Aging Contributes to Inflammation in Upper Extremity Tendons and Declines in Forelimb Agility in a Rat Model of Upper Extremity Overuse

We sought to determine if tendon inflammatory and histopathological responses increase in aged rats compared to young rats performing a voluntary upper extremity repetitive task, and if these changes are associated with motor declines. Ninety-six female Sprague-Dawley rats were used in the rat model of upper extremity overuse: 67 aged and 29 young adult rats. After a training period of 4 weeks, task rats performed a voluntary high repetition low force (HRLF) handle-pulling task for 2 hrs/day, 3 days/wk for up to 12 weeks. Upper extremity motor function was assessed, as were inflammatory and histomorphological changes in flexor digitorum and supraspinatus tendons. The percentage of successful reaches improved in young adult HRLF rats, but not in aged HRLF rats. Forelimb agility decreased transiently in young adult HRLF rats, but persistently in aged HRLF rats. HRLF task performance for 12 weeks lead to increased IL-1beta and IL-6 in flexor digitorum tendons of aged HRLF rats, compared to aged normal control (NC) as well as young adult HRLF rats. In contrast, TNF-alpha increased more in flexor digitorum tendons of young adult 12-week HRLF rats than in aged HRLF rats. Vascularity and collagen fibril organization were not affected by task performance in flexor digitorum tendons of either age group, although cellularity increased in both. By week 12 of HRLF task performance, vascularity and cellularity increased in the supraspinatus tendons of only aged rats. The increased cellularity was due to increased macrophages and connective tissue growth factor (CTGF)-immunoreactive fibroblasts in the peritendon. In conclusion, aged rat tendons were overall more affected by the HRLF task than young adult tendons, particularly supraspinatus tendons. Greater inflammatory changes in aged HRLF rat tendons were observed, increases associated temporally with decreased forelimb agility and lack of improvement in task success.     

Sedentary Time and Markers of Chronic Low-Grade Inflammation in a High Risk Population

Background

Sedentary behaviour has been identified as a distinct risk factor for several health outcomes. Nevertheless, little research has been conducted into the underlying mechanisms driving these observations. This study aimed to investigate the association of objectively measured sedentary time and breaks in sedentary time with markers of chronic low-grade inflammation and adiposity in a population at a high risk of type 2 diabetes mellitus.

Methods

This study reports data from an ongoing diabetes prevention programme conducted in Leicestershire, UK. High risk individuals were recruited from 10 primary care practices. Sedentary time (<25counts per 15s) was measured using Actigraph GT3X accelerometers (15s epochs). A break was considered as any interruption in sedentary time (≥25counts per 15s). Biochemical outcomes included interleukin-6 (IL-6), C-reactive protein (CRP), leptin, adiponectin and leptin:adiponectin ratio (LAR). A sensitivity analysis investigated whether results were affected by removing participants with a CRP level >10 mg/L, as this can be indicative of acute inflammation.

Results

558 participants (age = 63.6±7.7years; male = 64.7%) had complete adipokine and accelerometer data. Following adjustment for various confounders, sedentary time was detrimentally associated with CRP (β = 0.176±0.057, p = 0.002), IL-6 (β = 0.242±0.056, p = <0.001), leptin (β = 0.146±0.043, p = <0.001) and LAR (β = 0.208±0.052, p = <0.001). Associations were attenuated after further adjustment for moderate-to-vigorous physical activity (MVPA) with only IL-6 (β = 0.231±0.073, p = 0.002) remaining significant; this result was unaffected after further adjustment for body mass index and glycosylated haemoglobin (HbA_1c). Similarly, breaks in sedentary time were significantly inversely associated with IL-6 (β = −0.094±0.047, p = 0.045) and leptin (β = −0.075±0.037, p = 0.039); however, these associations were attenuated after adjustment for accelerometer derived variables. Excluding individuals with a CRP level >10 mg/L consistently attenuated the significant associations across all markers of inflammation.

Conclusion

These novel findings from a high risk population recruited through primary care suggest that sedentary behaviour may influence markers associated with inflammation, independent of MVPA, glycaemia and adiposity.     

Chronic Prostatic Infection and Inflammation by Propionibacterium acnes in a Rat Prostate Infection Model

Chronic inflammation in the prostate, seen as infiltration of inflammatory cells into the prostate gland in histological samples, affects approximately half the male population without indication of prostate disease, and is almost ubiquitous in patients diagnosed with benign prostate hyperplasia and cancer. Several studies have demonstrated the Gram-positive bacterium Propionibacterium acnes to be frequently present in prostate tissue from men suffering from prostate disease. P. acnes has been shown to be associated with histological inflammation in human prostatectomy specimens, and also to induce strong inflammatory response in prostate-derived tissue culture models. The present paper describes a rat model for assessment of the pathogenic potential of P. acnes in prostate. Prostate glands of Sprague Dawley rats (n = 98) were exposed via an abdominal incision and live P. acnes or, in control rats, saline were injected into the ventral and dorso-lateral lobes. Rats were sacrificed 5 days, 3 weeks, 3 months and 6 months post infection, and prostate tissue was analyzed for bacterial content and histological inflammation. Rat sera were assessed for levels of CRP and anti-P. acnes IgG. Live P. acnes could be recovered from the dorso-lateral lobes up to 3 months post infection, while the ventral lobes were cleared from bacteria at that time. In samples up to 3 months post infection, the dorso-lateral lobes exhibited intense focal inflammation. CRP and IgG levels were elevated throughout the span of the experiment, and reached maximum levels 3 weeks and 3 months post infection, respectively. We show that P. acnes have the potential to cause chronic infection in previously healthy prostate, and that the infection has potential to cause chronic histological inflammation in the infected tissue. The high prevalence of P. acnes in human prostate tissue calls for resolution of pathogenic details. The present rat model suggests that complications such as chronic inflammation may be induced by P. acnes infection.     

Precipitins to Dietary Proteins in Serum and Upper Intestinal Secretions of Coeliac Children

We have used precipitin tests to detect antibodies to 10 dietary proteins in the serum (71 cases) and intestinal secretions (51 cases) of a group of children. Thirty-three of the patients had untreated coeliac disease. Our aims were to find out if, in coeliac patients, there was intestinal secretion of antibodies to wheat proteins only or if, as in coeliac serum, antibodies to many food proteins were present; and to confirm that secretion of antibodies to wheat or gluten was specific for coeliac disease.Precipitins to one or more dietary antigens were detected in the intestinal secretions of 26 out of 30 coeliacs and of 11 out of 21 children who did not have coeliac disease. Most of the positive reactions were with the antigens wheat flour, gluten, oatmeal, and egg. Though precipitins to wheat flour or gluten were present in the intestinal secretions of 22 out of 30 coeliacs this was not specific for coeliac disease for these precipitins were also present in 8 out of 21 non-coeliac children.Serum precipitins were detected in 27 out of 33 coeliacs (to the antigens wheat flour, gluten, oatmeal, rice flour, milk, bovine calf serum, sheep serum, and egg) and in 5 out of 33 non-coeliacs (mainly to milk and calf serum, but two infants aged 3 and 5 months had precipitins to several antigens).     

Protective Effects of Anthocyanins from Blackberry in a Rat Model of Acute Lung Inflammation

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E₂ (PGE₂). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg_-1 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.     

Green Tea Extract Treatment Alleviates Ocular Inflammation in a Rat Model of Endotoxin-Induced Uveitis

Green tea extract (GTE) ingested by rats exerted anti-oxidative activities in various ocular tissues as shown in our previous studies. The present work investigated anti-inflammatory effects of GTE on endotoxin-induced uveitis (EIU). EIU was generated in adult rats by a footpad injection of 1 mg/kg lipopolysaccharide (LPS). Oral administration of GTE (550 mg/kg) was given one, two or four times after LPS injection. Twenty-four hours later, LPS produced severe hyperemia and edema in the iris. Immunocytochemical examinations showed an accumulation of infiltrating cells in the aqueous humor that were immunopositive for cluster of differentiation 43 (CD43) and CD68, markers for leucocytes and macrophages, respectively. Analyses of the aqueous humor showed an increase in pro-inflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). GTE treatments improved the clinical manifestations and reduced infiltrating cells and protein exudation in the aqueous humor, which were not observed under half dose of GTE (275 mg/kg). The number of CD68 positive macrophages residing in the iris and ciliary was also reduced. GTE suppressed production of TNF-α, IL-6 and MCP-1 in the aqueous humor, which was associated with a down-regulation of LPS receptor complex subunits, Toll-like receptor 4 (TLR-4) and CD14, and suppression of nuclear factor-kappa Bp65 (NF-κBp65) in the iris and ciliary body. Our findings show that GTE is a potent anti-inflammatory agent against the inflammation of EIU, and suggest a potential use in treatment of acute uveitis.     

盆腔结缔组织炎大鼠模型的层黏连蛋白表达

目的建立混合菌液致SD大鼠盆腔结缔组织炎模型,研究大鼠盆腔黏连组织的病理学改变与层黏连蛋白表达。方法用注射器将细菌混悬液0．5mL,注人大鼠盆腔,并结合子宫穿孔手术,建立SD大鼠盆腔结缔组织炎模型。采用Philips分级评分法对大鼠盆腔黏连程度分级评分,切取黏连组织,用多聚甲醛固定,进行病理检测,并采用免疫组化法测定层黏连蛋白表达情况。结果模型组大鼠盆腔黏连Philips分级评分以Ⅲ级为主,与正常对照组比较差异极显著。HE染色镜检模型组可见大量炎细胞浸润,脓肿形成,伴有多少不等的纤维组织增生。免疫组化染色镜检模型组可见层黏连蛋白高表达,纤维结缔组织增生程度越高,层黏连蛋白表达越多。结论盆腔注射混合菌液并结合子宫穿孔手术,可作为建立大鼠盆腔结缔组织炎模型的方法。层黏连蛋白在盆腔结缔组织炎发病中起重要作用,参与整个炎症过程并维持盆腔黏连组织纤维化,可作为判断盆腔结缔组织炎的炎症和纤维化程度的参考指标。     

Natural Killer Cells Promote Long-Term Hepatobiliary Inflammation in a Low-Dose Rotavirus Model of Experimental Biliary Atresia

ConclusionsLower inoculation of RRV-induced progressive liver injury and fibrosis via NK cells. These findings point to the potential use of NK cell-depleting strategies to block progression of liver disease in biliary atresia.     

Persistent Lung Inflammation and Fibrosis in Serum Amyloid P Component (Apcs-/-) Knockout Mice

Fibrosing diseases, such as pulmonary fibrosis, cardiac fibrosis, myelofibrosis, liver fibrosis, and renal fibrosis are chronic and debilitating conditions and are an increasing burden for the healthcare system. Fibrosis involves the accumulation and differentiation of many immune cells, including macrophages and fibroblast-like cells called fibrocytes. The plasma protein serum amyloid P component (SAP; also known as pentraxin-2, PTX2) inhibits fibrocyte differentiation in vitro, and injections of SAP inhibit fibrosis in vivo. SAP also promotes the formation of immuno-regulatory Mreg macrophages. To elucidate the endogenous function of SAP, we used bleomycin aspiration to induce pulmonary inflammation and fibrosis in mice lacking SAP. Compared to wildtype C57BL/6 mice, we find that in Apcs^-/- “SAP knock-out” mice, bleomycin induces a more persistent inflammatory response and increased fibrosis. In both C57BL/6 and Apcs^-/- mice, injections of exogenous SAP reduce the accumulation of inflammatory macrophages and prevent fibrosis. The types of inflammatory cells present in the lungs following bleomycin-aspiration appear similar between C57BL/6 and Apcs ^-/- mice, suggesting that the initial immune response is normal in the Apcs^-/- mice, and that a key endogenous function of SAP is to promote the resolution of inflammation and fibrosis.     

Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.     

Tourniquet-Applied Upper Limb Orthopaedic Surgery Results in Increased Inflammation and Changes to Leukocyte,Coagulation and Endothelial Markers

Purpose

During this pilot clinical study, patients scheduled for elective tourniquet-applied upper limb orthopaedic surgery were recruited to investigate the effects of surgery on various biological markers (n = 10 patients).

Methods

Three venous blood samples were collected from the arm at the ante-cubital fossa, at baseline (pre-operatively), 5 and 15 minutes after reperfusion (post-operatively). Neutrophil and monocyte leukocyte sub-populations were isolated by density gradient centrifugation techniques. Leukocyte activation was investigated by measuring the cell surface expression of CD62L (L-selectin), CD11b (Mac-1) and the intracellular production of hydrogen peroxide (H2O2), via flow cytometry. C-reactive protein (CRP) was measured using a clinical chemistry analyser. Plasma concentrations of protein C and von Willebrand factor (vWF) were measured using enzyme-linked fluorescent assays (ELFA).

Results

Following tourniquet-applied upper limb orthopaedic surgery, there was a decrease in neutrophil CD62L expression (p = 0.001), an increase in CD11b expression and in the intracellular production of H2O2 by neutrophils and monocytes (p<0.05). An increase in CRP concentration (p<0.001), a decrease in protein C concentration (p = 0.004), with a trend towards elevated vWF levels (p = 0.232) were also observed during this time.

Conclusions

Conventionally, patients undergoing orthopaedic surgery have been monitored in the peri-operative period by means of CRP, which is a non-specific marker of inflammation. This test cannot differentiate between inflammation due to current or pre-existing disease processes and the development of ischaemia-reperfusion injury surgery. The findings from this study suggest that markers such as CD11b, protein C and H₂O₂ may provide alternative ways of assessing leukocyte and coagulation activation peri-operatively. It is proposed that by allowing orthopaedic surgeons access to laboratory markers such as CD11b, protein C and H₂O₂, an accurate assessment of the extent of inflammation due to surgery per se could be made.     

Transient and Persistent Metabolomic Changes in Plasma following Chronic Cigarette Smoke Exposure in a Mouse Model

Cigarette smoke exposure is linked to the development of a variety of chronic lung and systemic diseases in susceptible individuals. Metabolomics approaches may aid in defining disease phenotypes, may help predict responses to treatment, and could identify biomarkers of risk for developing disease. Using a mouse model of chronic cigarette smoke exposure sufficient to cause mild emphysema, we investigated whether cigarette smoke induces distinct metabolic profiles and determined their persistence following smoking cessation. Metabolites were extracted from plasma and fractionated based on chemical class using liquid-liquid and solid-phase extraction prior to performing liquid chromatography mass spectrometry-based metabolomics. Metabolites were evaluated for statistically significant differences among group means (p-value≤0.05) and fold change ≥1.5). Cigarette smoke exposure was associated with significant differences in amino acid, purine, lipid, fatty acid, and steroid metabolite levels compared to air exposed animals. Whereas 60% of the metabolite changes were reversible, 40% of metabolites remained persistently altered even following 2 months of smoking cessation, including nicotine metabolites. Validation of metabolite species and translation of these findings to human plasma metabolite signatures induced by cigarette smoking may lead to the discovery of biomarkers or pathogenic pathways of smoking-induced disease.     

Telmisartan Attenuates Colon Inflammation,Oxidative Perturbations and Apoptosis in a Rat Model of Experimental Inflammatory Bowel Disease

Accumulating evidence has indicated the implication of angiotensin II in the pathogenesis of inflammatory bowel diseases (IBD) via its proinflammatory features. Telmisartan (TLM) is an angiotensin II receptor antagonist with marked anti-inflammatory and antioxidant actions that mediated its cardio-, reno- and hepatoprotective actions. However, its impact on IBD has not been previously explored. Thus, we aimed to investigate the potential alleviating effects of TLM in tri-nitrobenezene sulphonic acid (TNBS)-induced colitis in rats. Pretreatment with TLM (10 mg/kg p.o.) attenuated the severity of colitis as evidenced by decrease of disease activity index (DAI), colon weight/length ratio, macroscopic damage, histopathological findings and leukocyte migration. TLM suppressed the inflammatory response via attenuation of tumor necrosis factor-α (TNF-α), prostaglandin E₂ (PGE₂) and myeloperoxidase (MPO) activity as a marker of neutrophil infiltration besides restoration of interleukin-10 (IL-10). TLM also suppressed mRNA and protein expression of nuclear factor kappa B (NF-κB) p65 and mRNA of cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proinflammatory genes with concomitant upregulation of PPAR-γ. The alleviation of TLM to colon injury was also associated with inhibition of oxidative stress as evidenced by suppression of lipid peroxides and nitric oxide (NO) besides boosting glutathione (GSH), total anti-oxidant capacity (TAC) and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). With respect to apoptosis, TLM downregulated the increased mRNA, protein expression and activity of caspase-3. It also suppressed the elevation of cytochrome c and Bax mRNA besides the upregulation of Bcl-2. Together, these findings highlight evidences for the beneficial effects of TLM in IBD which are mediated through modulation of colonic inflammation, oxidative stress and apoptosis.     

A Novel Set of Hepatic mRNAs Preferentially Expressed during an Acute Inflammation in Rat Represents Mostly Intracellular Proteins

A cloning of hepatic cDNAs associated with the early phase of an acute, systemic inflammation was carried out by differential screening of arrayed cDNA clones from rat livers obtained at 4-8 h postchallenge with Freund's complete adjuvant. End sequencing of 174 selected clones provided three cDNA groups that coded for: (i) 23 known acute-phase proteins, (ii) 31 known proteins whose change in hepatic synthesis during an acute phase was so far unsuspected, and (iii) 36 novel proteins whose cDNAs were completely sequenced. For 16 proteins in the third group the hepatic mRNA could be detected and quantitated by Northern blot hybridization in Freund's adjuvant-challenged animals, and an extrahepatic expression in healthy animals was further investigated. Matching the open reading frames of the 36 novel proteins with general and specialized data libraries indicated the potential relationships of 16 of these proteins with known protein families/superfamilies and/or the presence of functional domains previously described in other proteins. Overall, our search for novel inflammation-associated proteins selected mostly known or as yet undescribed proteins with an intracellular or membrane location, which extends our knowledge of the proteins involved in the intracellular metabolism of hepatic cells during a systemic, acute-phase response. Finally, some of the cDNAs above allowed us to successfully identify hepatic mRNAs that are differentially expressed in acute vs chronic (polyarthritis) inflammatory conditions in rat.     

Prenatal Zinc Deficiency: Influence on Heart Morphology and Distribution of Key Heart Proteins in a Rat Model

The etiology of congenital heart disease is multifactorial, with genetics and nutritional deficiencies recognized as causative agents. Maternal zinc (Zn) deficiency is associated with an increased risk for fetal heart malformations; however, the contributing mechanisms have yet to be identified. In this study, we fed pregnant rats a Zn-adequate diet (ZnA), a Zn-deficient (ZnD), or a restricted amount of Zn adequate diet (RF) beginning on gestation day (GD) 4.5, to examine whether increased cell death and changes in cardiac neural crest cells (NCC) play a role in Zn deficiency-induced heart defects. Fetuses were collected on GD 13.5, 15.5, and 18.5 and processed for GATA-4, FOG-2, connexin-43 (Cx43), HNK-1, smooth muscle α-actin (SMA) and cleaved caspase-3 protein expression. Fetuses from ZnA-fed dams showed normal heart development, whereas fetuses from dams fed with the ZnD diet exhibited a variety of heart anomalies, particularly in the region of the outflow tract. HNK-1 expression was lower than normal in the hearts of GD13.5 and 15.5 ZnD fetuses, particularly in the right atrium and in the distal tip of the interventricular septum. Conversely, Cx43 immunoreactivity was increased throughout the heart in fetuses from ZnD dams compared to fetuses from control dams. The distribution and intensity of expression of SMA, GATA-4, FOG-2, and markers of apoptosis were similar among the three groups. We propose that Zn deficiency induced alterations in the distribution of Cx43 and HNK-1 in fetal hearts contribute to the occurrence of the developmental heart anomalies.     

Differential Phosphorylation of GluN1-MAPKs in Rat Brain Reward Circuits following Long-Term Alcohol Exposure

The effects of long-term alcohol consumption on the mitogen-activated protein kinases (MAPKs) pathway and N-methyl-D-aspartate-type glutamate receptor 1 (GluN1) subunits in the mesocorticolimbic system remain unclear. In the present study, rats were allowed to consume 6% (v/v) alcohol solution for 28 consecutive days. Locomotor activity and behavioral signs of withdrawal were observed. Phosphorylation and expression of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38 protein kinase and GluN1 in the nucleus accumbens, caudate putamen, amygdala, hippocampus and prefrontal cortex of these rats were also measured. Phosphorylation of ERK, but not JNK or p38, was decreased in all five brain regions studied in alcohol-drinking rats. The ratio of phospho/total-GluN1 subunit was reduced in all five brain regions studied. Those results suggest that the long-term alcohol consumption can inhibits GluN1 and ERK phosphorylation, but not JNK or p38 in the mesocorticolimbic system, and these changes may be relevant to alcohol dependence. To differentiate alcohol-induced changes in ERK and GluN1 between acute and chronic alcohol exposure, we have determined levels of phospho-ERK, phospho-GluN1 and total levels of GluN1 after acute alcohol exposure. Our data show that 30 min following a 2.5 g/kg dose of alcohol (administered intragastrically), levels of phospho-ERK are decreased while those of phospho-GluN1 are elevated with no change in total GluN1 levels. At 24 h following the single alcohol dose, levels of phospho-ERK are elevated in several brain regions while there are no differences between controls and alcohol treated animals in phospho-GluN1 or total GluN1. Those results suggest that alcohol may differentially regulate GluN1 function and ERK activation depending on alcohol dose and exposure time in the central nervous system.     

A Filtered Database Search Algorithm for Endogenous Serum Protein Carbonyl Modifications in a Mouse Model of Inflammation

During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum.During inflammation, activated phagocytes secrete reactive nitrogen species (RNS) and reactive oxygen species (ROS) that can eliminate infectious agents. If inflammation is chronic, RNS and ROS can also damage surrounding host tissue, resulting in protein modification in the form of protein-carbonyls (1). Total protein carbonylation has been used as a marker of oxidative stress and inflammation and increased levels have been seen in heart disease, lung disease, aging, neurodegenerative disorders, and inflammatory bowel disease (2–7). The carbonylation of proteins can result from the direct oxidation of protein side-chains, forming the glutamate and aminoadipate semialdehydes (Scheme 1) (8, 9), but can also occur from the indirect oxidation of polyunsaturated fatty acids (lipid peroxidation) and carbohydrates, leading to a variety of reactive aldehydes (Scheme 2) (10). These aldehydes covalently modify proteins through conjugate addition (often Michael addition) to nucleophilic amino acid side chains, creating protein-bound carbonyls (10, 11).Open in a separate windowScheme 1.Direct oxidative carbonylation of proteins to form glutamate and aminoadipate semialdehydes.Open in a separate windowScheme 2.Reactive aldehydes, arising from oxidation of polyunsaturated fatty acids and carbohydrates, can indirectly lead to protein carbonylation.In a previous study, DNA oxidative damage products, from tissues from the SJL mouse model of inflammation, were quantitated (12). Only the lipid peroxidation adducts increased in association with inflammation, which suggested an important role of lipids in inflammatory disease progression and established a direct correlation between inflammation and the increased formation of reactive aldehydes from oxidized lipids. Although DNA modification because of inflammation has been the focus of many animal and human studies, it is proteins that are considered most likely to be ubiquitously affected by disease, response, and recovery (13), and the biological consequences include more rapid protein turnover as well as novel signaling (14–16). The overall carbonylation of proteins has been well documented in other inflammatory animal models, which have shown significant increases in protein-carbonyls in the mucosal lining of rat colon (17) and mouse colon (5) whereas increased levels of protein carbonyls were observed in rat serum, along with a higher turnover of proteins from the inflamed tissue (18, 19). Furthermore, increased protein carbonyl modification has been reported in studies of the colon mucosal lining from patients diagnosed with inflammatory bowel disease (20, 21). Taken together, these observations suggest that an increase in carbonylated proteins is likely in the SJL mouse and that the extent and type of protein-carbonyls could potentially be a marker for inflammation and disease.The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to nitric oxide (NO) overproduction from inflammation (22). Injections of RscX lymphoma cells into these mice result in rapid tumor growth as well as host T-cell proliferation in lymph nodes, spleen, and liver, resulting in morbidity within 15 days. The induced macrophages create a 50-fold increase in NO production in spleen and lymph nodes and the post-translational modification 3-nitrotyrosine was highly elevated in spleen tissue.The identification of endogenously formed protein carbonyls in serum is challenging because of their low abundance and the large number of possible modifications (1, 2, 23), some of which are shown in Schemes 1 and 2. We recently identified proteins modified by the carbonyl 9,12-dioxo-10(E)-dodecenoic acid (DODE) in cells treated with the hydroperoxide of linoleic acid (13-HPODE) (24). This work used a technique first demonstrated by Maier and coworkers (25, 26). Protein carbonyls were derivatized with an aldehyde reactive probe (ARP),¹ a biotinylated hydroxylamine that reacts preferentially with aldehyde and keto groups (27), allowing for subsequent enrichment of the modified proteins by avidin affinity. DODE-modified proteins were also identified using an anti-DODE antibody and Western blots. Although a number of DODE modified proteins were identified, we were unable to definitively identify the carbonyl modified peptides by mass spectrometry due both to low abundance and to the interference of ARP-tag-specific fragment ions on database searching.In this current study, SJL mouse serum was screened for the presence of protein carbonyls endogenously formed during inflammation. Carbonyl-modified proteins were then identified using techniques previously established (24); first anti-DODE Western blotting followed by ARP derivatization/enrichment and two-dimensional liquid chromatography tandem MS (2D-LC-MS/MS). These proteins then formed a database of putative carbonyl-modified proteins from SJL mouse serum. To identify the type of carbonyl modification and the modified peptide, the ARP derivatized peptides were enriched and analyzed by mass spectrometry. To minimize the confounding effect of ARP fragmentation, an algorithm (in silico filter) was written that filtered the mass spectrometry data to select only those peptides containing the known ARP pattern of fragmentation. This alone effectively reduced the number of false positives. To further alleviate the interfering effects of ARP fragments on peptide identification by database searching, a de novo searching algorithm (Biotin Peptide Identification program, BPI) was written. Peptides were evaluated against the database of proteins that had been previously identified as potentially carbonyl modified. Because modified peptides were searched against a finite list of proteins and all results were manually evaluated, the BPI program did not calculate a statistical peptide score, which allowed the identification of lower abundant modified peptides that would not be considered significant by standard search engines such as Mascot. The BPI program was also written with the flexibility to evaluate a wide range of known carbonyl-adduct masses and could therefore screen for a large number of carbonyl adducts at one time. This should also allow the program to be used with modification/enrichment systems other than the one used here. The program thus selected a finite number of carbonyl modified peptides, resulting in the identification of a number of proteins that were endogenously carbonylated in serum from the SJL mouse inflammation model.     

Increased Serum Adipokines Implicate Chronic Inflammation in the Pathogenesis of Overactive Bladder Syndrome Refractory to Antimuscarinic Therapy

Objectives

Recent studies have shown that chronic inflammation is involved in overactive bladder (OAB) syndrome. OAB could be a subtype of neurogenic inflammation. This pilot study investigated serum adipokine levels in patients with OAB refractory to antimuscarinic therapy.

Methods

Thirty consecutive patients with OAB-dry (n = 16) or OAB-wet (n = 14) refractory to previous antimuscarinic treatment were prospectively enrolled in this study, a group of 26 normal subjects without lower urinary tract symptoms served as controls. Concentrations of serum C-reactive protein (CRP), nerve growth factor (NGF), and adipokines including interleukins ([IL], IL-1β, IL-6, IL-8), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, insulin, and leptin were quantified using a bead-based human serum adipokine panel B kit. Data were analyzed using the LX 200 platform. Patients were further classified as having dry or wet OAB and having medical diseases or not. The serum CRP, NGF, and adipokine levels were compared between OAB patients and the controls, and between OAB subgroups.

Results

The serum concentrations of CRP, NGF, IL-1β, IL-6, IL-8, and TNF-α in OAB-dry and OAB-wet patients were significantly higher than among the controls. There was no significant difference in adipokine levels between OAB-dry and OAB-wet, or between OAB patients with and without medical diseases. Serum CRP and NGF levels were significantly higher only in OAB-wet or OAB patients with medical diseases than among controls. The MCP-1 levels, on the other hand, were significantly higher in OAB-dry or OAB patients with disease, than the controls.

Conclusions

Both OAB-dry and OAB-wet patients showed increased serum CRP, NGF, and adipokine levels compared with the controls, suggesting chronic inflammation of the bladder involving both peripheral and central mechanisms in all OAB patients refractory to antimuscarinic therapy. The increased serum adipokine levels were not relevant to medical diseases.