The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21 days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

Time-Dependent Sequential Changes of IL-10 and TGF-ß1 in Mice with Deep Vein Thrombosis.

OBJECTIVES: To detect the expression changes of interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) during the development of deep vein thrombosis in mice, and to explore the application value of them in thrombus age estimation. METHODS: The mice in the experimental group were subjected to ligation of inferior vena cava. The mice were sacrificed by excessive anesthesia at 1 d, 3 d, 5 d, 7 d, 10 d, 14 d and 21 d after ligation, respectively. The inferior vena cava segment with thrombosis was extracted below the ligation point. The mice in the control group were not ligated, and the inferior vena cava segment at the same position as the experimental group was extracted. The expression changes of IL-10 and TGF-ß1 were detected by immunohistochemistry (IHC), Western blotting and real-time qPCR. RESULTS: IHC results revealed that IL-10 was mainly expressed in monocytes in thrombosis and TGF-ß1 was mainly expressed in monocytes and fibroblast-like cells in thrombosis. Western blotting and real-time qPCR showed that the relative expression levels of IL-10 and TGF-ß1 in each experimental group were higher than those in the control group. The mRNA and protein levels of IL-10 reached the peak at 7 d and 10 d after ligation, respectively. The mRNA expression level at 7 d after ligation was 4.72±0.15 times that of the control group, and the protein expression level at 10 d after ligation was 7.15±0.28 times that of the control group. The mRNA and protein levels of TGF-ß1 reached the peak at 10 d and 14 d after ligation, respectively. The mRNA expression level at 10 d after ligation was 2.58±0.14 times that of the control group, and the protein expression level at 14 d after ligation was 4.34±0.19 times that of the control group. CONCLUSIONS: The expressions of IL-10 and TGF-ß1 during the evolution of deep vein thrombosis present time-dependent sequential changes, and the expression levels of IL-10 and TGF-ß1 can provide a reference basis for thrombus age estimation.

Activation of dopamine D2 receptor promotes pepsinogen secretion by suppressing somatostatin release from the mouse gastric mucosa.

In vivo administration of dopamine (DA) receptor (DR)-related drugs modulate gastric pepsinogen secretion. However, DRs on gastric pepsinogen-secreting chief cells and DA D2 receptor (D2R) on somatostatin-secreting D cells were subsequently acquired. In this study, we aimed to further investigate the local effect of DA on gastric pepsinogen secretion through DRs expressed on chief cells or potential D2Rs expressed on D cells. To elucidate the modulation of DRs in gastric pepsinogen secretion, immunofluorescence staining, ex vivo incubation of gastric mucosa isolated from normal and D2R-/- mice were conducted, accompanied by measurements of pepsinogen or somatostatin levels using biochemical assays or enzyme-linked immunosorbent assays. D1R, D2R, and D5R-immunoreactivity (IR) were observed on chief cells in mouse gastric mucosa. D2R-IR was widely distributed on D cells from the corpus to the antrum. Ex vivo incubation results showed that DA and the D1-like receptor agonist SKF38393 increased pepsinogen secretion, which was blocked by the D1-like receptor antagonist SCH23390. However, D2-like receptor agonist quinpirole also significantly increased pepsinogen secretion, and D2-like receptor antagonist sulpiride blocked the promotion of DA. Besides, D2-like receptors exerted an inhibitory effect on somatostatin secretion, in contrast to their effect on pepsinogen secretion. Furthermore, D2R-/- mice showed much lower basal pepsinogen secretion but significantly increased somatostatin release and an increased number of D cells in gastric mucosa. Only SKF38393, not quinpirole, increased pepsinogen secretion in D2R-/- mice. DA promotes gastric pepsinogen secretion directly through D1-like receptors on chief cells and indirectly through D2R-mediated suppression of somatostatin release.

Impaired nitrergic relaxation in pyloric sphincter of the 6-OHDA Parkinson's disease rat.

Patients with Parkinson's disease (PD) often suffer from delayed gastric emptying, but the underlying mechanism remains unclear. We have shown previously that a PD rat model comprising bilateral substantia nigra destruction by 6-hydroxydopamine (6-OHDA rats) exhibits gastroparesis with alteration of neural nitric oxide synthase (nNOS) and acetylcholine in gastric corpus. However, changes in pyloric motility in the 6-OHDA rats have not been characterized. Solid gastric emptying test, immunofluorescence, Western blot, and in vitro pyloric motility recordings were used to assess pyloric motor function in the 6-OHDA rats. The 6-OHDA-treated rats displayed delayed solid gastric emptying and a lower basal pyloric motility index. In the 6-OHDA rats, high K+-induced transient contractions were weaker in pyloric sphincters. Electric field stimulation (EFS)-induced pyloric sphincter relaxation was lower in the 6-OHDA rats. NG-nitro-l-arginine methyl ester (l-NAME), a nonselective inhibitor of NOS, markedly inhibited the EFS-induced relaxation in both control and 6-OHDA rats. Pretreatment of tetrodotoxin abolished the effect of EFS on the pyloric motility. In addition, nNOS-positive neurons were extensively distributed in the pyloric myenteric plexus, whereas the number of nNOS-immunoreactive neurons and the protein expression of nNOS were significantly decreased in the pyloric muscularis of 6-OHDA rats. However, sodium nitroprusside-induced pyloric relaxations were similar between the control and 6-OHDA rats. These results indicate that the pyloric sphincters of 6-OHDA rats exhibit both weakened contraction and relaxation. The latter may be due to reduced nNOS in the pyloric myenteric plexus. The dysfunction of the pyloric sphincter might be involved in the delayed gastric emptying.NEW & NOTEWORTHY Reduced nitrergic neurons in pyloric myenteric plexus potently contributed to the attenuated relaxation in 6-hydroxydopamine (6-OHDA) rats, subsequently affecting gastric emptying. SNP could well improve the relaxation of pylori in 6-OHDA rats. The present study provides new insight into the diagnosis and treatment of delayed gastric emptying in patients with PD.

Gastric smooth muscle cells manifest an abnormal phenotype in Parkinson's disease rats with gastric dysmotility.

Gastroparesis is a common symptom in Parkinson's disease (PD) and whether any change occurs in gastric smooth muscle cells (SMCs) of PD patients is unclear. We previously reported that rats with bilateral substantia nigra lesions induced by 6-hydroxydopamine (6-OHDA), referred to as 6-OHDA rats, manifest typical gastroparesis. In the present study, we further investigate the underlying mechanism. By means of an organ bath system and an implantable radiotelemetry system, both a weakened contractile force of gastric circular smooth muscle and gastric myoelectric activity were detected in the 6-OHDA rats and phasic and tonic contractions elicited by carbachol or high concentration of potassium were significantly reduced in gastric circular muscle strips. A thickened smooth muscle layer was observed under a light microscope and an ultrastructure of hypertrophic SMCs, with increased caveolae and decreased dense bodies, was observed under transmission electron microscope. Furthermore, the mRNA and protein expression levels of contractile markers (myosin light chain 20, myosin heavy chain 11 and α-smooth muscle actin) and the transcription factor serum response factor (SRF) were significantly decreased, while the TNFα and IL-1ß content was increased in the 6-OHDA rats. These results suggest that the decreased contractile force in 6-OHDA rats may be associated with the phenotypic abnormality observed in SMCs, which is due to downregulated contractile proteins induced by decreased SRF expression in the inflammatory muscular microenvironment.

Annexin A1-derived peptide Ac2-26 facilitates wound healing in diabetic mice.

Impaired wound healing is a common complication of diabetes. In diabetic wounds, macrophages present dysfunctional efferocytosis and abnormal phenotypes, which could result in excessive neutrophil accumulation and prolonged inflammation, thereby eventually hindering wound repair. ANXA1 N-terminal peptide Ac2-26 exhibits a high potential in mitigating inflammation and improving repair; however, its efficacy in diabetic wound repair remains unclear. In this study, a cutaneous excisional wound model was built in genetically diabetic mice. Ac2-26 or a vehicle solution was employed locally in wound sites. Subsequently, wound zones were measured and sampled at different time intervals post-wounding. Using hematoxylin-eosin and Masson's trichrome staining, we observed the histopathological variations and collagen deposition in wound samples. Based on immunohistochemistry and immunofluorescence, the numbers of neutrophils, macrophages, and CD206-positive macrophages in the wound samples were determined. Cytokine expression in wound samples was studied by immunoblot assay. Results showed that Ac2-26 treatment could facilitate diabetic wound closure, down-regulate the number of neutrophils, and improve angiogenesis and collagen deposition. In addition, Ac2-26 application expedited macrophage recruitment and up-regulated the percentage of macrophages expressing CD206, which is a marker for M2 macrophages. Moreover, Ac2-26 inhibited the expressions of TNF-α and IL-6 and up-regulated the expressions of IL-10, TGF-ß, and VEGFA during diabetic wound healing. Hence, based on the aforementioned findings, Ac2-26 application in diabetic wounds could exert anti-inflammatory and pro-repair effects by reducing neutrophil accumulation and facilitating M2 macrophage development.

Expression of inflammatory cytokines in serum and peritoneal fluid from patients with different stages of endometriosis.

OBJECTIVE: To investigate the level of inflammatory cytokines in endometriosis patients, and explore the relationship between IL-37 concentration and endometriosis stages. METHODS: Inflammatory cytokine concentrations from 27 patients with different stages of endometriosis and 52 controls without endometriosis were examined by ELISA. Then, the specificity and sensitivity of cytokines for distinguishing from controls and the different stages of endometriosis were analyzed using the ROC curve. RESULTS: The difference in serum concentrations of IL-37, IL-17A, IL-10, and IL-2 between the endometriosis and control groups was statistically significant (p < .01). Compared with controls, significantly higher levels of serum IL-37 and IL-10, and significantly lower levels of serum IL-17A and IL-2 were detected in patients with endometriosis (p < .01). Furthermore, IL-2 concentration was significantly higher in peritoneal fluid (PF) in the endometriosis group (p = .0034), IL-10 concentrations in PF were significantly lower in the early-stages of endometriosis than in the more advanced groups (p = .0439), and IL-4 concentration in PF was significantly higher in more advanced endometriosis (p = .0228). The sensitivity and specificity of serum IL-37 for distinguishing endometriosis were 81.48% and 83.33%, respectively, and the cutoff concentration was 69.84 pg/ml. For IL-17A, the sensitivity and specificity were 96.30% and 100%, respectively, and the cutoff concentration was 57.54 pg/ml. For IL-10, the sensitivity and specificity was 92.59% and 100%, respectively, and the cutoff concentration was 3.301 pg/ml. For IL-2, the sensitivity and specificity were 74.07% and 93.75%, respectively, and the cutoff concentration was 1.813 pg/ml. For PF IL-2, the sensitivity and specificity were 29.73% and 100%, respectively, and the cutoff concentration was 1.06 pg/ml. CONCLUSIONS: IL-37, IL-17A, IL-10, and IL-2 may play a significant role in immune response in endometriosis. IL-37 levels may be used as a diagnostic marker for endometriosis.

Detection of RAGE expression and its application to diabetic wound age estimation.

With the prevalence of diabetes, it is becoming important to analyze the diabetic wound age in forensic practice. The present study investigated the time-dependent expression of receptor for advanced glycation end products (RAGE) during diabetic wound healing in mice and its applicability to wound age determination by immunohistochemistry, double immunofluorescence, and Western blotting. After an incision was created in genetically diabetic db/db mice and control mice, mice were killed at posttraumatic intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. Compared with control mice, diabetic mice showed the delayed wound healing. In control and diabetic wound specimens, RAGE immunoreactivity was observed in a small number of polymorphonuclear cells (PMNs), a number of macrophages, and fibroblasts. Morphometrically, the positive ratios of RAGE in macrophages or fibroblasts considerably increased in diabetic wounds during late repair, which exceeded 60% at 7 and 10 days post-injury. There were no control wound specimens to show a ratio of >60% in macrophages or fibroblasts. By Western blotting analysis, the ratios of RAGE to GAPDH were >1.4 in all diabetic wound samples from 7 to 10 days post-injury, which were >1.8 at 10 days after injury. By comparison, no control wound specimens indicated a ratio of >1.4. In conclusion, the expression of RAGE is upregulated and temporally distributed in macrophages and fibroblasts during diabetic wound healing, which might be closely involved in prolonged inflammation and deficient healing. Moreover, RAGE is promising as a useful marker for diabetic wound age determination.

Expression of KGF-1 and KGF-2 in Skin Wounds and Its Application in Forensic Pathology.

The expression of keratinocyte growth factor-1 (KGF-1) and keratinocyte growth factor-2 (KGF-2) in skin wounds in mice was studied using multiple methods. The dynamic expression of KGF-1 and KGF-2 for antemortem and postmortem injuries as well as the examination of antemortem injuries after death under different temperature and over varying time periods was studied. It demonstrates that skin KGF-1 resulting from an antemortem injury starts to rise at 6 hours, reaches its peak at 1 day, and starts to drop at 5 days. The expression of skin KGF-2 resulting from an antemortem injury starts to rise at 12 hours, reaches its peak at 7 days, and begins to drop at 10 days. Skin KGF-1 and skin KGF-2 after death stabilize within 7 days at 4°C and -20°C, within 5 days at 20°C, and within 1 day at 30°C. The application of KGF-1 and KGF-2 indicators in skin wound age determination is both feasible and reliable.

A Comprehensive Evaluation of the Bruker Biotyper MS and Vitek MS Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Systems for Identification of Yeasts, Part of the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Study, 2012 to 2013.

Among the 2,683 yeast isolates representing 41 different species (25 Candida and Candida-related species and 16 non-Candida yeast species) collected in the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program (2012 to 2013), the Bruker Biotyper MS matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system exhibited significantly higher accuracy rates than the Vitek MS system for identification of all yeast isolates (98.8% versus 95.4%, P <0.001 by Pearson's chi-square test) and for all Candida and Candida-related species isolates (99.4% versus 95.5%, P < 0.001).

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

[Detection of carbapenem-producing Enterobacteriaceae by MALDI-TOF MS].

OBJECTIVE: To evaluate a rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) assay in detecting carbapenemase-producing Enterobacteriaceae. METHODS: Thirty-four carbapenemase-producing Enterobacteriaceae and 50 carbapenem susceptible Enterobacteriaceae clinical isolates were tested. After a 2-hour incubation of bacteria with meropenem, the mixtures were centrifuged and the supernatants analyzed by MALDI-TOF MS. RESULTS: All spectra of carbapenem susceptible Enterobacteriaceae showed peaks of m/z 384, 406, 428 while those of carbapenemase-producing Enterobacteriaceae m/z 358, 380, 402, 424, 446, 468. And the assay of MALDI-TOF MS was highly consistent with sequencing (P = 1.000). CONCLUSION: MALDI-TOF MS can detect carbapenemase-producing Enterobacteriaceae rapidly and accurately.

[Antimicrobial resistance and virulence characteristics of Vibrio parahaemolyticus].

OBJECTIVE: To explore drug resistance and virulence characteristics of clinically isolated Vibrio parahaemolyticus (VP). METHODS: The patient records were reviewed. The laboratory information system was employed to count the quantity of bacterial diarrhea pathogens at our intestinal clinic from 2009 to 2012. The drug resistant phenotype was tested by the method of disk diffusion. Polymerase chain reaction (PCR) was used to detect thermo-stable direct hemolysin (TDH) gene (tdh), TDH-related hemolysin gene (trh) and type III secretion system 2 (T3SS2α, T3SS2ß). RESULTS: The VP infection rate was 37.90% (274/723). Susceptibility test showed that the resistance rates of ampicillin, cefalotin, cefuroxime and amikacin were 100% (216/216), 44.91% (97/216), 24.07% (52/216), 9.26% (20/216). The other 11 kinds of antibiotics were sensitive or intermediate. 96.76% (209/216) VP harbored tdh gene, 1.39% (3/216) trh gene and 100% T3SS2 gene, with 213 strains T3SS2α positive, 2 strains T3SS2ß positive and 1 strain both T3SS2α and T3SS2ß positive. CONCLUSION: The resistant rate of VP to ampicillin is the highest and VP has different levels of resistance against other antibiotics. Most clinical isolates of VP harbor tdh gene and T3SS2 plays an important role in the pathogenicity of VP.

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

OBJECTIVE: To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. METHODS: Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. RESULTS: Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. CONCLUSION: The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice.

Dynamic localization of CB2R and quantitative analysis of CB2R mRNA during skin wound healing in mice were performed. Co-localization of CB2R with F4/80 or α-SMA was detected by double-color immunofluorescence microscopy. A total of 110 male mice were divided into control, injury, and postmortem groups. Sixty-five mice were sacrificed, followed by sampling at 0.5 h-21 days post-injury. Five mice without incision were used as control. The other 40 mice that received incised wound were sacrificed at 5 days after injury. The samples were collected at 0 h-3 days postmortem. In the uninjured controls, CB2R immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands, dermomuscular layer, and vascular smooth muscle. In the incision groups, polymorphonulcear cells, macrophages, and myofibroblasts showed positive staining for CB2R. Morphometrically, the average ratios of CB2R-positive cells were more than 50 % at 5 days post-wounding, whereas it was <50 % at the other posttraumatic intervals. The average ratios of CB2R-positive macrophages maximized at 3 days post-wounding, and the average ratios of CB2R-positive myofibroblasts peaked at 5 days post-wounding. The relative quantity of CB2R mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >4.10, which was also confirmed by Western blotting. There was no significant change for CB2R protein within 6 h postmortem and for mRNA within 3 h postmortem as compared with the control group. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R is involved in modulating macrophages and myofibroblasts in response to inflammatory event and repair process in mouse skin wound healing, and CB2R is available as a marker for wound age determination.

[The number of circulating fibrocytes of skeletal muscle in rats after contusion].

OBJECTION: To investigate the time-dependent appearance of circulating fibrocytes of skeletal muscle in rats after contusion. METHODS: The model of skeletal muscle wound was established in rat. The circulating fibrocytes in contused skeletal muscle were detected by CD45 and procollagen I double immunofluorescence staining method. RESULTS: In the control group, CD45- and procollagen I-positive cells were not detected in skeletal muscle. A few CD45 cells were observed aged from 6 h to 1 d after contusion. A few CD45- and procollagen I-positive cells (fibrocytes) initially gathered in injury area 3d after injury. The ratio of positive fibrocytes significantly increased 5 d after injury. The ratio of fibrocytes was highest at 7 d after contusion and then decreased. The volume of fibrocytes showed bigger with injury time increase compared with 3 d group. The expression of procollagen I and CD45 were weakened at 14d after injury. CONCLUSION: The circulating fibrocytes are detected in contused skeletal muscle in time-dependent pattern. Circulating fibrocytes may be a marker in the wound age determination for contused skeletal muscle.

Nicotinic acetylcholine receptor α7 subunit is time-dependently expressed in distinct cell types during skin wound healing in mice.

Recent studies have shown that nicotinic acetylcholine receptor alpha7 subunit (nAChRα7) plays an important role in regulation of inflammation, angiogenesis and keratinocyte biology, but little is known about its expression after the skin is wounded. A preliminary study on time-dependent expression and distribution of nAChRα7 was performed by immunohistochemistry, Western blotting and RT-PCR during skin wound healing in mice. After a 1-cm-long incision was made in the skin of the central dorsum, mice were killed at intervals ranging from 6 h to 14 days post-injury. In uninjured skin controls, nAChRα7 positive staining was observed in epidermis, hair follicles, sebaceous glands, vessel endothelium and resident dermal fibroblastic cells. In wounded specimens, a small number of polymorphonuclear cells, a large number of mononuclear cells (MNCs) and fibroblastic cells (FBCs) showed positive reaction for nAChRα7 in the wound zones. Simultaneously, nAChRα7 immunoreactivity was evident in endothelial-like cells of regenerated vessels and neoepidermis. By morphometric analysis, an up-regulation of nAChRα7 expression was verified at the inflammatory phase after skin injury and reached a peak at the proliferative phase of wound healing. The expression tendency was further confirmed by Western blotting and RT-PCR assay. By immunofluorescent staining for co-localization, the nAChRα7-positive MNCs and FBCs in skin wounds were identified as macrophages, fibrocytes and myofibroblasts. A number of nAChRα7-positive myofibroblasts were also CD45 positive, indicating that they originated from differentiation of fibrocytes. The results demonstrate that nAChRα7 is time-dependently expressed in distinct cell types, which may be closely involved in inflammatory response and repair process during skin wound healing.

Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice.

The study investigated the expression of monoacylglycerol lipase (MGL) during the skin-incised wound healing in mice and applicability of the time-dependent expression of MGL to wound age determination by immunofluorescent staining, Western blotting, and real-time PCR. Furthermore, cell types were identified by double immunofluorescence. A total of 45 BALB/c male mice were used in this study. After a 1.5-cm-long incision in the central dorsum skin, mice were killed at intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. In the control, there was a low-level expression of MGL in the epidermis, hair follicles, and glandulae sebaceae. In the injured skin, MGL immunoreactivity was mainly detected in the neutrophils, macrophages, and myofibroblasts. Morphometrically, the average ratios of MGL-positive cells were more than 50% at 5 and 7 days post-wounding, whereas it was <50% at the other posttraumatic intervals. By Western blotting analysis, the average ratio of MGL protein expression was highest at 5 days after injury, which had a ratio of >2.30. Similarly, the relative quantity of MGL mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >2.54. In conclusion, MGL expression is detected in neutrophils, macrophages, and myofibroblasts and significantly up-regulated, suggesting that it may play roles in response to inflammation during skin-incised wound healing. From the viewpoint of forensic pathology, MGL detection is applicable to skin wound age determination.

[Correlation factor analysis of pan-drug resistant Acinetobacter baumannii strains in acquired infections].

OBJECTIVE: To explore the clinical factors, drug resistance and molecular epidemiology homologous characteristics of pan-drug resistant Acinetobacter baumannii (PDRAB) in acquired infections and analyze the correlation factor between epidemic characteristics and acquired infections. METHODS: A total of 60 PDRAB strains from nine acquired infections and related clinic data were collected from January 2009 to January 2011. The drug-resistant phenotype was tested by disk diffusion methods. The isolate identification and homology were studied by automation repetitive-element sequence-based (REP)-PCR typing platform from genes and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF MS) from proteins. RESULTS: All strains were resistant to 12 antibiotics except 2 strains to imipenem and meropenem. The strains in this study were divided into 12 types (A-L) by REP-PCR. And 60 strains were also clustered to a-e types by MALDI-TOF-TOF MS. Compared with MALDI-TOF-TOF MS, REP-PCR tended to be more accurate. Breathing machine carriage and cross transmission were the main reasons for a major epidemic outbreak at department of pulmonary medicine from July 2009 to October 2009. Hand transmission of medical care personnel was a key factor for SICU 2010 January to February. The contamination and transmission to environment of PDRAB in nasal pharynx or respiratory tract by superspreader were the main reasons for the other 7 epidemic outbreaks. Department of emergency medicine was the source of acquired infections. CONCLUSION: The key control measures of acquired infections are early identification and isolation of spreader, environment and instrument disinfection, hand washing and rational uses of antibiotics. MALDI-TOF-TOF MS will become a preferred tool of identification and classification of microorganisms because of its simple operation, affordable price and handling rapidity.

[The expression of GABA(A) receptor alpha1 and GABA(B) receptor 1 in medulla oblongata solitary nucleus and ambiguous nucleus in the cases of tramadol intoxication].

OBJECTIVE: To observe the expression of GABA(A) receptor alpha1 (GABA(A)alpha1) and GABA(B) receptor 1 (GABA(B)1) in human medulla oblongata solitary nucleus and ambiguous nucleus due to tramadol-induced death. METHODS: GABA(A)alpha1 and GABA(B)1 were detected by immunohistochemical SP method in tramadol-induced death group and control group. All results were evaluated by images analysis system. RESULTS: Low expression of GABA(A)alpha1 and GABA(B)1 were detected in solitary nucleus and ambiguous nucleus in the control brain tissue. In cases of tramadol-induced death, the expression of GABA(A)alpha1 and GABA(B)1 significantly increased. CONCLUSION: The mechanism of tramadol intoxication death could be caused by respiratory depression induced by over-expression of GABA(A)alpha1 and GABA(B)1 in medulla oblongata solitary nucleus and ambiguous nucleus.