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.

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.

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.

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.

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.

Memory B cell pool of autoimmune pulmonary alveolar proteinosis patients contains higher frequency of GM-CSF autoreactive B cells than healthy subjects.

The IgG-type neutralizing GM-CSF autoantibody (GMAb) is known to be the causative agent for autoimmune pulmonary alveolar proteinosis (APAP). Previous studies report that serum levels of IgG-GMAb are approximately 50-fold higher in APAP patients than in healthy subjects (HS). Serum levels of IgM-GMAb are also higher in APAP patients than in HS, but this has been assumed to be an etiological bystander. However, the mechanism for the excessive production of IgG-GMAb in APAP remains unclear. To investigate this, we detected putative GMAb-producing B cells (PGMPB) by inoculated B cells from the peripheral blood of APAP patients, HS, and umbilical cord blood mononuclear cells (UCBMNs) with Epstein-Barr virus. Both ELISA and ELISPOT assays showed that IgM-type GMAb was consistently and frequently present in all three groups, whereas IgG-type GMAb was high only in APAP patients, in whom it was exclusively produced in memory B cells and not in naive B cells. Since PGMPB in UCBMNs produced IgM-GMAb, but not IgG-GMAb, to the same extent as in HS and APAP patients, most IgM-GMAb reacted with GM-CSF in a non-specific manner. The memory B cell pool of APAP patients contain higher frequency of PGMPB than that of healthy subjects.

Histopathological comparison of pulmonary artery lesions between raccoon dogs (Nyctereutes procyonoides) and domestic dogs experimentally infected with Dirofilaria immitis.

Five raccoon dogs (Nyctereutes procyonoides) and two domestic dogs (Canis familiaris) were subcutaneously infected with 100 infective larvae (L3) of Dirofilaria immitis. Two and five worms, respectively, were collected from two of three raccoon dogs. Villous endarteritis was found in the raccoon dog with five worms and two dogs at 116 days after infection. The number of recovered worms in the raccoon dogs was significantly smaller than that of the domestic dogs having 22 and 29 worms, while histopathological features and the severity of the lesions in the raccoon dogs were similar to those in the domestic dogs. The vascular lesions in two chronically-infected raccoon dogs turned into much severe at 565 and 590 days after inoculation.

Expression of SART-1 mRNA in canine squamous cell carcinomas.

SART-1, a squamous cell carcinoma (SCC) antigen recognized by cytotoxic T lymphocytes, has been useful in human cancer therapy. The SART-1(259) peptide is a potential candidate for vaccine. The present study examined an orthologue of the mRNA coding this peptide in canine SCCs. Specimens were obtained from seven canine patients with SCC, and the mRNA was isolated from the samples. The SART-1 and beta-actin genes were amplified by reverse-transcription polymerase chain reaction, using the isolated mRNA as a template. Canine SART-1 was amplified in six of the seven specimens, while beta-actin was detected in all the samples. In dogs, carcinomas expressing SART-1 could be a target for cytotoxic T lymphocyte mediated immunotherapy.

Assay system development to measure the concentration of sargramostim with high specificity in patients with autoimmune pulmonary alveolar proteinosis after single-dose inhalation.

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 HEK293T-derived 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 ±â€¯2.85 pg/ml at a Tmax of 2 ±â€¯1.22 h.

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-alpha, 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.

Effects of inducing exercise on growing mice by means of three-dimensional structure in rearing environment.

We reared ICR mice during a growth period (3 to 10 weeks of age) and examined the effect of exercise induction, by enriching the rearing environment with obstacles such as ladders, compared to the standard environment. Environmental enrichment significantly increased the amount of exercise in both sexes (P<0.01). Enriched exercise mice had higher body weight than control mice at 6 to 9 weeks of age in males and 8 weeks of age in females (P<0.05). The sexual maturation of female enriched exercise mice was significantly advanced compared to the control (P<0.001). Enriched exercise mice showed decreased anxiety-like behavior in the open field test and lower plasma corticosterone levels in both sexes compared to the control, and differences were statistically significant in males (P<0.05). In both sexes, enriched exercise appeared to increase natural killer cells in blood compared to the control, but no statistical differences was detected. In conclusion, we confirmed that daily low-stress exercise could be induced using a three-dimensional rearing environment in growing mice. In addition, we suggest that exercise has beneficial effects on physical growth, sexual maturation and anxiety-like behavior. Furthermore, environmental enrichment might be more effective in male than female in group-housed mice.

Up-regulation of cluster of differentiation (CD) 11b expression on the surface of canine granulocytes with human granulocyte-macrophage colony-stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine, sharing a common beta subunit (CDw131) with interleukins 3 and 5. GM-CSF is important for its direct and indirect involvement in host defense. In veterinary medicine, human (h) GM-CSF has been used as a substitute for canine GM-CSF to stimulate canine granulocytes and macrophages. In this study, we compared the effects of three distinct hGM-CSFs produced by bacteria, yeasts and Chinese hamster ovary (CHO) cells with those of Escherichia (E) coli-produced canine GM-CSF on the cluster of differentiation 11b (CD11b) expression in canine granulocytes. The median effective dose (ED50) of hGM-CSFs from bacteria, yeasts and CHO cells was 3.09, 4.09 and 4.27 ng/ml, respectively, with no significant difference among three. In contrast, a significant difference was observed between ED50 of canine GM-CSF (0.56 ng/ml) and three hGM-CSFs according to the paired t-test (P<0.05). We conclude that hGM-CSF can activate canine granulocytes, but the average activity of the three rhGM-CSFs was approximately 15% of that of canine GM-CSF.

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.

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.

Mycoplasma pneumoniae extract induces an IL-17-associated inflammatory reaction in murine lung: implication for mycoplasmal pneumonia.

Mycoplasma pneumoniae (Mp) may cause immune cell reactions as pivotal aspects of this clinically common respiratory pathogen. Our aim is to determine if Mp extract induces a cellular immune response associated with interleukin (IL)-17, leading to lung inflammation and lung injury. BALB/c mice were immunized with Mp extract intraperitoneally followed by its intratracheal administration, to mimic repeated Mp infection found in humans (repeated inoculation, RI group). Those with a single inoculation were compared as single inoculation group (SI group). Analysis of bronchoalveolar lavage fluid (BALF) demonstrated that keratinocyte-derived cytokine, tumor necrosis factor-α, and IL-6 were produced and peaked on days 0.5 or 1, followed by IL-17 on day 2. Levels of these mediators in BALF were higher in RI group than SI group (P < 0.05). Further, significantly more neutrophils were recruited to the lungs of the RI group (P < 0.05). These observations suggest that IL-17 is involved in the prolonged induction of neutrophils in mice treated with Mp extract.

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.

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.

High avidity cytokine autoantibodies in health and disease: pathogenesis and mechanisms.

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.

Receptor-independent spread of a highly neurotropic murine coronavirus JHMV strain from initially infected microglial cells in mixed neural cultures.

Although neurovirulent mouse hepatitis virus (MHV) strain JHMV multiplies in a variety of brain cells, expression of its receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM 1) (MHVR) is restricted only in microglia. The present study was undertaken to clarify the mechanism of an extensive JHMV infection in the brain by using neural cells isolated from mouse brain. In contrast to wild-type (wt) JHMV, a soluble-receptor-resistant mutant (srr7) infects and spreads solely in an MHVR-dependent fashion (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In mixed neural cell cultures, srr7 infected a limited number of cells and infection did not spread, although wt JHMV induced syncytia in most of the cells. srr7-infected cells were positive for GS-lectin, a microglia marker. Fluorescence-activated cell sorter analysis showed that about 80% of the brain cells stained with anti-MHVR antibody (CC1) were also positive for GS-lectin. Pretreatment of those cells with CC1 prevented virus attachment to the cell surface and also blocked virus infection. These results show that microglia express functional MHVR that mediates JHMV infection. As expected, in microglial cell-enriched cultures, both srr7and wt JHMV produced syncytia in a majority of cells. Treatment with CC1 of mixed neural cell cultures and microglia cultures previously infected with wt virus failed to block the spread of infection, indicating that wt infection spreads in an MHVR-independent fashion. Thus, the present study indicates that microglial cells are the major population of the initial target for MHV infection and that the wt spreads from initially infected microglia to a variety of cells in an MHVR-independent fashion.