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1.
Induction of the proinflammatory cytokines interleukin-1 (IL-1) (alpha and beta), IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-alpha) in pulmonary alveolar macrophages (PAMs) was assessed following experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) and/or Mycoplasma hyopneumoniae by using in vivo and in vitro models. The in vivo model consisted of pigs infected with PRRSV and/or M. hyopneumoniae and necropsied at 10, 28, or 42 days postinfection. Pigs infected with both pathogens had a greater percentage of macroscopic lung lesions, increased clinical disease, and slower viral clearance than pigs infected with either pathogen alone. The pigs infected with both PRRSV and M. hyopneumoniae had significantly increased levels of mRNA for many proinflammatory cytokines in PAMs collected by bronchoalveolar lavage (BAL) at all necropsy dates compared to those in uninfected control pigs. Increased levels of IL-1beta, IL-8, IL-10, and TNF-alpha proteins in BAL fluid, as measured by enzyme-linked immunosorbent assay, confirmed the increased cytokine induction induced by the pathogens. An in vitro model consisted of M. hyopneumoniae-inoculated tracheal ring explants cultured with PRRSV-infected PAMs. PAMs were harvested at 6 or 15 h postinfection with either or both pathogens. The in vitro study detected increased IL-10 and IL-12 mRNA levels in PAMs infected with PRRSV at all time periods. In addition, IL-10 protein levels were significantly elevated in the culture supernatants in the presence of M. hyopneumoniae-inoculated tracheal ring explants. The increased production of proinflammatory cytokines in vivo and in vitro associated with concurrent M. hyopneumoniae and PRRSV infection may play a role in the increased rates of pneumonia associated with PRRSV infection. The increased levels of IL-10 may be a possible mechanism that PRRSV and M. hyopneumoniae use to exacerbate the severity and duration of pneumonia induced by PRRSV and modulate the respiratory immune response.  相似文献   

2.
Induction of the proinflammatory cytokines interleukin-1 (IL-1) (α and β), IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α) in pulmonary alveolar macrophages (PAMs) was assessed following experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) and/or Mycoplasma hyopneumoniae by using in vivo and in vitro models. The in vivo model consisted of pigs infected with PRRSV and/or M. hyopneumoniae and necropsied at 10, 28, or 42 days postinfection. Pigs infected with both pathogens had a greater percentage of macroscopic lung lesions, increased clinical disease, and slower viral clearance than pigs infected with either pathogen alone. The pigs infected with both PRRSV and M. hyopneumoniae had significantly increased levels of mRNA for many proinflammatory cytokines in PAMs collected by bronchoalveolar lavage (BAL) at all necropsy dates compared to those in uninfected control pigs. Increased levels of IL-1β, IL-8, IL-10, and TNF-α proteins in BAL fluid, as measured by enzyme-linked immunosorbent assay, confirmed the increased cytokine induction induced by the pathogens. An in vitro model consisted of M. hyopneumoniae-inoculated tracheal ring explants cultured with PRRSV-infected PAMs. PAMs were harvested at 6 or 15 h postinfection with either or both pathogens. The in vitro study detected increased IL-10 and IL-12 mRNA levels in PAMs infected with PRRSV at all time periods. In addition, IL-10 protein levels were significantly elevated in the culture supernatants in the presence of M. hyopneumoniae-inoculated tracheal ring explants. The increased production of proinflammatory cytokines in vivo and in vitro associated with concurrent M. hyopneumoniae and PRRSV infection may play a role in the increased rates of pneumonia associated with PRRSV infection. The increased levels of IL-10 may be a possible mechanism that PRRSV and M. hyopneumoniae use to exacerbate the severity and duration of pneumonia induced by PRRSV and modulate the respiratory immune response.  相似文献   

3.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory illness in infected pigs. It has been postulated that the ability of PRRSV to induce the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages of infected pigs would be important for PRRSV immunopathogenesis, although this property would be variable and might be dependent on the strain. Several strains were reported to induce IL-10 in infected macrophages while others would not. In this study, we analyzed the IL-10 expression during in vitro and in vivo infections by a well-characterized virulent strain of PRRSV, vFL12, which is derived from an infectious clone. Our results showed that the vFL12 strain did not up-regulate IL-10 at mRNA or protein levels in either infected macrophages or dendritic cells in vitro. Furthermore, immunofluorescence staining for IL-10 on tonsil sections of PRRSV-infected pigs did not produce any evidence of IL-10 induction in PRRSV-infected cells or in bystander cells of the lymphoid tissues. Hence, based on these results obtained with a well-characterized highly pathogenic PRRSV strain it may be concluded that the induction of IL-10 release is not a part of the PRRSV virulence mechanisms.  相似文献   

4.
An experimental respiratory model was used to investigate the interaction between Mycoplasma hyopneumoniae and swine influenza virus (SIV) in the induction of pneumonia in susceptible swine. Previous studies demonstrated that M. hyopneumoniae, which produces a chronic bronchopneumonia in swine, potentiates a viral pneumonia induced by the porcine reproductive and respiratory syndrome virus (PRRSV). In this study, pigs were inoculated with M. hyopneumoniae 21 days prior to inoculation with SIV. Clinical disease as characterized by the severity of cough and fever was evaluated daily. Percentages of lung tissue with visual lesions and microscopic lesions were assessed upon necropsy at 3, 7, 14, and 21 days following SIV inoculation. Clinical observations revealed that pigs infected with both SIV and M. hyopneumoniae coughed significantly more than pigs inoculated with a single agent. Macroscopic pneumonia on necropsy at days 3 and 7 was greatest in both SIV-infected groups, with minimal levels of pneumonia in the M. hyopneumoniae-only-infected pigs. At 14 days post-SIV inoculation, pneumonia was significantly more severe in pigs infected with both pathogens. However, by 21 days postinoculation, the level of pneumonia in the dual-infected pigs was similar to that of the M. hyopneumoniae-only-infected group, and the pneumonia in the pigs inoculated with only SIV was nearly resolved. Microscopically, there was no apparent increase in the severity of pneumonia in pigs infected with both agents compared to that of single-agent-challenged pigs. The results of this study found that while pigs infected with both agents exhibited more severe clinical disease, the relationship between the two pathogens lacked the profound potentiation found with dual infection with M. hyopneumoniae and PRRSV. These findings demonstrate that the relationship between mycoplasmas and viruses varies with the individual agent.  相似文献   

5.
An experimental model was used to investigate mRNA cytokine profiles in bronchoalvolar cells (BALC) from piglets, infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). The BALC's were analyzed for the cytokines TNF-alpha, IFN-gamma, IL-8, IL-10, and IL-12(p40) by real-time TaqMan polymerase chain reaction in 2-, 4-, and 6-week-old piglets, respectively. High levels of IFN-gamma mRNA was detected in all piglets, while IL-10 was upregulated in 2-week-old piglets, was at normal levels in 4-week-old piglets, and elevated again in 6-week-old piglets. IL-12 was weakly elevated in all three age groups. Virus was reduced by 50% in 4-week-old piglets and cleared by 6 weeks of age. The sustained expression of IFNgamma and reduction of IL-10 production indicate an important role for these cytokines in immunity to PRRSV.  相似文献   

6.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a respiratory virus of swine that plays an important role in multifactorial respiratory disease. European strains of PRRSV cause mild or no respiratory signs on their own, but can sensitize the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to bacterial lipopolysaccharides (LPS). The inflammatory effect of LPS depends on the binding to the LPS receptor complex. Therefore, we quantified the levels of CD14 expression and LPS-binding protein (LBP) in the lungs of pigs throughout a PRRSV infection. Twenty-four gnotobiotic pigs were inoculated intranasally with PRRSV (10(6) 50% tissue culture infectious doses per pig, Lelystad strain) or phosphate-buffered saline (PBS), and euthanized 1-52 days later. Lungs were examined for CD14 expression (immunofluorescence and image analysis), LBP (ELISA), and virus replication. PRRSV infection caused a clear increase of CD14 expression from 3 to 40 days post-inoculation (DPI) and LBP from 7 to 14 DPI. Both parameters peaked at 9-10 DPI (40 and 14 times higher than PBS control pigs, respectively) and were correlated tightly with virus replication in the lungs. Double immunofluorescence labelings demonstrated that resident macrophages expressed little CD14 and that the increase of CD14 expression in the PRRSV-infected lungs was probably due to infiltration of highly CD14-positive monocytes in the interstitium. As both CD14 and LBP potentiate the inflammatory effects of LPS, their increase in the lungs could explain why PRRSV sensitizes the lungs for the production of proinflammatory cytokines and respiratory signs upon exposure to LPS.  相似文献   

7.
The objective of these experiments was to examine the ability of Helicobacter pylori to stimulate interleukin-10 (IL-10) or IL-12 and select for either Th1 or Th2 cells. Gastric biopsy specimens were collected from patients who were categorized with respect to the presence of H. pylori and gastric disease as well as their age, gender, medications, and other factors. As Th1 and Th2 cells are selected by IL-12 and IL-10, respectively, biopsy specimens were screened for mRNA and protein for these cytokines. Although mRNA for IL-12 and IL-10 was detected in biopsy specimens obtained from both infected and uninfected patients, IL-12 protein predominated. Levels of IL-10 and IL-12 in gastric tissue did not change in response to infection. Moreover, gamma interferon (IFN-gamma)-producing T cells were found in both the infected and the uninfected gastric mucosa. Stimulation of peripheral blood leukocytes from either infected or uninfected donors with various concentrations of live or killed H. pylori induced immunoreactive IL-12 and IL-10. After stimulation with live H. pylori, IL-12 levels increased more than 30-fold, whereas IL-10 levels increased only 2- to 5-fold, compared to cells stimulated with medium alone. Interestingly, killed H. pylori induced significantly more IL-10 (P < 0.05) than live H. pylori, while recombinant urease only induced IL-10. These results demonstrate that live H. pylori selectively stimulates the induction of IL-12 and Th1 cells that produce IFN-gamma, whereas preparations used in oral vaccines induce more IL-10 and may favor Th2 cell responses.  相似文献   

8.
Parameters of humoral and cellular immunity were investigated in pigs experimentally infected with a modified-live European porcine reproductive and respiratory syndrome virus (PRRSV, strain DV). PRRSV was detected by real-time RT-PCR and PRRSV-specific antibodies by a commercial ELISA test-kit, respectively. Interleukins IL-1alpha, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-alpha) and interferon-gamma (IFN-gamma) as well as IL-2 receptor (IL-2R) were quantified at mRNA level using RT-PCR. Subpopulations of blood lymphocytes were assayed using flow cytometry. No significant changes neither in cytokine expression nor in shifts of CD4 and CD8 markers could be found, but similar curve diagrams concerning CD8 single positive T cells could be observed in all vaccinated animals with an initial decrease and an increase between post-infection days (PIDs) 7 and 14. In the vaccination group, TNF-alpha and IL-6 tended to be increased at PIDs 22 and 40, whereas no increase could be seen in IFN-gamma. When comparing the in vivo immune response to that being seen in in vitro experiments, similar shifts of CD4/CD8 lymphocyte subpopulations may be seen. Cytokine curve diagrams, however, do not reflect the in vitro findings to that extent.  相似文献   

9.
In mice, respiratory syncytial virus (RSV) infection during allergic provocation aggravates the allergic Th2 immune response, characterised by production of interleukin (IL)-4, IL-5, and IL-13, and eosinophilic inflammation. This enhancement of the Th2 response occurs simultaneously with a strong RSV-induced Th1 cytokine response (IL-12 and IFN-gamma). The present study investigated whether IFN-gamma and IL-12 are critically involved in this RSV-enhanced OVA allergy. Therefore, IFN-gammaR- and IL-12-deficient mice (both on a 129/Sv/Ev background) were sensitised and challenged with ovalbumin (OVA) and infected with RSV during the OVA challenge period. Neither gene deletion affected the development of ovalbumin-induced allergic inflammation in mice. However, when OVA-allergic IFN-gammaR deficient mice were infected with RSV, an increased pulmonary eosinophilic infiltrate and increased IL-4 and IL-13 mRNA expression in lung tissue were observed compared with identically treated wild-type mice. In contrast, deficiency of IL-12 did not aggravate the Th2 immune and inflammatory response in OVA/RSV-treated mice, compared with wild-type. In conclusion, the virus-induced IFN-gamma response diminishes the Th2 inflammatory response during OVA allergy but fails to prevent totally the enhancement of the OVA allergy by RSV. In contrast, IL-12 is not involved in inhibiting nor increasing the RSV-enhanced allergy in 129/Sv/Ev mice.  相似文献   

10.
The aim of this study was to characterize the immune response of dendritic cells derived from monocytes (Mo-DCs) in the porcine peripheral blood following infection with porcine reproductive and respiratory syndrome virus (PRRSV). Viral load assays indicated that PRRSV efficiently infected Mo-DCs but failed to replicate, whereas PRRSV infection of Mo-DCs decreased the expression of SLA-I, SLA-II, CD80 and CD40 compared with those of mock Mo-DCs. Furthermore, we analyzed the cytokine profiles using quantitative RT-PCR and ELISA. Results indicated apparent changes in IL-10 and IL-12 p40 expression but not in IFN-γ and TNF-α among Mo-DCs infected with PRRSV and uninfected Mo-DCs. Additionally, flow cytometry analysis of the altered Mo-DCs together with IL-4 and GM-CSF induction for 7days revealed the typical morphology and phenotype with 91.73% purity before infection with PRRSV. Overall, our data demonstrate that PRRSV impaired the normal antigen presentation of Mo-DCs and led to inadequate adaptive immune response by down-regulating the expression of SLA-I,SLA-II, CD80 and CD40. Enhanced Th2 -type cytokine IL-10 secretion and reduced Th1-type cytokines IL-12p40,IFN-γ and TNF-α secretion results in Th1/Th2 imbalance.  相似文献   

11.
The expression of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) was examined immunohistochemically in the lungs of pigs aged 21 days infected experimentally with Mycoplasma hyopneumoniae (Mh). Ten pigs were inoculated intranasally with Mh and killed in pairs weekly from 7 to 35 days post-infection (dpi). Immunolabelling for IL-12 and IFN-gamma was usually associated with inflammation, particularly in macrophages and lymphocytes in the thickened alveolar septa and in the hyperplastic bronchus-associated lymphoid tissue (BALT). Cells positive for both cytokines were detected at 7 dpi, their numbers increasing at 14 and 21 dpi, and slightly decreasing thereafter. The results suggest that IL-12 and IFN-gamma play a role in pulmonary defence mechanisms against Mh infection.  相似文献   

12.
We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory respiratory viral infection affects the overall immunity to a subsequent porcine respiratory coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-β) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of respiratory viral co-infections, and provide more definitive approaches for treatment.  相似文献   

13.
Porcine circovirus 2 (PCV2) replication is characterized by high variation among infected pigs. This study investigated the role of immunologic responses in causing this variation. Twelve gnotobiotic pigs were inoculated with PCV2. Four of these pigs were treated with cyclosporin A (CysA) to monitor the effect of the adaptive immunity on the development of the PCV2 infection. Through lymph node biopsies at 10, 15, and 21 days postinoculation (DPI), PCV2 replication in lymphoid tissues was monitored. The production of total PCV2-specific and PCV2-neutralizing antibodies was followed, together with interferon-gamma (IFN-gamma) mRNA expression levels in peripheral blood monocytes as a marker for cellular immunity. In general, the CysA-treated pigs showed the highest PCV2 titers, indicating that the adaptive immunity is necessary to restrain PCV2 replication. Three different PCV2 replication patterns were observed in non-CysA-treated pigs. Pattern 1: In two pigs, PCV2 was not detected. They had the highest neutralizing antibody titers, appearing from 15 DPI. In these pigs a good cellular response was indicated by a peak in IFN-gamma mRNA at 15 DPI. Pattern 2: Five pigs contained low to moderate PCV2 titers at 15 DPI, remaining constant or decreasing towards 21 DPI. Lower neutralizing antibody titers were observed and no rise in IFN-gamma was detected. Pattern 3: In one pig, a low PCV2 titer at 15 DPI dramatically increased toward 21 DPI. Although an antibody response against PCV2 was mounted, no PCV2-neutralizing antibodies were detected. This pig also showed no rise in IFN-gamma. The study findings indicate that variation in the onset of the adaptive immunity may account for variation in PCV2 replication among pigs. Absence of PCV2-neutralizing antibodies may be an important factor in the development of an increased virus replication.  相似文献   

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15.
Porcine reproductive-respiratory syndrome virus (PRRSV) is a key agent in multifactorial respiratory disease of swine. Intratracheal administration of bacterial lipopolysaccharides (LPSs) to PRRSV-infected pigs results in markedly enhanced respiratory disease, whereas the inoculation of each component alone results in largely subclinical disease. This study examines whether PRRSV-LPS-induced respiratory disease is associated with the excessive production of proinflammatory cytokines in the lungs. Gnotobiotic pigs were inoculated intratracheally with PRRSV and then with LPS at 3, 5, 7, 10, or 14 days of infection and euthanatized 6 h after LPS inoculation. Controls were inoculated with PRRSV or LPS only or with phosphate-buffered saline. Virus titers, (histo)pathological changes in the lungs, numbers of inflammatory cells, and bioactive tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 levels in bronchoalveolar lavage fluids were examined. All pigs inoculated with PRRSV-LPS developed severe respiratory disease, whereas the controls that were inoculated with PRRSV or LPS alone did not. PRRSV infection significantly enhanced cytokine production in response to LPS. Peak TNF-alpha, IL-1, and IL-6 titers were 10 to 100 times higher in the PRRSV-LPS-inoculated pigs than in the pigs inoculated with PRRSV or LPS alone; and the titers correlated with the respiratory signs. The levels of neutrophil infiltration and the pathological changes detected in the lungs of PRRSV-LPS-inoculated pigs resembled those detected when the effects of PRRSV and LPS inoculated alone are combined, but with no synergistic effects between PRRSV and LPS. These data demonstrate a synergism between PRRSV and LPS in the induction of proinflammatory cytokines and an association between induction of these cytokines and disease.  相似文献   

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18.
Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.  相似文献   

19.
We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted with Helicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-gamma, TNF-alpha, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-gamma secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3(+) T cells and significantly reduced frequencies of Helicobacter-reactive CD4(+) Th1 cells in MLN. In contrast, anti-IFN-gamma and/or anti-TNF-alpha had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-gamma double-deficient mice, we further show that IFN-gamma is not required for the development of colitis following H. hepaticus infection. MLN cells from infected IL-10/IFN-gamma KO animals secreted elevated amounts of IL-12 and TNF-alpha following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.  相似文献   

20.
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