Effects Of Potential Therapeutic Agents Research Articles

An in vitro Model to Study Placental Functions in Gestational Diabetes Mellitus

Background: Gestational diabetes mellitus (GDM) is a serious pregnancy complication that affects around 14 % of pregnancies worldwide. It not only causes short-term complications in the mother and child but also significantly increases the risks of several chronic diseases, including cardiovascular diseases and diabetes. An excess production of the soluble vascular endothelial growth factor (VEGF) receptor 1 in the placenta, which is a natural inhibitor of VEGF, has been linked to various pregnancy complications. However, placentas affected by GDM often exhibit increased vascularization, suggesting that the role of VEGF in GDM differs from its impact on typical pregnancy complications. Unfortunately, limited studies on placental functions in GDM have been conducted due to a lack of reliable culture models. Methods: In this study, we developed an in vitro model of GDM using the human choriocarcinoma trophoblast cell line BeWo and examined the effects of VEGF on glucose uptake. The BeWo cells were treated with a high glucose-containing (25 mM) syncytialization differentiation medium to produce insulin-resistant cells. Insulin resistance was assessed by a glucose uptake assay using the fluorescent glucose analog 2-NBDG and flow cytometry analysis. Results: The cells treated with high glucose exhibited a significant decrease in glucose uptake, suggesting the successful development of glucose resistance in these cells. However, VEGF treatment did not show a significant impact on glucose uptake in high glucose-treated cells. Conclusions: It is important to note that glucose homeostasis is a complex process involving multiple cell types, and further studies are necessary to fully understand the functions of VEGF on GDM placentas. Nevertheless, the in vitro model we have developed and validated will be a valuable tool for studying placental pathophysiology in GDM and evaluating the effectiveness of potential therapeutic agents. HIGHLIGHTS BeWo cells are the choriocarcinoma cell line which were cultured in the in vitro condition BeWo cells were differentiated using forskolin and 8-Br Camp Insulin resistant model of BeWo cells were prepared by high glucose (25 mM) treatment The effect of VEGF on glucose homeostasis in GDM model of BeWo cells was observed GRAPHICAL ABSTRACT

A Transgenic 5xFAD-M Line of Mice for Dendritic Spine Morphology Analysis in Alzheimer’s Disease

Cognitive impairments are closely related to synaptic loss in Alzheimer's disease (AD). Functional changes in synaptic contacts are reflected in dendritic spine morphology. Visualization of neurons for morphological studies in vivo is complicated by the fixed brain slice staining or expensive adeno-associated virus injections. We created a transgenic 5xFAD-M line of mice with AD-associated mutations and expressed GFP protein in single neurons of the brain. This mouse model of AD is a useful tool for the simplified visualization of the hippocampal neurons' morphology in vivo without additional staining manipulations. The progressive elimination of mushroom spines was demonstrated in 5xFAD-M mice between 4 and 5 months of age. Five-month-old 5xFAD-M male and female mice showed change both in the total density and the mushroom spines number compared to sex-matched control. We conclude 5xFAD-M mice can be a useful AD model for studying the mechanisms of synaptic pathology under neurodegenerative conditions and evaluating the effects of potential therapeutic agents on spine morphology as crucial aspect of memory loss in AD.

Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices.

Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics.

Hyperspectral evaluation of peritoneal fibrosis in mouse models.

Analysis of morphological changes of the peritoneal membrane is an essential part of animal studies when investigating molecular mechanisms involved in the development of peritoneal fibrosis or testing the effects of potential therapeutic agents. Current methods, such as histology and immunohistochemistry, require time consuming sample processing and analysis and result in limited spatial information. In this paper we present a new method to evaluate structural and chemical changes in an animal model of peritoneal fibrosis that is based on hyperspectral imaging and a model of light transport. The method is able to distinguish between healthy and diseased subjects based on morphological as well as physiological parameters such as blood and scattering parameters. Furthermore, it enables evaluation of changes, such as degree of inflammation and fibrosis, that are closely related to histological findings.

An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus

BackgroundMost bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; including phloem-limited plant pathogens like Candidatus Liberibacter asiaticus (CLas). CLas is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks an effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive.ResultsWe developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized tissue from CLas-infected ACP and a propidium monoazide (PMA) assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between intact and disrupted CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent PCR interference and identified a suitable positive control for nearly complete CLas disruption (0.1% Triton-X 100). Isolation buffer components were optimized with 72 mM salt mixture, 1 mM phosphate buffer and 1% glycerol serving to minimize unwanted interactions with treatment and PMA chemistries and to maximize recovery of intact CLas cells. The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity and identify which were most effective.ConclusionThis psyllid homogenate assay allows for a quick assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms. Use of an intact vs. disrupted assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies.

In vitro cyst formation of ADPKD cells.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the relentless growth of numerous fluid-filled cysts in the kidneys. Mutations in PKD1 and PKD2, genes that encode polycystin 1 and 2, respectively, are responsible for most cases of ADPKD. Currently, the cellular mechanisms responsible for cyst formation remain poorly understood. In vitro models have been used by researchers to investigate cellular processes for cyst formation in carefully controlled experimental conditions. Madin-Darby canine kidney (MDCK) cells, a distal tubule epithelial cell line, were first used to form 3-dimensional (3-D) cysts within a hydrated collagen gel. This method was applied to epithelial cells cultured from cysts of human ADPKD kidneys, allowing investigators to study cellular mechanisms for cyst growth using cells that harbor the genetic mutations responsible for ADPKD in humans. Studies using ADPKD in vitro cysts have provided insight into cellular processes regulating cell proliferation, fluid secretion, and cell polarity. These assays were used to demonstrate the central role of cAMP agonists, such as arginine vasopressin, on cyst growth; and to test the effectiveness of potential therapeutic agents, including tolvaptan. Results obtained from in vitro cyst experiments demonstrate the translational value of cell model systems for investigating the mechanisms for cyst formation in human ADPKD. In this chapter, we describe protocols for growing ADPKD cells in a 3-D in vitro cyst assay and measuring total cyst volume by microscopy and image analysis.

Regional gene expression analysis of multiple tissues in an experimental animal model of post-traumatic osteoarthritis

To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

12. Riluzole exerts short-term effects on cortical hyperexcitability in sporadic ALS

Riluzole is the only neuroprotective agent to date shown to be effective in amyotrophic lateral sclerosis (ALS). The mechanism by which riluzole exerts neuroprotective effects in is via antagonism of glutamate, and recent studies in ALS patients have established partial normalization of cortical hyperexcitability. Given the modest therapeutic benefits, the duration of riluzoles effect in ALS is probably short, although this remains to be fully elucidated. Consequently, the present study assessed longitudinal effects of riluzole on cortical excitability in a cohort of sporadic ALS patients, by utilising threshold tracking transcranial magnetic stimulation (TTTMS). Studies were longitudinally undertaken on 18 sporadic ALS patients, with cortical excitability assessed at baseline (prior to initiation of riluzole), at 4, 8, and 12 weeks post riluzole initiation. Motor evoked potentials were recorded over the right abductor pollicis brevis muscle. Statistical analysis was undertaken with a Wilcoxson paired analysis, using a Bonferroni correction. At baseline, cortical hyperexcitability was evident in the sporadic ALS cohort, as indicated by a marked reduction in SICI (3.5%). Riluzole therapy resulted in a marked increase in SICI at 4 weeks (8.4SEM%, P < 0.01), which was maintained at 8 weeks (9.4% SEM). Interestingly, at 12 weeks, SICI again reduced to baseline values (SICI12 week 4.6%) despite ongoing riluzole therapy. There were no significant changes in other TTMS parameters such as resting motor threshold, motor evoked potential amplitude or cortical silent period duration. The findings in the present study indicated that riluzole normalizes cortical excitability in ALS, although this cortical effect is limited. Given that cortical hyperexcitability is an important pathophysiological mechanism in ALS, the modest therapeutic benefits of riluzole could in part be explained by the short-term effects of riluzole on cortical hyperexcitability. Importantly, TTTMS may be utilised in future clinical trials to assess the effects of potential therapeutic agents on cortical function at early stages of drug development.

Detecting Behavioral Deficits Post Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI), ranging from mild to severe, almost always elicits an array of behavioral deficits in injured subjects. Some of these TBI-induced behavioral deficits include cognitive and vestibulomotor deficits as well as anxiety and other consequences. Rodent models of TBI have been (and still are) fundamental in establishing many of the pathophysiological mechanisms of TBI. Animal models are also utilized in screening and testing pharmacological effects of potential therapeutic agents for brain injury treatment. This chapter details validated protocols for each of these behavioral deficits post traumatic brain injury in Sprague-Dawley male rats. The elevated plus maze (EPM) protocol is described for assessing anxiety-like behavior; the Morris water maze protocol for assessing cognitive deficits in learning memory and spatial working memory and the rotarod test for assessing vestibulomotor deficits.

Recapitulation of metabolic defects in a model of propionic acidemia using patient-derived primary hepatocytes.

Propionic acidemia (PA) is a disorder of intermediary metabolism with defects in the alpha or beta subunits of propionyl CoA carboxylase (PCCA and PCCB respectively) enzyme. We previously described a liver culture system that uses liver-derived hemodynamic blood flow and transport parameters to restore and maintain primary human hepatocyte biology and metabolism utilizing physiologically relevant milieu concentrations. In this study, primary hepatocytes isolated from the explanted liver of an 8-year-old PA patient were cultured in the liver system for 10 days and evaluated for retention of differentiated polarized morphology. The expression of PCCA and PCCB was assessed at a gene and protein level relative to healthy donor controls. Ammonia and urea levels were measured in the presence and absence of amino acid supplements to assess the metabolic consequences of branched-chain amino acid metabolism in this disease. Primary hepatocytes from the PA patient maintained a differentiated polarized morphology (peripheral actin staining) over 10 days of culture in the system. We noted lower levels of PCCA and PCCB relative to normal healthy controls at the mRNA and protein level. Supplementation of branched-chain amino acids, isoleucine (5mM) and valine (5mM) in the medium, resulted in increased ammonia and decreased urea in the PA patient hepatocyte system, but no such response was seen in healthy hepatocytes or patient-derived fibroblasts. We demonstrate for the first time the successful culture of PA patient-derived primary hepatocytes in a differentiated state, that stably retain the PCCA and PCCB enzyme defects at a gene and protein level. Phenotypic response of the system to an increased load of branched-chain amino acids, not possible with fibroblasts, underscores the utility of this system in the better understanding of the molecular pathophysiology of PA and examining the effectiveness of potential therapeutic agents in the most relevant tissue.

Assessment of morphine-induced hyperalgesia and analgesic tolerance in mice using thermal and mechanical nociceptive modalities.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

Accumulation of C-terminal fragments of transactive response DNA-binding protein 43 leads to synaptic loss and cognitive deficits in human TDP-43 transgenic mice

Accumulation of the transactive response DNA-binding protein 43 (TDP-43) is a major hallmark of several neurodegenerative disorders, collectively known as TDP-43 proteinopathies. The most common TDP-43 proteinopathies, frontotemporal lobar degeneration with TDP–43-positive inclusions, and amyotrophic lateral sclerosis, share overlapping neuropathological and clinical phenotypes. The development and detailed analysis of animal models of TDP-43 proteinopathies are critical for understanding the pathogenesis of these disorders. Transgenic mice overexpressing mutant human TDP-43 (herein referred to as hTDP-43) are characterized by neurodegeneration and reduced life span. However, little is known about the behavioral phenotype of these mice. Here we report the novel finding that hTDP-43 mice develop deficits in cognition, motor performance, and coordination. We show that these behavioral deficits are associated with the accumulation of nuclear and cytosolic TDP-43 C-terminal fragments, a decrease in endogenous TDP-43 levels, and synaptic loss. Our findings provide critical insights into disease pathology, and will help guide future preclinical studies aimed at testing the effects of potential therapeutic agents on the onset and progression of TDP-43 proteinopathies.

Effect of potential therapeutic agents in reducing oxidative stress in pulmonary tissues of recurrent airway obstruction‐affected and clinically healthy horses

To determine and compare the reactive oxygen and nitrogen species (ROS and RNS) in pulmonary tissues of horses affected with recurrent airway obstruction (RAO) and clinically healthy horses, and to evaluate the effectiveness of potential therapeutic agents in reducing ROS and RNS in the tissues of these horses. We hypothesised that RAO-affected horses would have high levels of reactive species and that the test agents would reduce them. The objectives were as follows: 1) to determine the level of ROS and RNS in pulmonary tissues (bronchial and arterial rings) of RAO-affected and clinically healthy horses; and 2) to determine the ability of pentoxifylline, pyrrolidine-dithiocarbamate and a combined use of endothelin A and B receptor antagonists (BQ123 and BQ788, respectively) in reducing reactive species. Arterial and bronchial rings were collected from the diaphragmatic lung lobe of each horse immediately after euthanasia. The levels of ROS and RNS were measured in control tissues and those incubated with test agents, using an electron paramagnetic resonance instrument. The levels of ROS and RNS were significantly greater in arterial and bronchial tissues of RAO-affected than of clinically healthy horses. Pentoxifylline and endothelin antagonists reduced both ROS and RNS in tissues from RAO-affected horses. Basal levels of reactive species in clinically healthy horses were not affected by these agents. No difference in the level of reactive species was observed between arterial and bronchial tissues. Horses affected by RAO had higher ROS and RNS than clinically healthy horses. Pentoxifylline and endothelin antagonists effectively reduced ROS and RNS in pulmonary tissues of RAO-affected horses. The study suggested a potential use for pentoxifylline and endothelin antagonists in treating RAO-affected horses. As endothelin is involved in physiological functions, therapeutic use of its antagonists is cautioned.

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population(1,2). Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)(3) (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity. CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins. For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs(4). The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter(5,6). The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

PH Landscapes in a Novel Five-Species Model of Early Dental Biofilm

BackgroundDespite continued preventive efforts, dental caries remains the most common disease of man. Organic acids produced by microorganisms in dental plaque play a crucial role for the development of carious lesions. During early stages of the pathogenetic process, repeated pH drops induce changes in microbial composition and favour the establishment of an increasingly acidogenic and aciduric microflora. The complex structure of dental biofilms, allowing for a multitude of different ecological environments in close proximity, remains largely unexplored. In this study, we designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We then performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate.Methodology/Principal FindingsStrains of Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Streptococcus downei and Actinomyces naeslundii were employed in the model. Biofilms were grown in flow channels that allowed for direct microscopic analysis of the biofilms in situ. The architecture and composition of the biofilms were analysed using fluorescence in situ hybridization and confocal laser scanning microscopy. Both biofilm structure and composition were highly reproducible and showed similarity to in-vivo-grown dental plaque. We employed the pH-sensitive ratiometric probe C-SNARF-4 to perform real-time microscopic analyses of the biofilm pH in response to salivary solutions containing glucose. Anaerobic glycolysis in the model biofilms created a mildly acidic environment. Decrease in pH in different areas of the biofilms varied, and distinct extracellular pH-microenvironments were conserved over several hours.Conclusions/SignificanceThe designed biofilm model represents a promising tool to determine the effect of potential therapeutic agents on biofilm growth, composition and extracellular pH. Ratiometric pH analysis using C-SNARF-4 gives detailed insight into the pH landscape of living biofilms and contributes to our general understanding of metabolic processes in in-vivo-grown bacterial biofilms.

A 3 months mild functional test regime does not affect disease parameters in young mdx mice

To assess the effect of potential therapeutic agents in dystrophic mice it is useful to have a functional test regime that does not affect the natural disease progression of mdx mice with dystrophinopathy. We determined the effect of a 12 week test regime consisting of fore limb grip strength, rotarod analysis and two and four limb hanging wire tests on the disease progression of 4-week-old mdx mice. Mice performed the different functional tests on consecutive days on a weekly basis. No difference was found in serum creatine kinase levels between functionally active and sedentary mice. The percentage of fibrotic/necrotic areas assessed in a semi-automated way with colour deconvolution of skeletal muscles, heart and diaphragm did not vary within muscles or between groups, nor did the gene expression levels of disease-related genes. We conclude that this test regime may be suitable for short-term functional evaluation of therapeutic approaches in the mdx mouse.

Effect of Intranasal vs. Intraperitoneal Sensitization of Mice with House Dust Mite Antigen on Airway Hyperresponsiveness (AHR) and SOCS expression in the Lung

Increased density of Th2 cells with the secretion of IL-4, IL-5, IL-9, and IL-13 in the airways are hallmarks of allergic asthma, and induce AHR, airway inflammation, mucus hypersecretion, and goblet cell hyperplasia. These cytokines are modulated by a family of proteins called suppressor of cytokine signaling (SOCS). In this study, we investigated whether or not the route of sensitization with house dust mite induces different effects on the development of AHR and on SOCS expression in the lungs. Mice were sensitized either by intranasal injection (IN) or by intraperiotneal injection (IP) with house dust mite antigen (10 μg). Alum was used as an adjuvant only in IP sensitization. AHR to methacholine was determined by BUXCO. Staining of the lungs with H&E and tri-chrome was used to compare morphology and collagen deposition, respectively. Periodic Acid Schiff (PAS) staining was done to visualize goblet cell hyperplasia and mucus deposition and immunohistochemistry (IHC) was performed to visualize the expression of SOCS 1, 3, and 5. IN route of antigen sensitization induced a significantly increased AHR, airway inflammation, goblet cell hyperplasia, collagen deposition, and expression of SOCS1, 3, and 5 in the mouse lung compared to the IP route of antigen administration. Intranasal administration of the house dust mite antigen causes significantly increased induction of allergic airway response than by intraperitoneal route. This could serve as a better model to investigate the effects of potential therapeutic agents in allergic asthma.

Glucocorticoids Increase Repair Potential in a Novel in vitro Human Airway Epithelial Wounding Model

Airway epithelial damage is a cardinal feature of chronic asthma. Agents which enhance epithelial repair without triggering uncontrolled fibrosis of the mesenchyme would be predicted to be useful in the management of asthma. We have developed a repeat wound model using mucociliated human bronchial epithelial cell (HBEC) cultures to define the key pathways involved in airway epithelial repair, and to study the effects of potential therapeutic agents on epithelial repair in a chronic setting. We show that repair occurs primarily by cell migration to close a defect; this process requires activation of the EGF receptor (EGFR) and subsequent tyrosine kinase signalling. Migration is accompanied by up-regulation of CD44 in motile cells at the wound margins with proliferation of non-migrating cells adjacent to the wound area. In long-term studies beta2 adrenoceptor agonists and phosphodiesterase (PDE) inhibitors have no effect on repair potential, in contrast chronic treatment with the glucocorticoid dexamethasone extends the lifespan of repeatedly wounded differentiated cultures. We suggest part of the beneficial effects of glucocorticoids in asthma is related to this ability to prolong repair potential following repeated episodes of epithelial injury.

Circulatory Arrest as a Model for Studies of Global Ischemic Injury and Neuroprotection

Despite many programs aimed at better immediate care of cardiac arrest victims, the subsequent mortality rate is high, with myocardial and central nervous system (CNS) injuries as the most common causes of death. Preclinical research is badly needed to produce a sound base for future clinical trials and possible improvements in clinical outcome. In our laboratory, we use piglets weighing approximately 25 kg. Ventricular fibrillation is produced by an AC current and left without treatment for 8-12 min, after which cardiopulmonary resuscitation according to current human guidelines is undertaken. The heart is then defibrillated and restoration of spontaneous circulation induced. During the procedure, blood pressure and flow measurements are obtained in the systemic, pulmonary, and cerebral circulation. Peroxidation and inflammation are monitored by systemic and cerebral venous plasma concentrations of isoprostane (8-iso-PGF(2alpha)), an indicator of oxidative damage, and prostaglandin F(2alpha) metabolite (15-keto-dihydro-PGF(2alpha)), an indicator of cyclooxygenase-2 activity, respectively. Neurocellular damage is monitored by the jugular plasma concentration of protein S-100beta. Neurological outcome is assessed at >24 h after the incident. Our results show that plasma concentrations of 8-iso-PGF(2alpha) are greater after more extended periods of ischemia. PBN (alpha-phenyl-N-tert-butyl nitrone), a so-called spin-trap scavenger, has a neuroprotective effect since neurological outcome is enhanced, and the 8-iso-PGF(2alpha) concentration is decreased during reperfusion. Use of water-soluble sulfonated PBN (S-PBN) results in better autoregulation of cerebral cortical blood flow and less peroxidation of CNS lipids during reperfusion. These observations suggest that our model can be used to explore neuroprotective effects of potential therapeutic agents.