Activity Of Polymorphonuclear Cells Research Articles

Unraveling the immune and metabolic changes associated with metritis in dairy cows

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1β. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

DIVERSITY OF BACTERIA AND FUNGI AND PHAGOCYTIC PROPERTIES OF NEUTROPHILS IN SOME RESPIRATORY DISEASES

Respiratory diseases are considered serious diseases that affect all age groups and can lead, in most cases, to death. This study aimed to investigate the bacterial and fungal causes of some respiratory infections and the relationship between the phagocytic activity of polymorphonuclear cells and respiratory tract infection.146 clinical samples of sputum were collected from 50 patients with chronic tuberculosis, and pharyngeal swabs from 96 patients with respiratory infections other than tuberculosis, including chronic bronchitis (22 patients), allergic (66 patients), and asthma (5patients) pneumonia (3 patients). These samples were collected from patients referred to the Specialized National Centre for Chest and Respiratory Diseases in Baghdad for the period between 2018and 2019. It has been found that out of the total 146 patients, the number of infected males is 76 (52.1 %) and 70 females (47.9%). 174 bacterial isolates were isolated and identified, as well as it was noted that the percentage of respiratory infections caused by gram-positive bacteria was higher (66.1%) compared to the respiratory infections caused by gram-negative bacteria (33.9%), As for fungal infections, from 140 isolates showed that the percentage of respiratory infections caused by fungi was the highest (70.9%) compared to respiratory infections caused by yeasts (29%). The current study indicated that respiratory tract infections stimulated the phagocytic activity of polymorphonuclear cells for all age groups. Keywords:

MMP-2 salivary activity in type 2 diabetes mellitus patients

BackgroundType 2 diabetes mellitus (T2DM) and periodontitis are chronic inflammatory diseases with a bidirectional relationship. The uncontrolled levels of glucose in T2DM patients change the pathophysiology and balance of inflammatory mediators. Matrix Metalloproteinase-2 (MMP-2) is a zinc-dependent endopeptidase that is responsible for tissue remodeling and degradation of the extracellular matrix in periodontal tissue. Therefore, the uncontrolled levels of glucose in T2DM could lead to an imbalance in MMP-2 activity in saliva, favoring the development of periodontitis.MethodsNinety-seven T2DM patients from Hospital Dr. Donato Alarcon were included in the study. Following clinical examination, the patients were classified into four groups according to the presence and degree of periodontal disease and glycemic control. Blood and whole saliva samples (WSS) were collected from each patient. Blood samples were used for Hba1c and polymorphonuclear cells count determination, while WSS were used to determine MMP-2 activity, TIMP-1 and nitrite. MMP-2 activity was determined by zymography. TIMP-1 were determined by Western blotting, and nitric oxide (NO) levels were determined by the Griess method.ResultsOf the 97 patients with T2DM, 66 had periodontitis of different severities: 18 patients had mild periodontitis, 15 had moderate and 33 had severe. Salivary MMP-2 activity, HbA1c and TIMP-1 were positively correlated with the severity of periodontitis. On the other hand, the increase in HbA1c was negatively correlated with MMP-2 activity and quantity of TIMP-1 but was positively correlated with nitrite levels.ConclusionsT2DM with glycemic uncontrol conditions, distinct clinical alterations in periodontal tissue were identified, including a decrease in the gingival redness, increased the clinical attachment loss and imbalance of MMP-2/TIMP-1, as the possible causes of disorders promoting the progression of periodontitis. Accelerated periodontitis development with poor glycemic uncontrol likely results from the altered response of host defenses and decreased activity of polymorphonuclear cells. Taken together, these findings identify MMP-2 as a promising molecular market for periodontitis.

IMMUNOMODULATORY ACTIVITY OF NUTRACEUTICAL FORMULATION AND ITS POTENTIATION BY SELF-FORTIFICATION AND COW URINE DISTILLATE FORTIFICATION METHODS

Objective: This study prepared, evaluated immunomodulatory activity of nutraceutical formulation and studied the effect of self-mortification and cow urine distillate fortification methods on the immunomodulatory potential of nutraceutical formulation.Methods: Three types of nutraceutical formulations i.e. Nutraceutical formulation (NF), self fortitfied nutraceutical formulation (SFNF) and self fortitfied nutraceutical formulation fortified with cow urine distillate (SFNECUD) were prepared using fine powders of amla, apple, garlic, onion, wheat grass, papaya, turmeric and cow urine distillate by different methods. The immunomodulatory activity of nutraceutical formulations at a dose of 500 mg/kg was assessed by various immune function parameters like cell-mediated immunity (neutrophil adhesion, delayed type hypersensitivity (DTH) response and cyclophosphamide-induced neutropenia), humoral immunity (serum immunoglobulins level and haemoagglutination antibody titer), and phagocytic activity (carbon clearance and polymorphonuclear (PMN) cell activity).Results: Oral administration of NF, SFNF and SFNFCUD showed significant (p<0.01) increase in adhesion of neutrophils, potentiation of the DTH reaction and attenuation of cyclophosphamide-induced neutropenia. A significant increase in serum immunoglobulin levels and production of circulating antibody titer in response to sheep red blood cells (SRBCs) was also observed. In addition, an increase in the phagocytic index in carbon clearance assay and an increase in the phagocytic activity of PMN cells was observed.Conclusion: From the above results, it can be concluded that all three types of formulations showed significant immunostimulant activity. SFNF and SFNFCUD showed better immunomodulatory activity than NF suggesting the potentiation of immunomodulatory potential of NF activity by fortification methods.

Protective effect of cytosine-phosphate-guanosine oligodeoxynucleotide against experimental mastitis induced by Cryptococcus neoformans infection in goats

The present study investigated the effect of synthetic non-methylated oligonucleotides containing Cytosine-phosphate-guanosine dinucleotides (Cytosine-phosphate-guanosine oligodeoxynucleotide) on caprine mastitis with Heat Killed Cryptococcus neoformans Ag. 20 healthy local breed does were used with weight ranging of 25-30 Kg and free of mastitis by examination via California Mastitis Test and Somatic Cell Count. The does were allotted into four equal groups, the first group (G1) was treated intramammary with 100μg/kg of Cytosine-phosphate-guanosine oligodeoxy nucleotide on fifth day postpartum in the right mammary gland while the left mammary gland served as control and were infused with sterile phosphate buffered saline. On day 8 postpartum repeat dosages of Cytosine-phosphate-guanosine oligodeoxynucleotide and phosphate buffered saline were infused respectively. On day 9 pp the right mammary gland was infused with 2ml of 2x108 cell/ml of Heat Killed Cryptococcus neoformans Ag. The second group (G2) was infused at day 9 postpartum with 2ml of 2x108 cell/ml of Heat Killed Cryptococcus neoformans Ag in the right mammary gland only. The third group (G3) was left until the challenge test done after one week of immunization in the G1 and G2, by inoculation of 2ml of 5x106 viable C. neoformans in the right mammary gland. The fourth group (G4) was kept as a control receiving 2ml of sterile PBS. Blood samples were collected at 0, 5, 10, 20, 30 and 40 days of the study, to determine the antibody titer by passive haemagglutination assay, while the cell mediated immunity was evaluated by detecting the goat Interferon Gamma by ELISA test and Phagocytic index. Also the cell mediated immunity was determined by delayed type hypersensitivity test after 21 days of immunization. The results showed a significant variation (P≤0.05) between vaccinated groups (G1 and G2) and the control. However, there was a significant increase (P≤0.05) of skin thickness shown after 48 hrs in the G1compared to G2. High level of Interferon Gamma concentration was noticed in the G1 as compared with other groups. Moreover, cell mediated immunity developed effectively in the G1 which was noted by a significant increase (P≤0.05) of phagocytic activity of polymorphonuclear cells. The high level of antibody titer was observed in the G1 as compared with other groups. In conclusion: These results suggest that vaccination with Cytosine-phosphate-guanosine oligodeoxynucleotide plus Heat Killed Cryptococcus neoformans Ag intramammary lead to a good protection of caprine mammary glands against C. neoformans mastitis.

Impact of Probiotics for Reducing Infections in Veterans (IMPROVE): Study protocol for a double-blind, randomized controlled trial to reduce carriage of Staphylococcus aureus

BackgroundStaphylococcus aureus (S. aureus) is an organism of great public health importance, causing 20,000 deaths annually. Decolonization of patients with S. aureus may prevent infections, yet current options are limited to antimicrobials that promote antibiotic resistance and can cause adverse side effects. Probiotics have potential to reduce colonization of pathogenic bacteria, representing a promising alternative for S. aureus decolonization, but thus far lack rigorous evaluation. MethodsPotential subjects were recruited from inpatient and outpatient settings within a VA medical center and screened for S. aureus gastrointestinal (GI) or extra-GI colonization using swabs at multiple body sites. Positive, eligible, consenting participants were stratified by colonization site and randomized in a 1:1 ratio to 4-weeks of daily placebo or Lactobacillus rhamnosus (L. rhamnosus) HN001 probiotic treatment. Blood and stool samples, and treatment adherence reports were collected from each subject throughout the study, along with a final set of swabs at study completion to detect S. aureus carriage. The outcomes of this study are GI or extra-GI carriage by S. aureus at the end of 4weeks of therapy, change in phagocytic activity of polymorphonuclear cells from pre-intervention to post-intervention, and symptomatic S. aureus infection at any site during the study period. Conclusion114 participants have been recruited for this study. Analysis of outcomes is underway. This is the first clinical trial to examine the efficacy of L. rhamnosus HN001 for decolonization of S. aureus, and investigates the mechanism by which L. rhamnosus HN001 mediates its effect on S. aureus colonization.ClinicalTrials.govIdentifierNCT01321606

Evaluation of immunotherapeutic potential of Ocimum sanctumin bovine subclinical mastitis

Mastitis, the inflammation of mammary glands, is caused by a wide range of contagious and environmental microbes, whose clearance from the udder is generally dependent on the use of antibiotics. However, antibiotic therapy may result in development of drug-resistant microbes and depress the activity of polymorphonuclear cells. The present study was therefore undertaken to explore the immunotherapeutic potential of an herb, Ocimum sanctum, in mastitis in dairy cows. The study involved twenty HF × Sahiwal lactating dairy cows, each identified with at least one specific subclinical mastitis quarter as per International Dairy Federation criteria. The cows were divided into two equal groups: a control group and a group administered O. sanctum leaf powder at 600 mg/kg body weight daily divided into two doses orally for 7 days. The treatment could eliminate 69.23% of intramammary infections (χ 2 = 5.07; P ≤ 0.5) and resulted in a significant reduction in somatic cell count and ceruloplasmin concentration, thus subsiding udder inflammation and improving milk quality. The herb was also found to possess immunomodulation potential, as evidenced by the significant increase in phagocytic activity of milk neutrophils and enhanced lactoperoxidase and myeloperoxidase activities. Thus, the results indicated the immunotherapeutic potential of O. sanctum in treating bovine-specific subclinical mastitis.

Human neutrophil peptide-1 decreases during ageing in selected Mexican population.

Antimicrobial peptide innate immunity plays a central role in the susceptibility to infectious diseases, as has been described extensively in different settings. However, the role that these molecules play in the immunity mediated by polymorphonuclear phagocytes as part of the innate immunity of ageing individuals has not been described. In the present study, we addressed the question whether antimicrobial activity in polymorphonuclear cells from elderly individuals was altered in comparison with young adults. We compared phagocytosis index, bacterial killing efficiency, myeloperoxidase activity and cathelicidin expression. Results showed that there were no statistical differences among groups. However, human neutrophil peptide-1 (HNP-1) was decreased in the elderly individuals group. Results suggest that the decreased HNP-1 production in the polymorphonuclear phagocytes form elderly individuals might have an important participation in the increased susceptibility to infectious diseases.

Polymorphonuclear Cell Functional Impairment in Relapsing Remitting Multiple Sclerosis Patients: Preliminary Data

Multiple Sclerosis patients run an increased risk of microbial infections, which leads to high rates of hospitalization and infection-related mortality. Although immunotherapy may increase infection risk in some cases, data as to the relationship among microbial factors, immunotherapy and alterations in the innate immunity of these patients are still scanty. On these grounds, this interdisciplinary study aims at investigating the role the functional activity of polymorphonuclear cells (PMNs) play in relapsing remitting multiple sclerosis at different stages. The in vitro ability of PMNs from patients, either untreated or treated with immunosuppressant or immunomodulatory drugs to kill Klebsiella pneumonia or Candida albicans, were investigated and compared to PMNs from healthy subjects. The release of various cytokines was also assessed, as was the production of reactive oxygen species and their ability to regulate apoptosis after microbial stimulation. Our results indicate that although patients have a normal number of PMNs, they have a statistically significant (p<0.05) reduction in intracellular killing activity. Although variations are strongly related to the therapeutic management of patients, they are independent from their disease stage. As no statistically significant differences were observed between patients and controls in cytokine release values, reactive oxygen species production or apoptosis, we came to the conclusion that other factors may be involved. Supportive validation of these results from further studies might well help in identifying a subset of patients at high risk of infection who could benefit from a closer follow-up and/or antibiotic prophylaxis.

Staphylococcal Panton‐Valentine leucotoxin targets retina through the ganglion cell layer in a rabbit toxin‐induced endophthalmitis model

AbstractPurpose Staphylococcal leucotoxins form a cluster of bi‐component leucotoxins that are associated with tissue necrosis pathologies, such as necrotising pneumonia, osteomyelitis and endophtalmitis. Among them, Panton‐Valentine leucotoxine (PVL) is often associated with strains isolated in such diseases and seems to possess acute capabilities to promote necrosis. When injected into the rabbit vitreous, these toxins were proven to induce a huge inflammatory reaction that might concern the full eyeball. The purpose of this study was to identify the PVL target cells into the retina, using a rabbit model of endophtalmitis.Methods Three µg of LukS‐PV were injected in a series of rabbit eyes. After 1 hour, the eyes were enucleated. Frozen sections were prepared for immunofluorescence detection tests. PVL was immunolabelled using a specific antibody and a secondary fluorescent antibody. Co‐labelling with GFAP (Muller cells), RBPMS (ganglion cells), ChAT (starbust displaced amacrine cells) antibodies were realized. Left eyes were used as negative controls.Results LukS‐PV was present at the ganglion cells layer. A few dendrites in the inner plexiform layer were also noted. Co‐labeling experiments with the specific marker ChAT (acetylcholin transferase) segregated starbust displaced amacrine cells as a retinal target of LukS‐PV. 65% of the infected cells were ChAT positive.Conclusion At least, one retinal cell type, i.e. Starbust amacrine cells, can now be designated as a target of PVL and it will be important to explore how this cell fitness is sensitive to such a toxin compared with the activation of polymorphonuclear cells, and neurons that also were reported as possible targets.

Activation of polymorphonuclear cells releases the chemotactic factor Ecrg4 into conditioned media

Sentinel factors expressed on circulating cells monitor homeostasis and may initiate inflammatory signaling after injury. We identified Esophageal Cancer Related Gene‐4 (Ecrg4) as a candidate sentinel factor that resides on the surface of quiescent polymorphonuclear cells (PMNs) and responds to injury by releasing Ecrg4 protein from the cell surface. The objective of this study is to evaluate the kinetics of Ecrg4 release and the biological activity of Ecrg4 in chemotaxis migration assays. Peripheral blood was obtained from healthy volunteers and isolated cells were incubated with lipopolysaccharide (LPS) to characterize the release of Ecrg4 from the leukocyte surface. To establish the chemotactic potential of soluble recombinant Ecrg4, cells were subjected to in vitro migration assays using modified Boyden chambers. Ecrg4 was expressed at high levels on PMNs compared to other tissues surveyed, and is detected as a membrane‐anchored protein on the surface of resting PMNs. Exposure of cells to a dose curve of LPS led to shedding of Ecrg4 as determined by a reduction of Ecrg4+ PMNs and a concomitant increase in soluble Ecrg4 protein in conditioned media. Migration assays demonstrated that recombinant Ecrg4 increased the chemotaxis of primary fibroblasts. These results support a role for Ecrg4 as a novel chemokine expressed in a latent form on the surface of PMNs, and biologically active in a soluble form.

Abstract 170: Aldosterone Activates ex vivo Human Polymorphonuclear Leukocytes

An important role for the mineralocorticoid receptor (MR) in vascular inflammation has been proposed. Polymorphonuclear (PMN) leukocytes are critical players following an inflammatory response. We studied the role of aldosterone (ALDO) on ex vivo PMN function by isolation of cells from circulating human blood following density gradient sedimentation with polymorphprep from otherwise healthy subjects. These cells express MR as confirmed by western blot, qRT-PCR and 3H-aldosterone binding analyses. Incubation of ex vivo PMN cells with ALDO (1-10 nM) showed a rise in superoxide production (P&lt;0.01, n=9), an event that was blunted by pre-incubation with 500 nM canrenoic acid (CA), a MR antagonist (P&lt;0.03, n=6). Consistent with these results, we observed that 10 nM ALDO led to rapid increases in cytosolic Ca2+ levels using FURA-2AM fluorescence and was associated with decreases in cellular Mg2+ levels using MagFURA-2AM following 1.5 hr of ALDO. These events were associated with increases in VEGF secretion from PMN as determined by ELISA of supernatants (260.64±44.6 vs. 134.03±1.99 pg/ml, P&lt;0.01 when compared to vehicle, n=8) that was likewise sensitive to CA (P&lt;0.04, n=8); thus suggesting that ALDO-activated PMN cells increase factors involved in migration and endothelial cell activation. Indeed, we showed that 10 nM ALDO increased PMN cell migration in a time-dependent manner by CyQuant fluorescence that lasted up to 60 min and could be blocked by CA (P&lt;0.01, n=3). We then studied the effect of 10 nM ALDO-stimulated PMN cells followed by co-incubation of these cells with human endothelial cells for 4 hr. We observed that incubation of ALDO-stimulated PMNs vs vehicle treated cells led to a 2.1 fold increase in ICAM-1 expression levels using qRT-PCR but not VCAM-1 (P&lt;0.01, n=3) in endothelial cells. We observed similar findings in DMSO-differentiated HL-60 cells, a neutrophil-like human cell line. We detected the presence of MR by western blot and qRT-PCR in these cells. Incubation with 10 nM ALDO led to increases in cellular calcium, superoxide production (P&lt;0.01, n=3) and HL-60 migration (P&lt;0.03, n=4) when compared to vehicle. Thus our results suggest that activation of MR leads to PMN cell activation that may contribute to vascular inflammatory responses in vivo.

Abstract 3562: Overweight, inflammation of normal colorectal mucosa, and cancer risk.

Abstract Introduction: The relationship between chronic inflammation and risk of cancer is strongly supported by several observations on cancer in many tissues and organs. On the other hand, also an excess weight, resulting in accumulation of body fat, may be a risk factor for the development of some types of cancer. Several epidemiological studies have confirmed that an increase in Body Mass Index (BMI) is related to an increased risk of colorectal cancer. Some studies have shown that obesity can be considered a disease with a low-grade of inflammation. Myeloperoxydase (MPO) is one of the main enzymes characterizing polymorphonuclear cell activation, and may link the inflammatory response to metabolic alteration and neoplastic transformation. Aim: This study aimed at evaluating whether some anthropometric and clinical parameters in patients who underwent colonoscopy were related to the presence of colorectal tumors, and whether this relation may be explained by the level of inflammation of normal colorectal mucosa, assessed through the count of MPO-positive cells. Methods: We collected 210 samples of normal colorectal mucosa from 90 patients during colonoscopy, and fixed them in formalin and embedded in paraffin for histology. Thirty-four patients had colonic or rectal carcinoma, or one or more colorectal adenomas, 56 subjects had normal colonoscopy and were used as controls. For each patient, we measured: waist circumference, body mass index, blood pressure, and fasting glycemia. For each samples, we counted, after immunohistochemistry, the mean number of myeloperoxidase positive cells in at least twenty 100x optic fields on biopsies of normal colorectal mucosa. Results. Patients with waist circumference and BMI above or equal the threshold (for waist: 102 cm for men and 88 cm for women, for BMI 25) had higher percentages of colorectal tumors than subjects with parameters below the threshold (45% vs 33% for waist, and 42% vs 33% for BMI). Similar increases in percentages of lesions was observed according to blood pressure (41% above 140/90 and 35% below 140/90) and even more evident according to fasting glicemia (64% above or equal to 110mg/dl and 36% below). The mean number of MPO-positive cells in colorectal mucosa was strongly related to waist circumference, (2.7 ± 0.2, mean ± SEM, for waist equal or above the threesold according to sex vs 1.7 ± 0.2 for waist below the threesold, P&amp;lt;0.001), expecially in women. Similar results were obtained for BMI (2.5 ± 0.2 for BMI equal or above 25 vs 1.8 ± 0.2 for BMI below 25, P&amp;lt;0.001). No significant difference was observed according to blood pressure and fasting glicemia. Conclusions: Some anthropometric parameters related to overweight and body fat distribution are related to colorectal cancer risk and to the degree of mucosal inflammation. Thus, these easily measurable parameters may be useful for the identification of patients at high colorectal cancer risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3562. doi:1538-7445.AM2012-3562

Metallothionein deficiency exacerbates chronic inflammation associated with carcinogenesis in stomach of mice infected with &lt;i&gt;Helicobacter pylori&lt;/i&gt;

Metallothionein (MT), a low-molecular-weight protein with a high affinity for divalent heavy metal ions, is involved in many pathophysiological processes, including metal homeostasis, detoxification, cell proliferation and protection against oxidative damage. We previously found that MT in gastric mucosa plays a role in protecting against Helicobacter pylori (H. pylori)-induced gastritis at the early stage of infection. H. pylori-induced chronic gastric inflammation is shown to be associated with gastric carcinogenesis. Thus, to examine whether gastric MT contributes to protection against H. pylori-induced chronic inflammation, we compared histological changes in the gastric mucosa of MT-null and the wild-type mice at 53 weeks after inoculation three times with H. pylori SS1. As a result, we observed disruption of the gastric mucosa in MT-null mice, but not in the wild-type mice, even at the late stage of H. pylori-infection. Evaluation of pathological changes in gastric specimens by the updated Sydney grading system revealed that scores related to chronic inflammation and polymorphonuclear cell activity were higher in infected MT-null mice than those in the wild-type mice. Furthermore, a higher score for metaplasia was also observed in the MT-null stomach. These results suggested that MT might be involved in protecting against H. pylori-induced gastric chronic inflammation associated with carcinogenesis.

Long-term treatment with resveratrol attenuates oxidative stress pro-inflammatory mediators and apoptosis in streptozotocin-nicotinamide-induced diabetic rats

This study was designed to investigate the possible effectiveness of chronic resveratrol administration on redox state, inflammatory mediators and apoptosis rate in diabetic rats. Male Wistar rats were divided into four groups (n = 6): normal control, diabetic control, normal rats treated with resveratrol, and diabetic rats treated with resveratrol. Diabetes was induced by injection of streptozotocin (50 mg/kg; i.p.), 15 min after the prescription of nicotinamide (110 mg/kg; i.p.) in 12 h-fasted rats. Four-month oral resveratrol administration (5 mg/kg/day) significantly alleviated hyperglycemia, weight loss, enhancement of oxidative markers (lipid peroxidation index, nitrite/nitrate content and oxidized to reduced glutathione ratio) and superoxide dismutase activity in diabetic rats. Moreover, resveratrol administration to diabetic rats improved the elevated levels of plasma TNFα and IL-6 as well as NF-κB activity of polymorphonuclear cells. On the other hand, four months resveratrol administration decreased the apoptosis rate in the kidney, heart, retina, sciatic nerve and the polymorphonuclear cells of diabetic rats. These beneficial antidiabetic observations suggest that treatment with resveratrol may be considered as a therapeutic approach to reduce diabetic-related complications.

Serine protease autotransporters fromShigella flexneriand pathogenicEscherichia colitarget a broad range of leukocyte glycoproteins

The serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by pathogenic Gram-negative bacteria through the autotransporter pathway. We previously classified SPATE proteins into two classes: cytotoxic (class 1) and noncytotoxic (class 2). Here, we show that Pic, a class 2 SPATE protein produced by Shigella flexneri 2a, uropathogenic and enteroaggregative Escherichia coli strains, targets a broad range of human leukocyte adhesion proteins. Substrate specificity was restricted to glycoproteins rich in O-linked glycans, including CD43, CD44, CD45, CD93, CD162 (PSGL-1; P-selectin glycoprotein ligand 1), and the surface-attached chemokine fractalkine, all implicated in leukocyte trafficking, migration, and inflammation. N-terminal sequencing of proteolytic products revealed Pic (protease involved in colonization) cleavage sites to occur before Thr or Ser residues. The purified carbohydrate sLewis-X implied in inflammation and malignancy inhibited cleavage of PSGL-1 by Pic. Exposure of human leukocytes to purified Pic resulted in polymorphonuclear cell activation, but impaired chemotaxis and transmigration; Pic-treated T cells underwent programmed cell death. We also show that the Pic-related protease Tsh/Hbp, implicated in extraintestinal infections, exhibited a spectrum of substrates similar to those cleaved by Pic. In the guinea pig keratoconjunctivitis model, a Shigella pic mutant induced greater inflammation than its parent strain. We suggest that the class-2 SPATEs represent unique immune-modulating bacterial virulence factors.

Immunomodulatory effect of glucan on specific and nonspecific immunity after vaccination in puppies

The objective of the study was to determine the immunostimulatory effect of β-(1,3/1,6)-D-glucan in puppies. The effect exerted on the efficacy of vaccination, especially against canine parvovirus and rabies infection, was studied. The application of vaccine and glucan leads to significant increases in the nonspecific immunological parameters (phagocytic ability of leukocytes, blastogenic response of lymphocytes, metabolic and chemotactic activity of polymorphonuclear cells). The level of antibodies against canine parvovirus (Ab CPV) and rabies infection reached the most statistically significant values on the 28th day after the application of vaccine and a syrup containing β-(1,3/1,6)-D-glucan (Group GV) as compared to the control group (Group V, puppies receiving only vaccine). Dogs without glucan supplementation did not produce such significant levels of antibodies. We can conclude that glucan has relevant immunostimulatory effects in dogs with altered immunity. The glucan product tested in this study (PleraSAN V, PLEURAN, Bratislava, Slovakia) could be used in the small animal clinical practice.

Mitochondrial complex I impairment in leukocytes from type 2 diabetic patients

Diabetes is associated with oxidative stress. This study evaluated the rates of oxidative stress and mitochondrial impairment in type 2 diabetes patients. The study population consisted of 182 diabetic patients and 50 body-composition- and age-matched controls. We assessed anthropometric and metabolic parameters and mitochondrial function by evaluating mitochondrial oxygen (O2) consumption, reactive oxygen species (ROS) production, glutathione (GSH) levels, GSH/GSSG ratio, mitochondrial membrane potential, and mitochondrial complex I activity in polymorphonuclear cells from diabetes type 2 patients. We found an increase in waist circumference and augmented serum levels of triglycerides, proinflammatory cytokines (IL-6 and TNF-α), homocysteine, glycated hemoglobin, ultrasensitive C-reactive protein, glucose, insulin, and homeostasis model assessment of insulin resistance score in diabetic patients versus controls. There was an impairment of mitochondrial function in diabetic patients, evidenced by a decrease in mitochondrial O2 consumption, an increase in ROS production, decreased GSH/GSSG ratio, a drop in GSH levels, and an undermining of the mitochondrial membrane potential. Furthermore, an impairment of mitochondrial complex I was detected. This study supports the hypothesis of an association of type 2 diabetes and the rate of impaired mitochondrial function. We also propose that one of the targets of oxidative stress responsible for diabetes is mitochondrial complex I.

Increased Plasma Carbon Monoxide in Patients With Viral Cirrhosis and Hyperdynamic Circulation

Our aim was to measure plasma carbon monoxide (CO) in patients with liver cirrhosis and portal hypertension. In 36 cirrhotic patients (24 with ascites) and 9 healthy volunteers, we evaluated CO plasma levels and systemic hemodynamics (using ultra-trace gas chromatography and echocardiography, respectively). Heme oxygenase (HO) activity and expression were measured in isolated polymorphonuclear (PMN) cells. Plasma CO level (mean+/-s.d.) was 5.81+/-1.31 p.p.m. in healthy subjects (HS), significantly higher in non-ascitic patients (16.24+/-4.61 p.p.m., P<0.01), and even more high in ascitic patients (28.50+/-7.27 p.p.m., P<0.01 vs. the other two groups). HO activity in PMN cells was significantly greater in patients than in HS, with the highest levels being observed in patients with ascites. Western blot analysis showed enhanced expression of HO-1, but not HO-2. In the whole series of cirrhotic patients, plasma CO levels directly correlated with cardiac output, and inversely with systemic vascular resistance and mean arterial pressure. The HO/CO system is activated in patients with liver cirrhosis. This could contribute to the hyperdynamic circulatory syndrome observed in this condition.

Improvement of deficient natural killer activity and delayed bactericidal activity by a thiol proteinase inhibitor, E-64-d, in leukocytes from Chediak–Higashi syndrome patients in vitro

We previously reported that administration of a potent calpain inhibitor, E-64-d, which protects protein kinase C (PKC) from proteolysis, in a mouse model of Chediak–Higashi syndrome (CHS) (beige mice), decreases its susceptibility to Staphylococcus aureus infection. In the present study, we examined the in vitro effect of E-64-d on both deficient natural killer (NK) and delayed bactericidal activities of leukocytes from six CHS patients. Our results showed that pretreatment of peripheral blood mononuclear cells (PBMCs) obtained from CHS patients with E-64-d (1 μg/ml) significantly enhanced NK activity against K562 cells. The delayed bactericidal activity of polymorphonuclear cells (PMNs) against S. aureus also showed marked improvement. This was recovered to almost normal levels when PMNs were pretreated with E-64-d (1 μg/ml). On the other hand, the same concentration of E-64-d did not affect either the NK or bactericidal activity of normal controls. In addition, we confirmed that following E-64-d treatment, the abnormal down-regulation of PKC activity after concanavalin A (Con A) stimulation was eliminated in PBMCs obtained from CHS patients. To examine whether PKC is involved in the NK cell-mediated cytolysis and bactericidal activity of PMNs, two potent PKC inhibitors, chelerythrin and GÖ6976, were used. We found that chelerythrin inhibits NK activity of normal PBMCs in a dose-dependent manner, and GÖ6976 inhibits NK activity at doses that inhibit Ca 2+-dependent PKC isozymes. These inhibitors also suppressed the bactericidal activity of PMNs against S. aureus. Taken together, our findings suggested that E-64-d improved the compromised NK and bactericidal activity of leukocytes from CHS patients by reversing the down-regulation of PKC activity.