Cytoplasm Of Endothelial Cells Research Articles

Pseudomonas aeruginosa Exotoxin Y-Mediated Tau Hyperphosphorylation Impairs Microtubule Assembly in Pulmonary Microvascular Endothelial Cells

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly.

Ultrastructural pathology of vascular endothelial in patients with premalignant and malignant cervix lesions infected with papillomavirus

The purpose of this study was to determine the ultrastructural of vascular endothelial and relationship of the human papilloma virus (HPV) infection in patient with cervix lesions. Fifty-eight (58) tissue samples from the uterine cervix were obtained from patients with cervical intraepithelial neoplasia (CIN) and human papillomavirus infection at a public outpatient clinic in Aragua State, Venezuela. For the ultrastructural study, routine transmission electron microscopy techniques were used as well as polymerase chain reaction (PCR) for HPV. In the ultrastructural study (figure 3) NIC I sample with HPV type 6 infection. This section shows a capillary with reduplicated basement membrane and the endothelial cell cytoplasm widened (figure 4) Uterine cervix NIC II sample infected with HPV type 16. A mastocyte is shown. Note abundant collagen fibrils.( figure 5) NIC III sample with infection by HPV type 16. Capillary basement membrane is duplicated . Endothelial and pericyte cytoplasms are widened. Note in the pericyte the presence of pinocytotic vesicle.(figure 6) Invasive cervix cancer with HPV type 16. The widened endothelial cell cytoplasm shows prolongations into the lumen. Note the presence of primary lysosomes and pinocytotic vesivles.HPV11 was identified and both endothelial cytoplasm prolongations into the lumen and thickening of the endothelial wall were clearly seen. Changes of microvascular tissue in premalignant lesions and increased thickness of the endothelial capillary walls in malignant lesions of the uterine cervix indicated the activation of angiogenesis.

Tumor Endothelial Cell-Specific Drug Delivery System Using Apelin-Conjugated Liposomes

BackgroundA drug delivery system specifically targeting endothelial cells (ECs) in tumors is required to prevent normal blood vessels from being damaged by angiogenesis inhibitors. The purpose of this study was to investigate whether apelin, a ligand for APJ expressed in ECs when angiogenesis is taking place, can be used for targeting drug delivery to ECs in tumors.Methods and ResultsUptake of apelin via APJ stably expressed in NIH-3T3 cells was investigated using TAMRA (fluorescent probe)-conjugated apelin. Both long and short forms of apelin (apelin 36 and apelin 13) were taken up, the latter more effectively. To improve efficacy of apelin- liposome conjugates, we introduced cysteine, with its sulfhydryl group, to the C terminus of apelin 13, resulting in the generation of apelin 14. In turn, apelin 14 was conjugated to rhodamine-encapsulating liposomes and administered to tumor-bearing mice. In the tumor microenvironment, we confirmed that liposomes were incorporated into the cytoplasm of ECs. In contrast, apelin non-conjugated liposomes were rarely found in the cytoplasm of ECs. Moreover, non-specific uptake of apelin-conjugated liposomes was rarely detected in other normal organs.ConclusionsECs in normal organs express little APJ; however, upon hypoxic stimulation, such as in tumors, ECs start to express APJ. The present study suggests that apelin could represent a suitable tool to effectively deliver drugs specifically to ECs within tumors.

AB1301 Role of nuclear factor-κB and basic fibroblast growth factor in synovial pathology of rheumatoid synovitis

BackgroundRheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by erosive synovitis. Early changes in the synovium are marked by neovascularization, synoviocyte hyperplasia, and inflammatory cell infiltration, which produce...

Tadanafil increases microvascular permeability in mouse brain lymphoma models

Objective To study about Tadalafil increasing microvascular permeability in mouse brain lymphoma models.Methods Comparatively analyze the influence of tadalafil on cerebral microvascular permeability in the mouse brain lymphoma models,by the approaches of using Evan's Blue to test the microvascular permeability,testing concentrations of methotrexate (MTX) by HPLC,and studying ultrastructures by transmission electron microscope.Results The microvascular permeability in tumor-bearing mice treated with Tadalafil were significantly higher than that in the other groups (P ＜ 0.05).The concentration of MTX increased nearly 1.8 times in tumor tissue with Tadalafil pre-treatment.The increment were significantly higher than that in the other groups (P ＜ 0.05).Numerous pinocytotic vesicles in the cytoplasm of the endothelial cells were observed obviously in Tadalafil pre-treated mouse tumor.Conclusion Phosphodiesterase type 5 inhibitor,Tadalafil,could increased the capillary permeability of the tumor tissue in mouse brain lymphoma models. Key words: Microvascular permeability; Tadalafil; Evan's Blue; MTX; Lymphoma

Vagus nerve stimulation inhibits seizure activity and protects blood–brain barrier integrity in kindled rats with cortical dysplasia

AimsThis study investigates the effects of vagus nerve stimulation (VNS) on seizure severity and blood–brain barrier (BBB) integrity in kindled rats with cortical dysplasia (CD). Main methodsPregnant rats were exposed to 145cGy of gamma-irradiation on day 17 of pregnancy. In offsprings, kindling was induced by giving subconvulsive doses of pentylenetetrazole. Left VNS was performed for 48h at output currents of 0.5 or 1mA. Horseradish peroxidase (HRP) was used to study the BBB permeability. Immunohistochemistry for occludin and P-glycoprotein (P-gp) was also performed. Key findingsKindled rats with CD exhibited seizures with mean Racine's scores of 3.57±1.2 during video EEG recording. Kindled animals with CD receiving VNS at 0.5 and 1.0mA did not exhibit either clinical or electrophysiological signs of seizure. Immunostaining for occludin, a tight junction protein, in hippocampus remained relatively intact in all groups. VNS-treated and -untreated kindled animals with CD revealed intense immunostaining for P-gp in hippocampal formation (P<0.01). Electron microscopic observations revealed frequent transport vesicles containing electron-dense HRP reaction products in the cytoplasm of brain capillary endothelial cells in both cerebral cortex and hippocampus of kindled animals with CD. Those which were exposed to 1mA VNS were observed to have brain capillary endothelial cells largely devoid of HRP reaction products in both cerebral cortex and hippocampus. SignificanceThe results of this study suggest that VNS therapy at 1mA inhibits seizure activity and protects BBB integrity by limiting the enhancement of transcellular pathway in kindled animals with CD.

Cytoprotective Potential of Royal Jelly on Human Umbilical Vein Endothelial Cells against Nicotine Toxicity via Catalase

Aims: To examine the cytoprotective effects and mechanisms of a royal jelly extract in protecting the human umbilical vein endothelial cells (HUVECs) from nicotine toxicity. Study Design: Laboratory experimental tests. Place and Duration of Study: Department of Physiology and Department of Surgery, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand, between June 2011 and February 2012. Methodology: Cytotoxic assay of royal jelly to HUVECs was performed by using the 3(4,5-dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent. The cytoprotective effect was then investigated by examining the presence of vacuole-like structures in HUVECs exposed to nicotine 5 or 7.5 mM with and without royal jelly. Cells were stained with crystal violet and photographed under phase contrast microscope. mRNA levels of genes involved in intracellular antioxidant system, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GSR) were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Catalase Research Article European Journal of Medicinal Plants, 3(1): 88-98, 2013 89 activity was also determined by examining peroxidative function. Results: Vacuole-like structures were found in the cytoplasm of HUVECs exposed to 5 mM nicotine and higher. Royal jelly alone at the concentrations lower than 2 mg/ml did not affect the structure or the survival rate of HUVECs after 1, 4, and 7 days of treatment. For cytoprotective effect, royal jelly 1-4 mg/ml mixed with 5 mM nicotine could obviously decrease the numbers of cells containing vacuole-like structures in the cytoplasm of HUVECs with the doseand time-dependent fashion. The catalase mRNA levels and catalase activity in HUVECs exposed to 5 mM nicotine decreased significantly, but recovered when the cells were treated with royal jelly. Conclusion: Royal jelly can be safety applied to endothelial cells even at high doses. Royal jelly is able to attenuate the abnormal vacuole-like structures induced in endothelial cell cytoplasm when exposed to nicotine. Further investigation of antioxidant gene expression showed that the mechanism possibly involves a reduction of oxidative stress via an up-regulation of catalase. Besides the supplementary food, royal jelly could be useful for endothelial cell protection from nicotine toxicity found in smoking or nicotine addiction.

The Effect of Chlorpyrifos on Isolated Thoracic Aorta in Rats

This study investigated the effect of chlorpyrifos on thoracic aorta and on the level of NO in plasma and aorta. The effect of chlorpyrifos on thoracic aorta in organ bath was determined in 10 rats. Another 45 rats were assigned to 3 groups with 15 rats each: control group 1 received distilled water, control group 2 was given corn oil, and the last group was given 13.5 mg/kg chlorpyrifos dissolved in corn oil every other day for 8 weeks orally. Chlorpyrifos (10−10 M–10−5 M) showed no effect on isolated thoracic aorta. Plasma AChE activity was decreased, while LDH, ALT, GGT, and AST activities were increased in chlorpyrifos group compared to control groups. Plasma NO level was increased in chlorpyrifos group compared to control groups. iNOS expression was present in all groups in the cytoplasm of the endothelia and in the smooth muscle cells of aorta. According to semiquantitative histomorphological analysis, iNOS immunopositive reactions were seen in the decreasing order in chlorpyrifos, control 2, and control 1 groups. eNOS immunopositive reactions were observed in the endothelial cell cytoplasm, rarely in the subintimal layer, and the smooth muscle cells of aorta. There were no differences among the groups in terms of eNOS immunostaining. In conclusion, chlorpyrifos induced NO production in aorta following an increase in NOS expression.

Expression of Lymphangiogenic Vascular Endothelial Growth Factor Family Members in Bovine Corpus Luteum

The aim of this study was to evaluate mRNA expression, protein concentration and localization of the assumedly important lymphangiogenic factors VEGFC and VEGFD and the receptor FLT4 in bovine corpora lutea (CL) during different physiological stages. In experiment 1, CL were collected in a slaughterhouse and stages (days 1-2, 3-4, 5-7, 8-12, 13-16, >18) of oestrous cycle and month <3, 3-5, 6-7 and >8 of pregnancy. In experiment 2, prostaglandin F2α (PGF)-induced luteolysis was performed in 30 cows, which were injected with PGF analogue on day 8-12 (mid-luteal phase), and CL were collected before and 0.5, 2, 4, 12, 24, 48 and 64 h after PGF injection. The mRNA expression was characterized by RT-qPCR. All three factors were clearly expressed and showed significant changes during different groups and periods examined in both experiments. Protein concentrations of VEGFD and FLT4 measured by ELISA were not detectable in early cyclic CL but increased to higher plateau levels during pregnancy. After PGF-induced luteolysis FLT4 protein showed an increase within 2-24 h after the injection. FLT4 localization by immunohistochemistry in the cytoplasm of luteal cells was relatively weak in early CL. It increased in late CL and especially in CL during pregnancy. During pregnancy, a positive FLT4 staining in both the nucleus and cytoplasm of lymphatic endothelial cells in peripheral tissue was observed. In conclusion, our results lead to the assumption that lymphangiogenic factors are produced and regulated in CL and may be involved in mechanisms regulating CL function, especially during pregnancy.

Amorphous Silica Nanoparticles Promote Monocyte Adhesion to Human Endothelial Cells: Size‐Dependent Effect

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO(2)-NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co-cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO(2)-NPs at non-cytotoxic concentrations for 12 h. A significant increase (up to 2-fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO(2)-NPs induces protein expression of adhesion molecules (ICAM-1 and VCAM-1) as well as significant up-regulation in mRNA expression of ICAM-1 in both endothelial cell types. Experiments performed with fluorescent-labelled monodisperse amorphous SiO(2)-NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co-cultured cells.

Increased caveolae density and caveolin-1 expression accompany impaired NO-mediated vasorelaxation in diet-induced obesity

Diet-induced obesity induces changes in mechanisms that are essential for the regulation of normal artery function, and in particular the function of the vascular endothelium. Using a rodent model that reflects the characteristics of human dietary obesity, in the rat saphenous artery we have previously demonstrated that endothelium-dependent vasodilation shifts from an entirely nitric oxide (NO)-mediated mechanism to one involving upregulation of myoendothelial gap junctions and intermediate conductance calcium-activated potassium channel activity and expression. This study investigates the changes in NO-mediated mechanisms that accompany this shift. In saphenous arteries from controls fed a normal chow diet, acetylcholine-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylyl cyclase inhibitors, but in equivalent arteries from obese animals sensitivity to these agents was reduced. The expression of endothelial NO synthase (eNOS) and caveolin-3 in rat saphenous arteries was unaffected by obesity, whilst that of caveolin-1 monomer and large oligomeric complexes of caveolins-1 and -2 were increased in membrane-enriched samples. The density of caveolae was increased at the membrane and cytoplasm of endothelial and smooth muscle cells of saphenous arteries from obese rats. Dissociation of eNOS from caveolin-1, as a prerequisite for activation of the enzyme, may be compromised and thereby impair NO-mediated vasodilation in the saphenous artery from diet-induced obese rats. Such altered signaling mechanisms in obesity-related vascular disease represent significant potential targets for therapeutic intervention.

Vascular-targeted nanotherapy for obesity: Unexpected passive targeting mechanism to obese fat for the enhancement of active drug delivery

We previously reported that nanoparticles (NPs) modified with a prohibitin-homing peptide ligand via a short PEG2kDa-spacer could deliver its pay-load into the cytoplasm of endothelial cells in murine adipose tissue and escape from endosomes/lysosomes in vitro. We herein report, for the first time, on a dual-targeting strategy for mediating the enhanced targeting activity of NPs to adipose endothelial cells in diet-induced obesity (DIO). The targeted accumulation of prohibitin-targeted nanoparticles (PTNP), modified with a peptide ligand via a long PEG-linker, was significantly increased in white fat vessels of normal healthy mice compared to the other non-PEGylated targeted NPs, whereas the undesired accumulation of PTNP in the liver was considerably reduced. These results demonstrate that the PEGylation of targeted NPs is a critical factor in maximizing the in vivo targeted delivery of NPs and can be attributed to a significant decrease in recognition by the reticuloendothelial system. After systemic administration to DIO mice, PTNP exclusively accumulated in both adipose vessels and angiogenic clusters of obese fat cells. Surprisingly, PEGylated NPs with no active targeting moieties also accumulated in these clusters, demonstrating that the nanoscaled carriers passively accumulate in clusters via a mechanism similar to that for the enhanced permeability and retention effect, as has been well established in tumor targeting. Therefore, the enhanced delivery of PTNP appears to be mediated by both passive accumulation to angiogenic regions and active recognition by endothelial cells. Thus, the systemic administration of a proapoptotic peptide with the delivery via PTNP significantly reduced the body weight of DIO mice, as evidenced by the targeted ablation of adipose endothelial cells. These findings are potentially useful in terms of the design and development of vascular-targeted nanotherapy in the effective control of obesity.

CD36 expression in the brains of SAMP8

SAMP8, senescence accelerated mice with age-related deficits in memory and learning, are known to show age-related increases of amyloid precursor protein (APP) and immunopositivity for amyloid-β (Aβ) proteins, and moreover to be under elevated oxidative stress. The elevated expression of class B scavenger receptor CD36, which is the receptor of oxidized LDL and also one of efflux transporters of Aβ proteins in the cerebral vessels, is thought to mediate free radical production in cerebral ischemia and induce oxidative stress. Accordingly, by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, we examined whether the expression of CD36 was increased in the brains of 10–12-week-old SAMP8 with elevated oxidative stress. Ten to 12-week-old SAMR1 mice were used as controls without the features. The gene and protein expression of CD36 was significantly higher in the brains of SAMP8 than those of SAMR1. Confocal microscopic examination revealed that the CD36 immunoreactivity was seen in the cytoplasm of endothelial cells and F4/80-positive perivascular cells of the brains. These findings indicate that the expression of CD36 in the brains of SAMP8 is increased compared with that of SAMR1.

Cys–X–Cys ligand 9 might be an immunological factor in the pathogenesis of oral submucous fibrosis and its concomitant oral lichenoid lesion

Oral submucous fibrosis (OSF) is a chronic oral precancerous disease primarily caused by betel quid chewing. Some OSF patients are concomitant with oral lichenoid lesion (OLL), a white-streak lesion with a higher risk for cancerization, in OSF mucosa. Immunological reaction has been considered as one of their common pathogenic mechanisms. Cys-X-Cys ligand 9 (CXCL9) is an important factor to recruit effector neutrophils and lymphocytes in immunological reactions. However, the expression levels of CXCL9 in OSF and OLL remain unclear. We investigated the expression levels of CXCL9 in 10 normal buccal mucosa (NBM) samples and 56 OSF concomitant with OLL patients, and evaluated the possible mechanism of CXCL9 on their pathogenesis. Our results showed NBM demonstrated negative CXCL9 expression. OSF stained positive CXCL9 mainly in the cytoplasm of inflammatory cells and endothelial cells throughout the superficial layer of connective tissue, while its concomitant OLL showed much stronger CXCL9 in all mononuclear cells of subepithelial inflammatory infiltration (p = 0.0006). There was an upregulated trend of CXCL9 expression from NBM to OSF to OLL. However, no significant association between CXCL9 expression and clinicopathologic parameters of patients was found. In conclusion, CXCL9 was found for the first time to contribute to the immunological pathogenesis for both OSF and its concomitant OLL, indicating a continuously enhanced intensity of immunoreactivity in their pathogenic process. CXCL9 might be a useful tool to monitor the phase and disease severity of OSF and OLL, and a potential target for further clinical therapy for both lesions.

Immunohistochemical localization of beta defensins in the endometrium of rat uterus during the postpartum involution period

β-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production. This study was conducted to determine the possible changes in proportions and localization of β-defensin 1-4 in the rat uterus at the 1st, 3th, 5th, 10th and 15th days of postpartum and at the period of diestrus using immunohistochemical techniques. In the present study, it was determined that β-defensin 1-4 were generally found in all structural components of the endometrium (luminal and glandular epithelium, stromal cells and blood vessels) in both the nucleus and the cytoplasm of cells during the involution period and diestrus. Suprisingly, immunoreaction of β-defensin 2 was also observed in the lateral membrane of the luminal and glandular epithelial cells on the 10th day of involution and immunostaining of β-defensin 4 was also localized in the apical membrane of the luminal and glandular epithelial cells. The current study demonstrated β-defensin 1-4 immunoreactivities in the endothelium of blood vessels were stronger throughout the involution period. Although β-defensins 2 and 3 were localized in both the nuclei and the cytoplasm of endothelial cells, β-defensins 1 and 4 were present in only cytoplasm. These results show that the most component of rat endometrium expresses human β-defensin 1-4 in a involution-dependent manner. Therefore it may be asserted that these molecules constitute a organised protection to prevent uterus from probable infections during the involution process.

ALPHAB-CRYSTALLIN EXPRESSION IN EPIRETINAL MEMBRANE OF HUMAN PROLIFERATIVE DIABETIC RETINOPATHY

To examine the expression of alphaB-crystallin and its colocalization with vascular endothelial growth factor in the epiretinal membrane of human proliferative diabetic retinopathy. Ten epiretinal membranes of proliferative diabetic retinopathy and three normal retinas surgically excised were included in this study. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with alphaB-crystallin, vascular endothelial growth factor, and CD31 antibodies. AlphaB-crystallin was expressed in all epiretinal membranes examined. The immunolocalization of alphaB-crystallin was detected in the cytoplasm of CD31-positive endothelial cells, but not in normal retinal blood vessels. Furthermore, alphaB-crystallin immunoreactivity was colocalized in vascular endothelial growth factor-positive endothelial cells in proliferative diabetic retinopathy membranes. AlphaB-crystallin was expressed in proliferative diabetic retinopathy membranes, and colocalized with vascular endothelial growth factor-positive neovessels. AlphaB-crystallin may play a potential role in the pathogenesis of epiretinal membranes in proliferative diabetic retinopathy, together with vascular endothelial growth factor.

G-protein-coupled Receptor Kinase Interactor-1 (GIT1) Is a New Endothelial Nitric-oxide Synthase (eNOS) Interactor with Functional Effects on Vascular Homeostasis

Endothelial cell nitric-oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in the vasculature, undergoes extensive post-translational modifications that modulate its activity. Here we have identified a novel eNOS interactor, G-protein-coupled receptor (GPCR) kinase interactor-1 (GIT1), which plays an unexpected role in GPCR stimulated NO signaling. GIT1 interacted with eNOS in the endothelial cell cytoplasm, and this robust association was associated with stimulatory eNOS phosphorylation (Ser(1177)), enzyme activation, and NO synthesis. GIT1 knockdown had the opposite effect. Additionally, GIT1 expression was reduced in sinusoidal endothelial cells after liver injury, consistent with previously described endothelial dysfunction in this disease. Re-expression of GIT1 after liver injury rescued the endothelial phenotype. These data emphasize the role of GPCR signaling partners in eNOS function and have fundamental implications for vascular disorders involving dysregulated eNOS.

Expression of pentraxin 3 in the lungs of human, horse, cattle and pig

Bacterial lung diseases cause significant mortality and morbidity, causing enormous economic damage to the animal industry and cost billions of dollars in human health care. The innate immune system plays a critical role in the initiation of immune response in the lung. Pentraxin 3 (PTX3) is an acute phase protein that is produced at sites of inflammation by many cell types upon stimulation by proinflammatory cytokines, and agonists such as endotoxins. PTX3 recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in normal and inflamed lungs of the horse, pig, human, and cattle using light and electron microscopic immunochemistry. We show PTX3 staining in bronchial epithelial cells and vascular endothelium in normal and inflamed lungs. Alveolar macrophages and inflammatory cells recruited into the lungs of bacteria‐infected animals stain intensely for PTX3. Immuno‐gold electron microscopy on horse, and calf lungs showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages. These data show PTX3 is expressed in normal lungs and its expression is increased in inflamed lungs.Grant Funding Source: Alberta Livestock and Meat Agency

Intravenous immunoglobulins prevent the breakdown of the blood-brain barrier in experimentally induced sepsis

The effects of immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M on blood-brain barrier integrity and survival rates in septic rats were comparatively investigated. Sepsis was induced by cecal ligation and perforation in Sprague-Dawley rats. The animals were divided into the following groups: Sham, cecal ligation and perforation, cecal ligation and perforation plus immunoglobulin G (250 mg/kg, intravenous), and cecal ligation and perforation plus immunoglobulins enriched with immunoglobulin A and immunoglobulin M (250 mg/kg, intravenous). Immunoglobulins were administered 5 mins before cecal ligation and perforation and the animals were observed for behavioral changes for 24 hrs following cecal ligation and perforation. Blood-brain barrier permeability was functionally and structurally evaluated by determining the extravasation of Evans Blue and horseradish peroxidase tracers, respectively. Immunohistochemistry and Western blotting for occludin were performed. The high mortality rate (34%) noted in the septic rats was decreased to 15% and 3% by immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M, respectively (p < .01). Both immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M alleviated the symptoms of sickness behavior in the septic rats, with the animals becoming healthy and active. Increased extravasation of Evans Blue into the brain tissue of the septic rats was markedly decreased with the administration of both immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M (p < .01). Occludin expression remained essentially unchanged in all groups, including the cecal ligation and perforation group. In the cecal ligation and perforation group, increased luminal and abluminal vesicles containing electron-dense horseradish peroxidase-reaction product were noted in the cytoplasm of endothelial cells located in the hippocampus and the cerebral cortex. Tight junction was ultrastructurally intact, suggesting that the transcellular pathway is responsible for the blood-brain barrier breakdown in sepsis. Following immunoglobulin G or immunoglobulins enriched with immunoglobulin A and immunoglobulin M treatment, no ultrastructural evidence of leaky capillaries in the brain was observed in the septic rats, indicating the blockade of the transcellular pathway by immunoglobulins administration. Our study suggests that immunoglobulin G and immunoglobulins enriched with immunoglobulin A and immunoglobulin M improve the integrity of the blood-brain barrier and inhibits cecal ligation and perforation-induced symptoms of sickness behavior in rats.

Histopathological diagnosis of Japanese spotted fever using formalin-fixed, paraffin-embedded skin biopsy specimens Usefulness of immunohistochemistry and real-time PCR analysis

Japanese spotted fever (JSF) is caused by Rickettsia japonica, and lethal cases are reported yearly in southwest Japan. We thus established the method of diagnosing JSF by immunohistochemistry (IHC) and real-time PCR (RT-PCR) using formalin-fixed, paraffin-embedded skin biopsy specimens. Two monoclonal antibodies were used for IHC, and the 17k genus common antigen gene served as the target of RT-PCR. We collected skin biopsy (n = 61) and autopsy (n = 1) specimens from 50 patients clinically suspected of JSF. Immunohistochemically, the rickettsial antigens were localized as coarse dots in the cytoplasm of endothelial cells and macrophages. Thirty-one seropositive cases plus one autopsy case (group A) and nine seronegative cases but with positive IHC and/or RT-PCR (group B) were judged as JSF. Nine cases were regarded as non-JSF disorders based on negative serology, IHC and RT-PCR (group C). Of 50 biopsies (eschar 34, eruptions 10, and scabs 6) from groups A and B, IHC and RT-PCR positivities were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, and scabs were insufficient. Unexpectedly, 18 biopsies happened to be fixed in 100% formalin, and this lowered the detection rate by RT-PCR, but IHC was tolerant. Sequence analysis using five skin biopsy specimens confirmed a 114 bp DNA stretch homologous to that reported for the target gene of R. japonica. In 26 (84%) of the 31 seropositive patients, the diagnosis was made by IHC and/or RT-PCR earlier than serology.