牦牛AIF-1蛋白表达及对巨噬细胞炎性因子mRNA的影响

旨在对牦牛同种移植炎症因子-1(allograft inflammatory factor-1,AIF-1)蛋白进行原核表达、纯化,并探讨其对巨噬细胞炎性因子的影响。采用q-PCR检测AIF-1基因在牦牛5种组织中的表达量,构建原核表达载体表达纯化AIF-1蛋白,q-PCR检测小鼠巨噬细胞4种炎性因子的表达量。结果表明,AIF-1基因在麦洼牦牛脾中表达水平最高,极显著高于其它组织(P<0.01)。表达并纯化出约29.47 ku的AIF-1重组蛋白,1.0、10.0、100.0μg·mL^-1 AIF-1蛋白均能促进小鼠巨噬细胞IL-1β、IL-6、TNF-α和iNOS的表达。这表明AIF-1在巨噬细胞免疫应答中发挥着一定作用,为深入研究牦牛AIF-1功能提供参考。     

Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.     

Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs

Despite accumulating knowledge of porcine macrophages and dendritic cells (DCs) from in vitro studies, information regarding monocytes/macrophages and DCs in lymphoid tissues of enteric pathogen-infected neonatal animals in vivo is limited. In this study we evaluated the influence of commensal bacterial [two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and L. reuteri] colonization and rotavirus infection on distribution and frequencies of monocytes/macrophages and conventional DCs (cDCs) in ileum, spleen and blood. Gnotobiotic pigs were inoculated with LAB and virulent Wa strain human rotavirus (HRV) (LAB+HRV+), HRV only (LAB-HRV+), LAB only (LAB+HRV-) or mock (LAB-HRV-). The cDCs were characterized as SWC3(+)CD11R1(+), whereas monocytes/macrophages were identified as SWC3(+)CD11R1(-) by flow cytometry in the gnotobiotic pigs at 10 days of age. Infection with HRV alone activated/recruited significantly more monocytes/macrophages to the intestine than LAB colonization and 56% versus 28% of these cells expressed CD14. Colonization with LAB alone also significantly increased the frequencies of monocytes/macrophages and cDCs and the CD14 expression on monocytes/macrophages in ileum and spleen compared to the controls. LAB colonization plus HRV infection significantly reduced macrophage and cDC frequencies in spleen compared to LAB colonization or HRV infection alone, suggesting that LAB colonization down-regulated HRV- infection-induced monocyte/macrophage activation/recruitment at the systemic lymphoid tissue. These results illustrated the distribution of porcine monocytes/macrophages and cDCs and the frequencies of CD14 expression on these cells in intestinal and systemic lymphoid tissues in the early stage of immune responses to intestinal colonization by LAB versus infection by an enteric pathogen HRV and will facilitate further in vivo studies on functional characterization of these immune cells in neonates.     

Monoclonal antibodies against porcine macrophages

Two mouse monoclonal antibodies (MAB) (clones 2G6 and 2B10) directed against porcine macrophages are described that are suitable for use in immunohistochemistry, FACS analysis and western blot. As immunogen, porcine cells from bronchoalveolar lavage (BAL) were used. The MABs obtained belonged to the mouse IgG1 subclass. The molecular weights of the corresponding antigens were detected by western blot under non-reducing conditions (2G6: 140-150kDa; 2B10: 140-145kDa). For specificity screening, porcine snap-frozen tissues of lung, lung lymph node, tonsil, spleen, thymus, brain, liver, gut and kidney were used. The MABs were able to identify cell populations of the mononuclear phagocytic system in these organs. While MAB 2G6 detected tissue macrophages (sinusoidal lymph node macrophages, red pulp spleen macrophages, Kupffer cells, Langerhans cells, thymus macrophages, macrophages of lung and macrophages of kidney), MAB 2B10 stained cells scattered in the lymph node (subsinusoidal, interfollicular and follicular macrophages) and in the lung interstitium. Additionally, it showed reactivity with Kupffer cells, spleen and kidney macrophages. An immunoreactivity of the MABs could be established also for human but not for bovine and avian macrophages. By flow cytometric analysis, MAB 2B10 reacted with a subpopulation of BAL and peritoneal cells. Antibody 2G6 detected macrophages of the BAL and the peritoneal fluid as well as peripheral blood monocytes.     

Immunohistochemical characterization of macrophage and dendritic cell subpopulations of the spleen,thymus, tongue and heart in cyclophosphamide-induced immunosuppressed rat

This study was undertaken to investigate the immunohistochemical characterization of different subpopulations of macrophages and dendritic cells (DCs) of the spleen, thymus, tongue and heart in cyclophosphamide (CY)-induced immunosuppressed rat. After CY treatment, remarkably, ED1+, ED2+ and ED3+ macrophage subpopulations, in general exhibited signs of cellular activation such as an increase in number and size of cell, and an upregulation of the ED1, ED2 and ED3 reactive surface molecule expression in all the organs studied, except for some macrophage subpopulations including ED1+ macrophages in the non-lymphoid tissues. Subpopulations of DCs showed a differential sensitivity to CY. Lymphoid DCs were more sensitive to CY than non-lymphoid interstitial DCs. CY induced a conspicuous upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in the vascular endothelial cells, splenic marginal zone and thymic cortex. In this study, we demonstrated the in vivo effects of CY treatment on subpopulations of macrophages and DCs as well as on ICAM-1 expression in the rat spleen, thymus, tongue and heart. Moreover, our results shed more light on the activation effects of CY on certain subpopulations of macrophages, on the differential sensitivity of DCs to CY between the immature and mature ones, on the functional role of different subpopulations of macrophages, and on the significance of upregulated ICAM-1 expression in the splenic marginal zone and thymic cortex after CY treatment.     

Interleukin 1 alpha mRNA-expressing cells on the local inflammatory response in feline infectious peritonitis.

By in situ hybridization with biotinylated human interleukin 1 alpha (IL-1 alpha) cDNA probe, distribution of feline IL-1 alpha mRNA-expressing cells was examined in the tissues from 49 cases diagnosed as feline infectious peritonitis (FIP) by pathological examination. IL-1 alpha mRNA-expressing cells were found in visceral peritoneum, lymphoid organs, liver, kidney, pancreas, digestive tract, lung, pleura, brain, palpebral conjunctiva, and bone marrow. Hybridization signals for IL-1 alpha mRNA were mostly located in the local inflammatory lesions on the serosal surface of various organs and omentum, which were frequently involved in the lesions of FIP (27.8 +/- 5.1 cells/mm2). Morphological examination suggested that they were infiltrated macrophages. However, few IL-1 alpha mRNA-expressing macrophages were in the lesions of other organs. These data suggested that IL-1 alpha produced from macrophages in the local inflammatory sites might participate in the initiation and development of the lesions on the visceral peritoneum in FIP.     

IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪不同组织和细胞中的表达特征分析

白细胞介素1受体拮抗剂（interleukin 1 receptor antagonist,IL1RN）是一种重要的宿主免疫调节因子,是天然存在的IL-1拮抗剂。为了解IL1RN基因在断奶仔猪不同组织和细胞中表达的特征,利用Trizol法提取晋汾白猪仔猪和新山西黑猪仔猪的心脏、肝脏、脾脏、淋巴细胞、巨噬细胞等18种组织和细胞的总RNA,采用RT-PCR方法检测IL1RN基因在不同组织和细胞中的表达情况,应用SPSS 19.0软件分析IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪4种免疫组织和细胞（脾脏、淋巴结、巨噬细胞、白细胞）中的表达差异情况。结果显示：IL1RN基因在晋汾白猪仔猪和新山西黑猪仔猪的脂肪组织中均不表达,在其余各组织和细胞中均表达;IL1RN基因在晋汾白猪仔猪的巨噬细胞与淋巴结、脾脏、白细胞之间的表达差异极显著（P<0.01）,且在淋巴结与脾脏、白细胞之间的表达差异极显著（P<0.01）;IL1RN基因在新山西黑猪仔猪的脾脏、淋巴结、巨噬细胞、白细胞的表达两两之间均呈极显著差异（P<0.01）;IL1RN基因在2个猪种的脾脏、巨噬细胞、白细胞之间的表达呈极显著差异（P<0.01）,在淋巴结中的表达呈显著差异（P<0.05）。该研究结果为进一步揭示2个猪种的IL1RN基因在疾病抵抗方面的作用及相关机制提供了依据。     

Pathology and virus tissue distribution of Turkey origin reoviruses in experimentally infected Turkey poults

The pathogenesis of 4 isolates of turkey-origin reovirus (NC/SEP-R44/03, NC/98, TX/98, and NC/85) and 1 chicken-origin reovirus (1733) was examined by infecting specific pathogen free (SPF) poults. These turkey-origin reovirus (TRV) isolates were collected from turkey flocks experiencing poult enteritis and are genetically distinct from previously reported avian reoviruses. Microscopic examination of the tissues collected from the TRV-infected poults revealed different degrees of bursal atrophy characterized by lymphoid depletion and increased fibroplasia between the bursal follicles. To understand the relationship between virus spread and replication, and the induction of lesions, immunohistochemical staining (IHC) for viral antigen, in situ hybridization (ISH) for the detection of viral RNA, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for the detection of apoptosis in affected tissues was performed. Both IHC and ISH revealed viral antigen and RNA in the surface epithelial cells of the bursa, in macrophages in the interstitium of the bursa and, to lesser degree, in splenic red pulp macrophages and intestinal epithelial cells. Increased apoptosis of bursal lymphocytes and macrophages was observed at 2 and 5 days postinoculation. No lesions were found in tissues from poults inoculated with the virulent chicken-origin strain, however viral antigen was detected in the bursa and the intestine. Although all TRVs studied displayed similar tissue tropism, there were substantial differences in the severity of the lesions produced. Poults inoculated with NC/SEP-R44/03 or NC/98 had moderate to severe bursal atrophy, whereas poults inoculated with TX/98 or NC/85 presented a mild to moderate bursal lymphoid depletion. The lymphoid depletion observed in the bursa appears to be the effect of an indirectly induced apoptosis and would most likely result in immune dysfunction in poults infected with TRV.     

Comparison of in vitro replication and cytopathology caused by strains of canine distemper virus of vaccine and field origin

Three biological properties of canine distemper virus were examined to determine if any would consistently differentiate field from vaccine strains of the virus. The properties were the ability to (1) infect macrophages and epithelial cells, (2) produce distinct cytopathologic effect in alveolar and peritoneal macrophages and Vero cells, and (3) produce pocks on the chorioallantoic membrane of embryonated chicken eggs. Four vaccine strains and 5 field isolates were used in the study. The 5 field isolates were obtained directly from canine tissues. Of the 3 properties studied, only the comparison of the ability of the viruses to infect macrophages and epithelial cells was a consistent marker of virus origin. Virulent field isolates would only infect macrophage cultures, whereas the vaccine strains infected both types of cells. One avirulent field isolate from a case of old dog encephalitis reacted more like a vaccine strain by infecting both cell types.     

Relationship between Mycobacterium avium subspecies paratuberculosis, IL-1alpha, and TRAF1 in primary bovine monocyte-derived macrophages

Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular pathogen that resides in host macrophage cells. Presently, little is known about how MAP is able to subvert the normal bacteriocidal functions of infected macrophages. Previously, we reported that ileal tissues from MAP infected cattle contained high levels of interleukin-1 alpha (IL-1alpha) and tumor necrosis factor receptor-associated factor 1 (TRAF1), relative to ileal tissues from uninfected cattle. High-level expression of these two proteins could have profound effects on macrophage function, intracellular signaling, and apoptosis. We now demonstrate that high levels of TRAF1 protein are located primarily within macrophages infiltrating areas of MAP infection. We have also utilized cultured bovine monocyte-derived macrophage cells (MDM) either infected with live MAP or stimulated with recombinant IL-1alpha (rIL-1alpha) to determine if there is a relationship between IL-1alpha and TRAF1 expression. These studies have identified a dose dependent increase in TRAF1 protein levels in bovine MDM in response to infection with live MAP or following treatment with rIL-1alpha. Sustained TRAF1 protein expression was dependent upon interaction of rIL-1alpha with it's receptor and rIL-1beta was also able to enhance TRAF1 gene expression. Our results suggest that MAP may use the IL-1-TRAF1 system to enhance TRAF1 protein expression in infected bovine MDM. These novel results provide evidence for a new avenue of research on the effect of MAP and other intracellular pathogens on macrophage signaling and apoptosis.     

Blood smear from a pregnant cat that died shortly after partial abortion

A 1-year-old, 5- to 6-week pregnant cat was presented with a history of aborting 3 kittens the previous night. During the examination, the cat began to seizure and died. At necropsy, formalin-fixed tissues and blood smears prepared from an EDTA blood sample collected via cardiac puncture were submitted to a diagnostic laboratory. Examination of the blood smears revealed numerous large (15-75 microm) clumped macrophages containing dark blue intracytoplasmic organisms consistent with Cytauxzoon merozoites. Scattered erythrophagocytic macrophages were also observed. Within several erythrocytes, 1-2 small, round, ring-like piroplasms consistent with Cytauxzoon were observed. Histologic examination revealed numerous large, schizont-laden macrophages filling the blood vessels of multiple organs. The cytologic and histologic findings were diagnostic for Cytaxuzoon felis infection. This case was of particular interest because of the unusually large number of organism-laden macrophages in the cardiac blood sample, an uncommon finding in peripheral blood. Although the cat was presented for complications of abortion, it remains uncertain whether C felis organisms crossed the placenta and infected the fetuses or lead to the abortions.     

Changes in gene expression profiles and cytokine secretions in peripheral monocytes by treatment with small extracellular vesicles derived from a canine lymphoma cell line

Interactions between tumor and immune cells within the tumor microenvironment play an important role in tumor progression, and small extracellular vesicles (EVs) derived from these tumor cells have been shown to exert immunomodulatory effects on various immune cells, including macrophages and lymphocytes. Although the immunomodulatory effects of small EVs derived from human cancer cells have been intensively investigated, few studies have investigated the effects of lymphoma-derived small EVs on macrophages in both human and veterinary medicine. Here, we evaluated the effects of canine lymphoma-derived small EVs on canine primary monocytes, which are the major source of macrophages in neoplastic tissues. Comprehensive gene expression analysis of these treated monocytes revealed their distinct activation via the Toll-like receptor (TLR) and NF-κβ signaling pathways. In addition, treatment with lymphoma small EVs increased the secretion of MCP-1, which induces the infiltration and migration of monocytes and lymphocytes in neoplastic and cancer tissues. The results of this study indicate that canine lymphoma small EVs activate monocytes, possibly through the activation of TLR and NF-κβ signaling pathways, and induce monocytes to secrete of MCP-1, which might contribute to immune cell infiltration within the tumor microenvironment.     

Lentiviruses are etiological agents of chronic diseases in animals and acquired immunodeficiency syndrome in humans.

Lentiviruses are species-specific, exogenously transmitted retroviruses that have a unique ability to replicate continuously but at a restricted rate in host tissues. This property is thought to be related to the retroviral nature of the replication process (RNA to DNA to RNA) and to the ability of the viruses to do this in cells of the macrophage lineage. The viral genomes are expressed only in certain populations of macrophages and this is dependent on a number of interactive factors including the genus of the host, the age of the host, maturation/differentiation factors in macrophages, the strain of virus and regulatory factors in the virus and the regulatory factors in the virus and the macrophages. Macrophages permissive for virus replication are found in specific tissues and virus replication in the cells causes development of lesions in the particular tissues. The nature of the lesions varies from virus induced necrosis to immunopathology to possible toxic infects of monokines produced by the infected macrophages. Cats and primates have further complicating diseases caused by the remarkable sensitivity of their helper T lymphocytes to infection with their lentiviruses. Elimination of these cells leads to onset of various local and systemic diseases caused by opportunistic agents. Whereas equidae and small ruminant animals develop diseases related to infection in macrophage populations, felines, macaques and humans develop diseases related to both infection in their macrophages and elimination of their T lymphocytes.     

Distribution of immunoglobulin-bearing leukocytes in bovine mammary tissue infected chronically with Staphylococcus aureus

Mammary parenchymal and test end tissues from cows with chronic Staphylococcus aureus mastitis were examined to determine the distribution of immunoglobulin (Ig) G1- and IgG2-bearing leukocytes. Leukocytes bearing IgG2 predominated in S. aureus infected quarters, with highest numbers observed at the Furstenberg's rosette followed by streak canal and parenchymal tissue areas. Significantly more IgG1- and IgG2-bearing leukocytes were observed at the Furstenberg's rosette and significantly more IgG2-bearing leukocytes were observed at the streak canal of S. aureus infected quarters compared to uninfected quarters. Receptors for cytophilic IgG on neutrophils and macrophages may increase efficiency of phagocytosis and improve the antimicrobial effectiveness of these cells in treat end tissues.     

Characterization of cytoplasmic lysozyme immunoreactivity as a histiocytic marker in normal canine tissues

The immunoreactive lysozyme distribution in normal canine tissues was determined to assess the value of lysozyme as a marker of histiocytic differentiation. Lysozyme was largely confined to monocytes, neutrophils, macrophages, serous cells in mucosal associated exocrine glands, and renal proximal tubular epithelium. Macrophages in the majority of tissues stained intensely for lysozyme, although in lymphoid tissues not all acid phosphatase and nonspecific esterase positive cells contained lysozyme. In particular, dendritic antigen presenting cells, including Langerhans cells, follicular dendritic cells, and interdigitating reticulum cells, lacked immunoreactive lysozyme; hence lysozyme appeared to represent a discriminatory marker with respect to these cells. Also, a small proportion of non-dendritic macrophages appeared to lack lysozyme. It was concluded that the demonstration of immunoreactive lysozyme was a useful adjunct to conventional morphological techniques for the identification of tissue macrophages provided that due caution was exercised in the interpretation of the results of lysozyme staining.     

Lectin histochemistry of Peyer's patches in the porcine ileum

Lectin staining pattern in Peyer's patches of porcine ileum was studied using twenty one biotinylated-labeled lectins as cell markers which were visualized with avidin-biotin-peroxidase complex method (ABC). WGA appears to be a selective marker for tingible body macrophages in the porcine germinal centers. ConA may be a positive marker for the lymphoid tissues, whereas 9 lectins (DBA, SBA, SJA, s-WGA, PNA, ECL, UEA-I, PHA-E, and PHA-L) may be negative markers for the lymphoid tissues in all areas.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

Detection of porcine circovirus 2 (PCV-2) DNA by nested PCR from formalin-fixed tissues of post-weaning multisystemic wasting syndrome (PMWS) pigs in Thailand

A retrospective study for the detection of porcine circovirus 2 (PCV-2) DNA was conducted by nested PCR method in 16 cases of swine post-weaning multisystemic wasting syndrome (PMWS) in Thailand. Histopathology showed characteristic lesions of PMWS and intracytoplasmic viral inclusion bodies in macrophages infiltrating in lymphoid tissues. PCV-2 DNA was detected from formalin-fixed and/or formalin-fixed paraffin-embedded tissues from all pigs with PMWS. The amplified products were digested with Hae III.     

Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in-situ hybridisation

Non-radioactive digoxigenin (DIG)-labelled probes that can differentiate porcine circovirus (PCV) 1 from PCV2 in formalin-fixed, paraffin-wax-embedded tissues by in-situ hybridisation were developed. A 349 base pair (bp) DNA fragment from open reading frame (ORF) 1 of PCV1 and a 481 bp DNA fragment from ORF2 of PCV2 generated by polymerase chain reaction (PCR) were used as PCV1 and PCV2 probes, respectively. A specific DIG-labelled PCV1 DNA probe did not hybridise with PCV2-infected PK-15 cells and vice versa. From the 40 field cases with postweaning multisystemic wasting syndrome tested by in-situ hybridisation, 30 (75 per cent) cases were PCV2-positive only and 10 (25 per cent) cases were positive for both PCV1 and PCV2. PCV1 and PCV2 DNAS were detected mainly in the macrophages of lymph nodes and spleens. Positive cells typically exhibited a dark brown to black reaction product mainly in the cytoplasm but also occasionally in the nucleus. In-situ hybridisation together with the differential probes developed in the present study represent an additional tool capable of differentiating of both types of PCV in formalin-fixed, paraffin-wax-embedded tissues.