Demonstrating the effectiveness of intra-articular prolotherapy combined with peri-articular perineural injection in knee osteoarthritis: a randomized controlled trial.

BACKGROUND: This study aimed to compare the efficacy of intra-articular prolotherapy (IG) combined with peri-articular perineural injection (PG) in the management of knee osteoarthritis (KOA). METHODS: A total of 60 patients with the diagnosis of KOA were included in this double-blinded randomized controlled clinical trials. The inclusion criteria were as follow: (1) 48-80 years old; (2) the diagnose of KOA; (3) the grade 2 and 3 of the Kellgern-Lawrence grading scale; (4) the pain, crepitation, and knee joint stiffness continuing for 3 months at least. The main exclusion criteria were as follow: (1) any infection involving the knee skin; (2) history of any Influencing factors of disease. All patients were divided into three groups and received either IG, PG and I + PG under the ultrasound guidance and the 2, 4 and 8 weeks follow-up data of patients were available. (IG n = 20 or PG n = 20, I + PG n = 20). Visual Analogue Scale (VAS), The Western Ontario McMaster University Osteoarthritis Index (WOMAC) and the pressure pain threshold (PPT) were used as outcome measures at baseline, 2, 4 and 8 weeks. RESULTS: There were no statistically significant differences in terms of age, sex, BMI, duration of current condition and baseline assessments of pain intensity, WOMAC scores and PPT. After treatment, the improvement of VAS activity, WOMAC and PPT values was showed compared with pre-treatment in all groups (p < 0.05). At 4 and 8 weeks after treatment, the VAS and WOMAC scores of the I + PG were significantly lower than those of the PG or IG, and the difference was statistically significant (p < 0.05). The PPT values of PG and I + PG were significantly improved compared to IG at 2, 4, and 8 weeks after treatment. CONCLUSION: The ultrasound guided I + PG of 5% glucose seem to be more effective to alleviate pain and improve knee joint function than single therapy in short term. Clinical rehabilitators could clinically try this combination of I + PG to improve clinical symptoms in patients with KOA.

New insights into a microvascular invasion prediction model in hepatocellular carcinoma: A retrospective study from the SEER database and China.

Background and Aims: The prognosis of liver cancer is strongly influenced by microvascular infiltration (MVI). Accurate preoperative MVI prediction can aid clinicians in the selection of suitable treatment options. In this study, we constructed a novel, reliable, and adaptable nomogram for predicting MVI. Methods: Using the Surveillance, Epidemiology, and End Results (SEER) database, we extracted the clinical data of 1,063 patients diagnosed with hepatocellular carcinoma (HCC) and divided it into either a training (n = 739) or an internal validation cohort (n = 326). Based on multivariate analysis, the training cohort data were analyzed and a nomogram was generated for MVI prediction. This was further verified using an internal validation cohort and an external validation cohort involving 293 Chinese patients. Furthermore, to evaluate the efficacy, accuracy, and clinical use of the nomogram, we used concordance index (C-index), calibration curve, and decision curve analysis (DCA) techniques. Results: In accordance with the multivariate analysis, tumor size, tumor number, alpha-fetoprotein (AFP), and histological grade were independently associated with MVI. The established model exhibited satisfactory performance in predicting MVI. The C-indices were 0.719, 0.704, and 0.718 in the training, internal validation, and external validation cohorts, respectively. The calibration curves showed an excellent consistency between the predictions and actual observations. Finally, DCA demonstrated that the newly developed nomogram had favorable clinical utility. Conclusions: We established and verified a novel preoperative MVI prediction model in HCC patients. This model can be a beneficial tool for clinicians in selecting an optimal treatment plan for HCC patients.

SGLT2 inhibition and renal urate excretion: role of luminal glucose, GLUT9, and URAT1.

Inhibitors of the Na+-glucose cotransporter SGLT2 enhance urinary glucose and urate excretion and lower plasma urate levels. The mechanisms remain unclear, but a role for enhanced glucose in the tubular fluid, which may interact with tubular urate transporters, such as the glucose transporter GLUT9 or the urate transporter URAT1, has been proposed. Studies were performed in nondiabetic mice treated with the SGLT2 inhibitor canagliflozin and in gene-targeted mice lacking the urate transporter Glut9 in the tubule or in mice with whole body knockout of Sglt2, Sglt1, or Urat1. Renal urate handling was assessed by analysis of urate in spontaneous plasma and urine samples and normalization to creatinine concentrations or by renal clearance studies with assessment of glomerular filtration rate by FITC-sinistrin. The experiments confirmed the contribution of URAT1 and GLUT9 to renal urate reabsorption, showing a greater contribution of the latter and additive effects. Genetic and pharmacological inhibition of SGLT2 enhanced fractional renal urate excretion (FE-urate), indicating that a direct effect of the SGLT2 inhibitor on urate transporters is not absolutely necessary. Consistent with a proposed role of increased luminal glucose delivery, the absence of Sglt1, which by itself had no effect on FE-urate, enhanced the glycosuric and uricosuric effects of the SGLT2 inhibitor. The SGLT2 inhibitor enhanced renal mRNA expression of Glut9 in wild-type mice, but tubular GLUT9 seemed dispensable for the increase in FE-urate in response to canagliflozin. First evidence is presented that URAT1 is required for the acute uricosuric effect of the SGLT2 inhibitor in mice.

Identification and function of adenosine A3 receptor in afferent arterioles.

Adenosine plays an important role in regulation of renal microcirculation. All receptors of adenosine, A1, A2A, A2B, and A3, have been found in the kidney. However, little is known about the location and function of the A3 receptor in the kidney. The present study determined the expression and role of A3 receptors in mediating the afferent arteriole (Af-Art) response and studied the interaction of A3 receptors with angiotensin II (ANG II), A1 and A2 receptors on the Af-Art. We found that the A3 receptor expressed in microdissected isolated Af-Art and the mRNA levels of A3 receptor were 59% of A1. In the isolated microperfused Af-Art, A3 receptor agonist IB-MECA did not have a constrictive effect. Activation of A3 receptor dilated the preconstricted Af-Art by norepinephrine and blunted the vasoconstrictive effect of both adenosine A1 receptor activation and ANG II on the Af-Art, respectively. Selective A2 receptor antagonist (both A2A and A2B) had no effect on A3 receptor agonist-induced vasodilation, indicating that the dilatory effect of A3 receptor activation is not mediated by activation of A2 receptor. We conclude that the A3 receptor is expressed in the Af-Art, and activation of the A3 receptor dilates the Af-Art.

Aldosterone stimulates superoxide production in macula densa cells.

Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O(2)(-)) and nitric oxide (NO) by macula densa (MD) cells. MD O(2)(-) inactivates NO; however, among the factors that increase MD O(2)(-) release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O(2)(-) production due to upregulation of cyclooxygenase-1 (COX-2) and NOX-2, and NOX-4, isoforms of NAD(P)H oxidase. Studies were performed on MMDD1 cells, a renal epithelial cell line with properties of MD cells. RT-PCR and Western blotting confirmed the expression of MR. Aldosterone (10(-8) mol/l for 30 min) doubled MMDD1 cell O(2)(-) production, and this was completely blocked by MR inhibition with 10(-5) mol/l eplerenone. RT-PCR, real-time PCR, and Western blotting demonstrated aldosterone-induced increases in COX-2, NOX-2, and NOX-4 expression. Inhibition of COX-2 (NS398), NADPH oxidase (apocynin), or a combination blocked aldosterone-induced O(2)(-) production to the same degree. These data suggest that aldosterone-stimulated MD O(2)(-) production is mediated by COX-2 and NADPH oxidase. Next, COX-2 small-interfering RNA (siRNA) specifically decreased COX-2 mRNA without affecting NOX-2 or NOX-4 mRNAs. In the presence of the COX-2 siRNA, the aldosterone-induced increases in COX-2, NOX-2, and NOX-4 mRNAs and O(2)(-) production were completely blocked, suggesting that COX-2 causes increased expression of NOX-2 and NOX-4. In conclusion 1) MD cells express MR; 2) aldosterone increases O(2)(-) production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O(2)(-). The resulting balance between O(2)(-) and NO in the MD is important in modulating TGF.

NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa.

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O(2)(-)) and angiotensin II (ANG II). We have reported that NaCl-induced O(2)(-) in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O(2)(-) in MD is unknown. Thus we determined the pathways by which ANG II increased O(2)(-) in the MD by measuring O(2)(-) in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O(2)(-) levels to increase by more than twofold (P < 0.01). This increase was blocked by losartan (AT(1) receptor blocker) but not PD-123319 (AT(2) receptor antagonist). Apocynin (a NOX inhibitor) decreased O(2)(-) by 86% (P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O(2)(-) was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O(2)(-), whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O(2)(-) production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O(2)(-) production in the MD primarily via AT(1)-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

In vitro suppression of xenoimmune-mediated macrophage activation by human CD4+CD25+ regulatory T cells.

BACKGROUND: Macrophages are important effector cells in T cell-mediated xenograft rejection. The aim of this study was to determine whether CD4+CD25+ regulatory T cells (Tregs) were capable of suppressing macrophage activation in vitro. METHODS: Porcine cell or xenoantigen-primed human peripheral blood mononuclear cells, CD4+ T cell-depleted peripheral blood mononuclear cells, or CD14+ macrophages plus autologous CD4+CD25- T cells were cultured with or without expanded autologous Tregs. Transwell cultures were used to separate the various components to determine the need for cell-cell contact. RESULTS: Pig cell primed CD14+ macrophages required the presence of CD4+CD25- T cells for activation and increased expression of CD40, interleukin-12, and tumor necrosis factor-alpha. This up-regulated expression of macrophage activation markers was reduced substantially in the presence of autologous Tregs. Coculture with Tregs did not alter macrophage viability but reduced the capacity of macrophages to stimulate proliferation of responder T cells. Tregs required direct contact with CD4+CD25- T cells to inhibit macrophage activation but activated macrophage phenotype was not altered by separating the stimulated human peripheral blood mononuclear cells or CD14+ macrophages from Tregs in a transwell system. Macrophages did not require direct cell contact with porcine stimulator cells for full activation by CD4+CD25- T cells. CONCLUSIONS: Human Tregs were able to suppress xenoantigen-primed and CD4+ T-cell-mediated macrophage activation and antigen-presenting cell function. However, Tregs had no direct effect on macrophages in vitro.

Selective rejection of porcine islet xenografts by macrophages.

BACKGROUND: Our previous study has shown that porcine antigen-primed and CD4+ T cell-activated macrophages are capable of recognition and rejection of porcine xenografts after adoptive transfer. However, whether this is an absolute xenograft specific rejection remains to be confirmed. METHODS: Mouse islet allografts and neonatal porcine islet cell cluster (NICC) xenografts were admixed and transplanted under the left kidney capsule, and NICC xenografts alone were transplanted under the right kidney capsule of strepotozotocin-induced diabetic NOD-SCID mice. After achievement of normoglycemia, the NOD-SCID recipients were transferred with macrophages purified from NICC transplant NOD-SCID mice reconstituted with CD4+ T cells. Five weeks after macrophage transfer the left kidney with the admixed grafts were removed. Graft survival and function following macrophage transfer was assessed by blood glucose measurement and immunohistochemistry. RESULTS AND CONCLUSIONS: Adoptive transfer with activated macrophages did not affect the normalized blood glucose levels in NOD-SCID recipients of admixed grafts until left nephrectomy 5 weeks post-macrophage transfer. Insulin-positive and porcine C-peptide-negative mouse islets were detected in the admixed grafts. The surviving mouse islets in the admixed grafts were surrounded but not infiltrated by macrophages. The nephrectomized recipients demonstrated sustained hyperglycemia and completely destroyed NICC xenografts in their remaining right kidneys 8 weeks after macrophage transfer. Taken together, these data provide direct evidence of porcine islet xenograft specific rejection by activated macrophages.

Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine.

It was reported recently that histamine induced Toll-like receptor (TLR)2 and TLR4 expression in endothelial cells and enhanced their sensitivity to Gram-positive and Gram-negative bacteria; and that TLRs were expressed in airway epithelial cells and that several inflammatory mediators modulated their expression. However, little is known of potential influence of histamine on TLRs in pulmonary epithelial cells. In the present study, effects of histamine on expression of TLRs in both human A549 and NCI-H292 cell lines were examined by using real-time quantitative RT-PCR analysis, flow cytometry and immunofluorescent staining. The results revealed that both cell types constitutively expressed mRNAs for TLR1-TLR10. Histamine up-regulated the expression of TLR3 mRNA by 12.3- and 11.6-fold, respectively in both cell types. The time course showed that histamine induced TLR3 mRNA expression was initiated at 30 min, nearly reached peak levels after 2 h and was sustained at least until 12 h. Histamine also induced TLR3 protein expression in A549 and NCI-H292 cells. Histamine and poly (I:C), a specific TLR3 ligand stimulated interleukin (IL)-8 secretion from both cell types. Moreover, histamine enhanced poly (I:C)-induced IL-8 secretion and phosphorylation of NF-kappaB in the two cell types, and histamine H1 receptor antagonists inhibited the action of histamine. In conclusion, histamine selectively up-regulated expression of TLR3, and stimulated IL-8 secretion from the cells. Histamine also enhanced poly (I:C) induced IL-8 secretion and phosphorylation of NF-kappaB. These observations suggest that histamine might play an important role in enhancing the innate immune responses of airway to viral infection.

[Histamine release properties of human basophils in response to various stimuli].

AIM: To investigate the effects of protease activated receptor-2 (PAR-2) agonist, heparin and other stimuli on histamine release from human basophils. METHODS: Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. The PBMCs were then resuspended in complete Hank's balanced salt solution (HBSS) and challenged with various stimulating agents. Histamine level in samples was determined by ELISA. RESULTS: 10 mg/L trypsin could induce histamine release from basophils. However, its stimulatory effect on basophils was weaker than that of anti-IgE, calcium ionophore (CI), F-Met-Leu-Phe (FMLP), C5a and substance P. PAR-2 agonist SLIGKV-NH(2) failed to activate basophils. Heparin, C5 and adenosine did not induce any histamine release at concentrations tested, but heparin enhanced histamine release induced by C5a and substance P. CONCLUSION: Trypsin, anti-IgE, CI, FMLP, C5a and substance P can induce histamine release from baosophils, but PAR-2 agonist can not. Heparin can greatly enhance the ability of C5a and substance P to stimulate histamine release, which may be a novel mechanism of amplification of basophil activation signal.