Mycobacterium avium subsp. paratuberculosis Antigens Elicit a Strong IgG4 Response in Patients with Multiple Sclerosis and Exacerbate Experimental Autoimmune Encephalomyelitis.

Neuroinflammation can be triggered by microbial products disrupting immune regulation. In this study, we investigated the levels of IgG1, IgG2, IgG3, and IgG4 subclasses against the heat shock protein (HSP)70533-545 peptide and lipopentapeptide (MAP_Lp5) derived from Mycobacterium avium subsp. paratuberculosis (MAP) in the blood samples of Japanese and Italian individuals with relapsing remitting multiple sclerosis (MS). Additionally, we examined the impact of this peptide on MOG-induced experimental autoimmune encephalomyelitis (EAE). A total of 130 Japanese and 130 Italian subjects were retrospectively analyzed using the indirect ELISA method. Furthermore, a group of C57BL/6J mice received immunization with the MAP_HSP70533-545 peptide two weeks prior to the active induction of MOG35-55 EAE. The results revealed a significantly robust antibody response against MAP_HSP70533-545 in serum of both Japanese and Italian MS patients compared to their respective control groups. Moreover, heightened levels of serum IgG4 antibodies specific to MAP antigens were correlated with the severity of the disease. Additionally, EAE mice that were immunized with MAP_HSP70533-545 peptide exhibited more severe disease symptoms and increased reactivity of MOG35-55-specific T-cell compared to untreated mice. These findings provide evidence suggesting a potential link between MAP and the development or exacerbation of MS, particularly in a subgroup of MS patients with elevated serum IgG4 levels.

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund's adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10. M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne's disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP) strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

A mucosal immune response induced by oral administration of heat-killed Mycobacterium avium subsp. paratuberculosis exacerbates EAE.

Findings in humans and animals have demonstrated a potential role for Mycobacterium avium subsp. paratuberculosis (MAP) antigenic components in encephalitogenic T cell activation. Here we reported that oral administration of MAP activates the mucosal immunity and exacerbates active experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice, modulating the immune cell traffic from secondary lymphoid organs to central nervous system. The detection of antigenic mycobacterial components by intestinal antigen-presenting cells may modulate the immune system and the subsequent inflammatory status through various signaling mechanisms, including the synthesis of pro-inflammatory cytokines involved in EAE pathogenesis.

Seroprevalence of Immunoglobulin G Antibodies Against Mycobacterium avium subsp. paratuberculosis in Dogs Bred in Japan.

In this study, the seroprevalence of immunoglobulin G (IgG) antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in dogs bred in Japan was evaluated. Ninety-two non-clinical samples were obtained from three institutes and fifty-seven clinical samples were obtained from a veterinary hospital in Japan. Serum titers of total IgG, IgG1 and IgG2 isotype antibodies against MAP were measured using an indirect enzyme-linked immunosorbent assay (ELISA). The IgG antibodies against MAP in non-clinical serum obtained from three institutes was observed to be 2.4%, 20% and 9.0%. Similarly, the IgG1 antibodies titers against MAP were observed to be 7%, 20% and 0%. Lastly, the IgG2 antibodies against MAP were observed to be 7%, 20% and 4.4%. No significance differences in these titers were observed among the three institutes. The IgG, IgG1 and IgG2 antibodies in serum obtained from a veterinary hospital were observed to be 55.3%, 42% and 42%, respectively. Significant differences were found between the non-clinical and clinical samples. The titers in the clinical samples showed a high degree of variance, whereas low variance was found in the non-clinical samples. The IgG antibody levels were thought to be induced following exposure to MAP-contaminated feed. The difference in titers between the clinical and non-clinical samples is likely to be related to the amount of MAP antigen contamination in dog foods.

Author Correction: Altered humoral immunity to mycobacterial antigens in Japanese patients affected by inflammatory demyelinating diseases of the central nervous system.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Adjuvant and antigenic properties of Mycobacterium avium subsp. paratuberculosis on experimental autoimmune encephalomyelitis.

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease in ruminants, has been linked as a possible risk factor for human multiple sclerosis. In the current study we investigated the adjuvant effect of MAP on experimental autoimmune encephalomyelitis (EAE). Groups of C57BL/6 mice were actively immunized with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide emulsified in incomplete Freund's adjuvant modified containing heat-killed MAP (MIFA). MOG-MIFA immunized mice showed an early disease onset and more severe clinical scores in comparison with MOG-CFA immunized mice, demonstrating for the first time the adjuvant effect of MAP on EAE development.

Erratum: Kuribayashi, T. et al. Seroprevalence of Immunoglobulin E Antibodies against Japanese Cedar Pollen Allergens Cry j1 and Cry j2 in Dogs Bred in Japan. Vet. Sci. 2018, 5, 79.

Due to an error during production, the column title of Figure 2 and Figure 3 are misaligned in the Results section of the published paper [...].

Anti-Mycobacterial Antibodies in Paired Cerebrospinal Fluid and Serum Samples from Japanese Patients with Multiple Sclerosis or Neuromyelitis Optica Spectrum Disorder.

Local synthesis of antibodies and presence of oligoclonal bands in the cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS). We investigated the frequency of antibodies against mycobacterial and relevant human epitopes in the CSF of patients with MS or neuromyelitis optica spectrum disorder (NMOSD) and whether these antibodies differed from those present in the serum. Matched serum and CSF samples from 46 patients with MS, 42 patients with NMOSD, and 29 age-matched and sex-matched control subjects were screened retrospectively for the presence of antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) pentapeptide (MAP_5p), MAP_2694295⁻303, and myelin basic protein (MBP)85⁻98 peptides by using indirect ELISA. Serum levels of anti-MAP_5p and anti-MAP_2694295⁻303 antibodies were highly prevalent in patients with MS when compared to patients with NMOSD and controls. Several patients with MS had detectable anti-MAP_5p and anti-MAP_2694295⁻303 antibodies in the CSF. Furthermore, a group of patients with MS showed intrathecally restricted production of antibodies against these peptides. Women appeared to mount a stronger humoral response to mycobacterial peptides than men. No significant difference in the frequency of anti-MBP85⁻98 antibodies was found between patients with MS and those with NMOSD. These data highlight the zoonotic potential of MAP, which suggests its involvement in MS etiopathogenesis.

Seroprevalence of Immunoglobulin E Antibodies against Japanese Cedar Pollen Allergens Cry j 1 and Cry j 2 in Dogs Bred in Japan.

Levels of Japanese cedar pollen (Cryptomeria japonica) have increased in Japan and cedar pollinosis caused by Japanese cedar pollen has been reported in dogs. Serum levels of immunoglobulin E (IgE) against Cry j 1 and Cry j 2 in dogs raised in institutes and treated at veterinary hospitals in Japan were thus investigated. A total of 71 sera obtained from two institutes and 87 sera obtained from veterinary hospitals in the Hyogo and Kanagawa Prefectures were analyzed in this study. Serum levels of IgE were measured using the enzyme-linked immunosorbent assay with commercial purified Cry j 1 and Cry j 2. IgE against Cry j 1 and Cry j 2 in sera obtained from the two institutes were detected, despite the dogs being bred in enclosed areas. Moreover, significant differences were noted in the serum levels of IgE against Cry j 1 and Cry j 2 between the two institutes. The number of samples showing Cry j 1 or Cry j 2 levels above the cut-off values was greater in the Kanagawa Prefecture than in the Hyogo Prefecture. In total, 14 dogs showed Cry j 1 and Cry j 2 levels greater than the cut-off values in the Hyogo Prefecture, and only three such dogs were seen in the Kanagawa Prefecture. A significant correlation between serum levels against both allergens was observed (r² = 0.6931, p < 0.0001).

Altered humoral immunity to mycobacterial antigens in Japanese patients affected by inflammatory demyelinating diseases of the central nervous system.

Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis (BCG) have been associated to several human autoimmune diseases such as multiple sclerosis (MS), but there are conflicting evidence on the issue. The objective of this study is to evaluate their role in Japanese patients affected by inflammatory demyelinating disorders of the central nervous system (IDDs). A total of 97 IDDs subjects including 51 MS and 46 neuromyelitis optica spectrum disorder (NMOSD) patients, and 34 healthy controls (HCs) were tested for the detection of IgG, IgM and IgA against mycobacterial antigens by indirect ELISA. The levels of anti-MAP IgG were higher in MS patients compared to NMOSD patients (AUC = 0.59, p = 0.02) and HCs (AUC = 0.67, p = 0.01), and the anti-MAP antibodies were more prevalent in MS patients treated with interferon-beta (OR = 11.9; p = 0.004). Anti-BCG IgG antibodies were detected in 8% of MS, 32% of NMOSD and 18% of HCs, the difference between MS and NMOSD groups was statistically significant (AUC = 0.66, p = 0.005). Competition experiments showed that nonspecific IgM were elicited by common mycobacterial antigens. Our study provided further evidence for a possible association between MAP and MS, while BCG vaccination seemed to be inversely related to the risk of developing MS.

Humoral response against host-mimetic homologous epitopes of Mycobacterium avium subsp. paratuberculosis in Japanese multiple sclerosis patients.

Several works have demonstrated the existence of a link between Mycobacterium avium subsp. paratuberculosis (MAP) and MS in Italy. In this study, we analyzed the serology of MAP in a Japanese population while looking at several markers of MAP. Fifty MS patients, 12 clinically isolated syndrome (CIS) patients, 30 other neurological disorders (OND) patients, and 50 healthy controls (HCs) were tested using ELISA for the presence of IgG antibodies toward immunodominant epitopes MAP_0106c121-132, homologues MBP85-98, homologues IRF5424-432, MAP_402718-32, and MAP_2694295-303. MAP-positive patients were also analyzed in relation to their clinical/demographic characteristics. Amongst all peptides, only antibodies against MAP_2694295-303 were more prevalent in MS patients (30%), as compared to OND patients (3%) (p = 0.009; area under roc curve (AUC) = 0.61) and HCs (2%) (p = 0.0004; AUC = 0.65) and in CIS patients (25%) compared to HCs (p = 0.023; AUC = 0.55). Logistic regression analysis showed a higher frequency of anti-MAP_2694295-303 antibodies in the sera of oligoclonal bands positive MS patients (p = 0.2; OR = 2, 95%CI: 0.55-7.7). These findings support the view that MAP could act as a risk factor or a triggering agent of MS in some Japanese patients with a genetic susceptibility to the mycobacterium.

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)-amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH2 (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.

Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.

Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p<0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans.

An immuno-epidemiological model for Johne's disease in cattle.

To better understand the mechanisms involved in the dynamics of Johne's disease in dairy cattle, this paper illustrates a novel way to link a within-host model for Mycobacterium avium ssp. paratuberculosis with an epidemiological model. The underlying variable in the within-host model is the time since infection. Two compartments, infected macrophages and T cells, of the within-host model feed into the epidemiological model through the direct transmission rate, disease-induced mortality rate, the vertical transmission rate, and the shedding of MAP into the environment. The epidemiological reproduction number depends on the within-host bacteria load in a complex way, exhibiting multiple peaks. A possible mechanism to account for the switch in shedding patterns of the bacteria in this disease is included in the within-host model, and its effect can be seen in the epidemiological reproduction model.

Elimination Half-Lives of Acute Phase Proteins in Rats and Beagle Dogs During Acute Inflammation.

The half-lives of typical acute phase proteins in rats and beagle dogs during acute inflammation were investigated. Acute inflammation was induced by injection of turpentine oil in rats and administration of indomethacin in beagle dogs. Serum concentrations of α2-macroglobulin (α2M) and C-reactive protein (CRP) were measured by enzyme-linked immunosorbent assay and α1-acid glycoprotein (AAG) was measured by single radial immunodiffusion. Half-life was calculated as 0.693/elimination rate constant (K). The mean half-lives in the terminal elimination phase of α2M and AAG were 68.1 and 164.8 h, respectively. The half-life of AAG was significantly longer than that of α2M. Mean half-lives in the terminal elimination phase of CRP and AAG were 161.9 and 304.4 h, respectively. The half-life of AAG was significantly longer than that of CRP in beagle dogs. No significant differences in the half-life of AAG were observed between rats and beagle dogs. Furthermore, serum concentrations in the terminal elimination phase could be simulated with the K data acquired in this study.

Molecular pathogenesis of bovine paratuberculosis and human inflammatory bowel diseases.

Paratuberculosis (Ptb), caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic enteritis that affects many ruminants and other wild animals worldwide. Ptb is a great concern in animal health and in etiology of human Crohn's disease (CD). In the present study, we detected Map-specific insertion sequence IS900 of DNA in tissue sections surgically removed from lesions of patients with CD (29 samples), ulcerative colitis (UC) (17 samples), and non-inflammatory bowel disease (IBD) (20 samples). We then compared the histopathological findings of 29 CD and 17 UC cases with those of 35 cases of bovine Ptb, since few comparative pathological studies of human IBD and Ptb have been conducted. The QPCR examination indicated positive results in 13.37% of CD cases, 3.57% of UC cases, and 10% of non-IBD cases. Human CD tissues typically exhibited destructive full thickness enteritis with severe lympho-plasma infiltration and scattered additional granulomas; UC lesions exhibited much less inflammation than CD lesions. Non-IBD control samples did not exhibit pathological changes. Human CD and UC lesions were very different from Ptb lesions that are characterized by predominant granuloma formation. Immunohistochemistry for Map antigen and acid-fast staining were negative in all human IBD cases but were always positive in Ptb cases. Our present comparative study strongly suggests that we reconsider the previous hypothesis that "Map infection" causes CD, even though human intestines were considered to have been exposed to the Map antigen containing the DNA.

Impairment of α(2)-macroglobulin synthesis in experimental hepatopathic rats treated with turpentine oil.

The aim of this study was to investigate the synthesis of α(2)-macroglobulin (α2M) in hepatopathic rats injected with turpentine oil to induce acute inflammation. Hepatopathy was induced by oral administration of acetaminophen at a dose of 1 g/kg daily for 2 weeks or a 25% solution of carbon tetrachloride (CCl(4)) at 2 ml/kg body weight three times per week for 7 weeks. Acute inflammation was induced by intramuscular injection of turpentine oil at a dose of 1.0 ml/kg body weight. Serum concentrations of α2M were measured by enzyme-linked immunosorbent assay. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total protein differed significantly between acetaminophen or CCl(4)-induced hepatopathic rats and acetaminophen control (AA-control) or CCl(4) control (CC-control) rats. Furthermore, pathological examination confirmed hepatopathy in rat livers. Peak serum concentrations and area under the time-concentration curve for α2M showed significant differences between hepatopathic rats and AA-control or CC-control rats. Thus, serum concentrations of α2M did not increase when compared with nontreated rats.

Epidemiological situation and control strategies for paratuberculosis in Japan.

Paratuberculosis (Ptb), caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic and progressive granulomatous enteritis that affects many livestock and wild animals worldwide. The clinical disease is called Johne's disease (JD). In Japan, all dairy cattle (harf million head) are examined for Ptb every five years. About 1000 the officially examined cattle are diagnosed annually as positive for Ptb, but most of these exhibit only minor or no clinical signs and typical lesions in recent years. In contrast to the situation in Japan, the disease prevalence in western countries is very high. We have used ELISA and a culture examination of Map, and recently real-time PCR to diagnose this disease. In this review, the author outlines the history of the epidemic and national practical strategies to control paratuberculosis in Japan.

Mycobacterium avium subsp. paratuberculosis lipophilic antigen causes Crohn's disease-type necrotizing colitis in Mice.

BACKGROUND: A 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model was developed to investigate the pathogenesis and to evaluate a method of treating human Crohn's disease. This experimental model rapidly induces colitis similar to human Crohn's disease lesion in a reproducible manner. However, natural exposure of the human digestive tract to TNBS is unrealistic. A novel animal model based on realistic data is eagerly anticipated in future research on pathogenesis of CD. METHOD: We evaluated the potency of Map antigen molecules in an effort to develop a novel colitis model using a more realistic source than TNBS. We prepared the Map antigen by ethanol extraction and developed a mouse model in a manner similar to that of the well-known TNBS-induced colitis in mice. In the experiment, seven days after subcutaneous (SC) injection of the antigen into normal C57BL/6 mice, the same antigen in 50% ethanol was injected into the colon by the transanal route with a fine cannula. RESULTS: On the fifth day after the transanal injection, histopathological examination revealed full-thickness necrotizing colitis with erosion and ulcers; severe infiltration with neutrophils, lymphocytes, macrophages, and perforation. However, no change was detected with each single Map-antigen injection. CONCLUSION: The present results provide a novel animal model for research on CD and may be the key to clarifying the relationship between CD and Map. This is the first evidence that mycobacterium antigen induces necrotizing colitis.

Prostagladin D2 is a mast cell-derived antiangiogenic factor in lung carcinoma.

It is well established that prostaglandins (PGs) are involved in tumor angiogenesis and growth, yet the role of prostaglandin D(2) (PGD(2)) remains virtually unknown. Here, we show that host hematopoietic PGD(2) synthase (H-PGDS) deficiency enhances Lewis lung carcinoma (LLC) progression, accompanied by increased vascular leakage, angiogenesis, and monocyte/mast cell infiltration. This deficiency can be rescued by hematopoietic reconstitution with bone marrow from H-PGDS-naive (WT) mice. In tumors on WT mice, c-kit(+) mast cells highly express H-PGDS. Host H-PGDS deficiency markedly up-regulated the expression of proangiogenic factors, including TNF-α in the tumor. In mast cell-null Kit(W-sh/W-sh) mice, adoptive transfer of H-PGDS-deficient mast cells causes stronger acceleration in tumor angiogenesis and growth than in WT mast cells. In response to LLC growth, H-PGDS-deficient mast cells produce TNF-α excessively. This response is suppressed by the administration of a synthetic PGD(2) receptor agonist or a degradation product of PGD(2), 15-deoxy-Δ(12,14)-PGJ(2). Additional TNF-α deficiency partially counteracts the tumorigenic properties seen in H-PGDS-deficient mast cells. These observations identify PGD(2) as a mast cell-derived antiangiogenic factor in expanding solid tumors. Mast cell-derived PGD(2) governs the tumor microenvironment by restricting excessive responses to vascular permeability and TNF-α production.