Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ-coupled two-dimensional LC-MS/MS.

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ-coupled 2D LC-MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25-fold at p < 0.05) and 55 proteins were downregulated (<0.8-fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen-like (CD5L), hyaluronan-binding protein 2 (HABP2), and retinol-binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.

Serum complement C4b, fibronectin, and prolidase are associated with the pathological changes of pulmonary tuberculosis.

BACKGROUND: Mycobacterium tuberculosis infection can activate the immune system, leading to characteristic pathological changes such as inflammatory granuloma, caseous necrosis, and cavity formation. METHODS: Clinical data of 187 cases of pulmonary tuberculosis (PTB) were analyzed using statistical methods, while serum levels of complement C4b (C4b), fibronectin (FN), and prolidase (PEPD) were detected using the ELISA method among the control, minimal PTB, moderate PTB, and advanced PTB groups. RESULTS: We found significantly higher levels of serum C4b and PEPD (P = 0.018, P = 0.003), and significantly lower levels of serum FN (P < 0.001) in PTB patients. Furthermore, the serum levels of 3 proteins were significantly different among 3 PTB groups. FN level was significantly higher in the moderate PTB group, compared with patients in the minimal and advanced PTB groups (P < 0.05, P < 0.01). PEPD level was significantly higher in the moderate PTB group, compared with the minimal PTB group (P < 0.05). Analysis of clinical data showed that serum albumin, C-reactive protein (CRP), prealbumin, and C4 were significantly higher (P < 0.05), while serum globulin was significantly lower in patients with PTB (P < 0.001). A significant negative correlation was found between C4b and albumin, prealbumin. On the other hand, a significant positive correlation was found between C4b and globulin, CRP, PEPD, as well as between PEPD and CRP (P < 0.05). CONCLUSIONS: Our study showed that C4b, FN, and PEPD are associated with tissue damage, granuloma formation, and cavity formation, respectively, in patients with PTB. The present study provides a new experimental basis to understand the pathogenesis and pathological changes of PTB.

[Effects of nitric oxide on peritoneal lymphatic stomata and lymph drainage via NO-cGMP-Ca2+ pathway].

To study the cell signal transduction mechanism of nitric oxide (NO) on the peritoneal lymphatic stomata and lymph drainage in the rat, cGMP content were measured by a commercially available radioimmunoassay kit, and the [Ca(2+)](i) were observed by a confocal laser scanning microscope in the cultured peritoneal mesothelial cell. Animal experiment was practiced to study the effect of NO-cGMP-Ca(2+) pathway on the lymphatic stomata and lymph absorption. The results showed that: (1) Sper/NO increased cGMP of the rat peritoneal mesothelial cell (RPMC) in a dose-dependent manner (P<0.01) compared to the control group. This effect was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) (P<0.05), a specific inhibitor of soluble guanylyl cyclase (sGC). The level of [Ca(2+)](i) in single RPMC decreased by adding Sper/NO (P<0.05). Pretreatment with ODQ for 10 min blocked the Sper/NO-induced decrease in [Ca(2+)](i). L-typed calcium channel blocker nifedipine induced an immediate and marked decrease in [Ca(2+)](i) (P<0.05).. After [Ca(2+)](i) reached a balance again, adding Sper/NO could not change [Ca(2+)](i) (P>0.05). (2) Sper/NO increased the area of the stomata (P<0.01) and the quantity of the tracer in a dose-dependent manner (P<0.05) compared to the control group. Pretreatment with ODQ significantly inhibited Sper/NO-induced change of lymphatic stomata and lymph drainage (P<0.01). Nifedipine increased the opening area of the lymphatic stomata (P< 0.01) and the concentration of absorbed trypan blue of the diaphragm (P<0.05). Sper/NO could not make a further change in the samples pretreated by nifedipine (P> 0.05). The results indicate that NO can decrease [Ca(2+)](i) in the RPMC through the NO-cGMP pathway. This procession is related with the L- type voltage-gated Ca(2+) channel. NO enlarges the opening area of the lymphatic stomata and enhances the lymph drainage of tracer by NO-cGMP-[Ca(2+)](i) pathway.

Hydronephrosis due to ureteral endometriosis in women of reproductive age.

OBJECTIVE: The aim of the present study was to improve the understanding of ureteral endometriosis, and remind the clinics to be highly suspicious of it in women of reproductive age with hydronephrosis without evidence of stones and malignancy. METHODS: A retrospective analysis was performed on a database of 82 patients who underwent surgery for hydronephrosis due to ureteral endometriosis between Jan. 2007 and Apr. 2014. RESULTS: All patients evaluated in this study were divided into three groups: Group A consisted of patients between 20-30 years (n = 12), Group B comprised of patients between 31-40 years (n = 29), Group C consisted of patients between 41-50 years (n = 41). Patients in Group C had a greater prevalence of pelvic pain compared with patients in Group A and Group B (P < 0.05). However there were no differences with respect to the prevalence of other non-specific genitourinary symptoms and the urinary symptoms. Infertility was found to occur more frequently in patients in Group A compared with patients in Group B and Group C (P < 0.05). Because of the lack of specific symptoms, ureteral endometriosis was diagnosed (20.1 ± 10.3) months on average after the patients suffered from mild hydronephrosis or mild loin pain. Preoperative examinations showed different degree of hydronephrosis, but lack of specificity. All patients underwent surgery by laparotomy or laparoscopy, such as ureterectomy with ureteroureterostomy or ureterocystoneostomy. The pathological examination confirmed the diagnosis of ureteral endometriosis. CONCLUSION: The diagnosis of ureteral endometriosis is elusive and relies heavily on clinical suspicion. Hence, women in the reproductive age, especially with infertility and pelvic pain, who have hydronephrosis without evidence of stones and malignance, should be adequately assessed via imaging techniques or diagnostic laparoscopy or cystoscopy to highly suspect the diagnosis of ureteral endometriosis.

Ultrastructure and three-dimensional study of the lymphatic stomata in the costal pleura of the rabbit.

The aim of this report was to investigate the ultrastructure and three-dimensional organization of the pleural lymphatic stomata in the adult rabbit costal pleura by electron microscopy. A computer image processing system attached to the scanning electron microscopy was used to get statistical evaluation of the dimensions of pleural lymphatic stomata. Mesothelial cells were digested by 2 mol/L NaOH solution in order to expose the submesothelial connective tissue with macula cribriformis. Two kinds of mesothelial cells were observed on the costal pleura: the flattened cells and the cuboidal ones. Both had microvilli on their surface. Pleural lymphatic stomata were located only in the regions of cuboidal mesothelial cells. The average area of a stoma was 7.20 +/- 3.69 microm2 and their average density 121 +/- 72/mm2. Pleural cavity is connected with the lympo-vascular system by lymphatic stomata and the interstitial layer with a dense network of lamina cribriformis. The macula cribriformis (7-60 microm in diameter) were found in subpleural connective tissue below the cuboidal mesothelial cells. Consequently on cross-section, the pathway represents a channel consisting of the stoma, the connective tissue space, and the gap between endothelial cells of the lymphatics. Closed lymphatic stomata and the milky spots composed of macrophages could be observed on the costal pleura. Our results suggest that the pleural cavity is connected with the lymphatic capillaries through the lymphatic stomata and the subpleural channel. This is the only "highway" from the pleural cavity to the vessels. This pathway may be importantly involved in the material exchange and the immunity of the pleura cavity.

Ultrastructural study of pleural lymphatic drainage unit and effect of nitric oxide on the drainage capacity of pleural lymphatic stomata in the rat.

The objective of this study was twofold: first to investigate the ultrastructure of the lymphatic drainage unit on the costal pleura of rats by electron microscopy, and secondly to examine the effect of nitric oxide on the pleural lymphatic stomata and fluid absorption from the pleural cavity. The lymphatic drainage unit of the rat costal pleura is composed of three special components: the lymphatic stomata between the mesothelial cells, the initial part of the lymphatic vessels and the underlying connective tissue containing many foramina. The unit is the main passage to drainage fluid, particles and cells in the pleural space. To investigate the regulator of the lymph drainage, nitric oxide synthase inhibitor and nitric oxide donor were injected into the peritoneal cavity of the rats, respectively. Trypan blue was used as tracer. The ultrastructural changes of pleural lymphatic stomata were observed under scanning electron microscope and analyzed by a computer image processing system. It turned out that the area and density of the pleural lymphatic stomata were positively correlated with the nitric oxide quantity (p < 0.05). After the tracer was injected into the pleural cavity, the nitric oxide donor group exhibited a higher trypan blue concentration than the control group (p < 0.05). The ability of the pleura to absorb trypan blue was enhanced because of the larger opening of the lymphatic stomata (p < 0.05). It is suggested that nitric oxide can increase lymphatic absorption of the pleura by opening pleural lymphatic stomata.

[Effects of nitric oxide on pleural lymphatic stomata and lymphatic drainage in the rat].

To investigate the effects of nitric oxide (NO) on the pleural lymphatic stomata and lymph absorption from the pleural cavity, the NOS (nitric oxide synthase) inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME) and the NO donor isosorbide dinitrate (ISDN) were injected into the peritoneal cavity of the rats respectively. Trypan blue was used as a tracer. Then the concentrations of NO and trypan blue in the blood serum were measured, and the ultrastructural changes in pleural lymphatic stomata were observed under a scanning electron microscope (SEM) and studied by a computer image processing system attached to SEM. It turned out that the concentration of NO in the serum was 49.34+/-18.47 micromol/L, and the area and density of the pleural lymphatic stomata were 6.80+/-1.13 microm(2) and 170.24+/-66.60 /0.1 mm(2) respectively in the NO donor group. The concentration of NO reduced to 17.72+/-6.58 micromol/L, and the area and density of the pleural lymphatic stomata were 5.72+/-1.54 microm(2) and 61.71+/-12.73/0.1 mm(2) in the NOS inhibitor group. We found that the area and density of the pleural lymphatic stomata were positively correlated with the NO quantity. After the tracer was injected into the pleural cavity, the NO donor group exhibited a higher trypan blue concentration than the control group. The ability of the pleura to absorb trypan blue was enhanced because of the large opening of the stomata. It is suggested that NO can increase lymph absorption of the pleura by relaxing pleural lymphatic stomata.

Cell signal transduction mechanism for nitric oxide regulating lymphatic stomata and its draining capability.

We have previously demonstrated that nitric oxide (NO) is involved in the regulation of the lymphatic stomata. However, the related mechanisms are still unknown. The present study was designed to test the hypothesis that NO-cyclic guanosine monophosphate (cGMP) -mediated cytosolic Ca(2+) concentration ([Ca(2+)]i) signaling may contribute to the regulation of the lymphatic stomata and lymph drainage. Using trypan blue as a tracer, the effects of NO-cGMP-Ca(2+) signal cascade on the lymphatic stomata and lymph absorption were examined by means of scanning electron microscopy. Then, the role of NO in cGMP and [Ca(2+)]i of rat peritoneal mesothelial cells (RPMCs) was measured by radioimmunoassay and a confocal laser scanning microscope. Our results showed that NO-donor spermine/nitric oxide complex (Sper/NO) could broaden the opening area of the lymphatic stomata and enhance lymph absorption in a dose-dependent manner. These NO-mediated changes could be blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylyl cyclase, and mimicked by calcium channel blocker nifedipine. Furthermore, Sper/NO enhanced the cGMP level and lessened [Ca(2+)](i) in RPMCs, which was completely abrogated at the presence of ODQ. Nifedipine induced an immediate and marked decrease of [Ca(2+)](i) in the RPMCs, which was not attenuated by addition of Sper/NO, indicating that the Sper/NO-cGMP signaling system induced [Ca(2+)](i) change was related to the L-type voltage-gated calcium channel in the RPMCs. Our results suggest that NO enlarges the opening area of the lymphatic stomata to strengthen the lymph drainage of tracer by means of NO-cGMP-[Ca(2+)]i signal transduction pathway in the RPMCs.