediate-term stimulation (24h) 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 (72-168h) was higher at low concentrations of crhGM-CSF (15-30pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72h 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.