A porcine alveolar macrophage cell line stably expressing CD163 demonstrates virus replication and cytokine secretion characteristics similar to primary alveolar macrophages following PRRSV infection.

The in vitro investigation of cytokine secretion induced by porcine reproductive and respiratory syndrome virus (PRRSV) requires porcine alveolar macrophages (PAMs) and their interaction with immunocytes. However, immortalized monoclonal PAMs (mPAMs) are non-permissive for PRRSV infection. The porcine CD163 receptor isolated from primary PAMs (pPAMs) confers susceptibility to PRRSV infection; thus, this approach could be used to establish a novel cell line to facilitate the exploration of PRRSV infection kinetics. Here, we amplified the coding region of the CD163 gene from pPAMs and integrated it into an mPAM line using a lentivirus expression system. After verification, the monoclonal PAM cell line stably expressing CD163 (mPAM-CD163-GFP) was infected with either the highly pathogenic PRRSV strain JXA1 or the classical PRRSV strain SD1, which produced high infectious titers of progeny virus reaching > 109 copies/mL or a 50 % tissue culture infective dose of 105.5 over at least 100 cell generations. We also investigated cytokine and Toll-like receptor expression in infected mPAM-CD163-GFP cells and pPAMs. The mPAM-CD163-GFP cell line showed similar patterns of viral replication and cytokine secretion compared with pPAMs, so it may be extremely useful for replacing primary cells for in vitro investigations of the mechanisms of cytokine secretion and interactions between PRRSV-infected PAMs and immunocytes.

Decreased synovial fluid pituitary adenylate cyclase-activating polypeptide (PACAP) levels may reflect disease severity in post-traumatic knee osteoarthritis after anterior cruciate ligament injury.

BACKGROUND: It has been demonstrated that anterior cruciate ligament (ACL) injury-induced cartilage degeneration is the key risk factor for post-traumatic knee osteoarthritis (PTKOA).Pituitary adenylate cyclase-activating polypeptide (PACAP), a common neuropeptide exerting a wide spectrum of functions, has been proved to inhibit inflammation and prevent cartilage degeneration. OBJECTIVE: The current study was performed to investigate circulating and synovial fluid PACAP concentrations in ACL injury patients to determine their relationship with the disease progression of the severity of post-traumatic knee osteoarthritis (PTKOA). METHODS: 72 ACL injury patients receiving arthroscopical examination and surgery were enrolled in the study. Meanwhile, 60 gender-and-age non-traumatic patellar dislocation patients were enrolled as controls. The VAS score, Lysholm Score and International Knee Documentation Committee (IKDC) score were all recorded to evaluate the clinical severity. Serum and synovial fluid (SF) PACAP levels were investigated by enzyme-linked immunosorbent assay (ELISA).The IL-1ß and TNF-α levels were also investigated. The degree of meniscus injury was assessed by MR imaging. The modified Mankin score was recorded to examine the cartilage histopathological alternations. Receiver operating characteristic (ROC) curve was performed to discuss the diagnostic value of PACAP levels for the prediction of the radiographic grading in comparison with IL-1ß and TNF-α. RESULTS: Serum PACAP levels between PTKOA patients and patellar dislocation did not reach significant differences. However, SF PACAP levels were significantly lower in PTKOA patients than controls. In addition, SF PACAP levels were negatively associated with MRI imaging grade for meniscus injury and VAS score, and were positively associated with Lysholm and IKDC scores. In addition, SF PACAP levels were negatively related to Mankin score as well as the expressions of IL-1ß and TNF-α. ROC analysis curve showed that attenuated PACAP may serve as a favorable marker for the diagnosis of MRI for meniscus injury. CONCLUSIONS: SF PACAP concentrations showed an independent and negative association with disease severity in PTKOA following ACL injury. Local treatment with PACAP may act as a possible adjuvant therapy for delaying the process of PTKOA.

The synovial fluid neuropeptide PACAP may act as a protective factor during disease progression of primary knee osteoarthritis and is increased following hyaluronic acid injection.

The correlation of serum and synovial fluid (SF) pituitary adenylate cyclase-activating polypeptide (PACAP) levels with disease progression of primary knee osteoarthritis (OA) was explored. Radiographic severity of OA was determined by Kellgren-Lawrence (K-L) grades. PACAP levels were measured by ELISA before treatment, and 4 and 8 wk following hyaluronic acid (HA) injection. Levels of IL-1ß and MMP-3 were also detected. The numeric pain scale (NPS), revised Oxford Knee Score (OKS), and American Knee Society Score (AKSS) were employed to evaluate to symptomatic severity. Receiver-operating-characteristic (ROC) curve analysis was carried out to compare the diagnostic value of PACAP, IL-1ß, and MMP-3 for the K-L grade. PACAP concentrations in SF but not serum were significantly lower in OA patients compared with controls. SF PACAP levels were negatively associated with K-L grades and higher NPS as well as worse AKSS and OKS. Further analysis demonstrated that PACAP concentration in SF was negatively correlated with expressions of IL-1ß as well as MMP-3 and may act as a marker for radiographic progression along with MMP-3. Last, we found SF PACAP levels exhibited an incremental trend after HA injection. These findings confirmed the crucial role of PACAP deficiency in the development of primary knee OA.

[Over-expression of platelet-derived growth factor-BB in degenerative hypertrophied ligamentum flavum].

OBJECTIVE: To investigate the role of platelet-derived growth factor-BB (PDGF-BB) in the degeneration of the hypertrophied ligamentum flavum (LF) in the lumbar spine. METHODS: Surgical specimens of degenerative hypertrophied LF were obtained from 11 patients with lumbar spinal stenosis (LSS, mean age 57.8 years), with those from 10 age-matched patients with lumbar disc herniation (LDH, mean age 53.5 years) as the normal controls. The thickness of the LF was measured preoperatively by axial T1-weighted magnetic resonance imaging (MRI) at the facet joint level. Immunohistochemistry was employed to determine the expression of PDGF ß and PDGF-BB in the LF. The mRNA and protein expressions of PDGF-BB in the LF were detected using real-time PCR and Western blotting, respectively. RESULTS: The thickness of the LF was 5.30±1.12 mm in the degenerative group and 2.80±1.53 mm in the control group, showing a significant difference between the two groups (P<0.001). PDGF-ß and PDGF-BB were positive in the fibroblasts in hypertrophied LF. The mRNA and protein expressions of PDGF-BB were significantly higher in the degenerative group than in the control group (P=0.013 and 0.023, respectively). CONCLUSION: The high expression of PDGF-BB in the hypertrophied LF suggests its important role in the development of hypertrophy of LF in lumbar spinal canal stenosis.