Local Tissue Response Research Articles

Intranasal Live Influenza Vaccine Priming Elicits Localized B Cell Responses in Mediastinal Lymph Nodes.

Pandemic live attenuated influenza vaccines (pLAIV) prime subjects for a robust neutralizing antibody response upon subsequent administration of a pandemic inactivated subunit vaccine (pISV). However, a difference was not detected in H5-specific memory B cells in the peripheral blood between pLAIV-primed and unprimed subjects prior to pISV boost. To investigate the mechanism underlying pLAIV priming, we vaccinated groups of 12 African green monkeys (AGMs) with H5N1 pISV or pLAIV alone or H5N1 pLAIV followed by pISV and examined immunity systemically and in local draining lymph nodes (LN). The AGM model recapitulated the serologic observations from clinical studies. Interestingly, H5N1 pLAIV induced robust germinal center B cell responses in the mediastinal LN (MLN). Subsequent boosting with H5N1 pISV drove increases in H5-specific B cells in the axillary LN, spleen, and circulation in H5N1 pLAIV-primed animals. Thus, H5N1 pLAIV primes localized B cell responses in the MLN that are recalled systemically following pISV boost. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination.IMPORTANCE We have previously shown that pandemic live attenuated influenza vaccines (pLAIV) prime for a rapid and robust antibody response on subsequent administration of inactivated subunit vaccine (pISV). This is observed even in individuals who had undetectable antibody (Ab) responses following the initial vaccination. To define the mechanistic basis of pLAIV priming, we turned to a nonhuman primate model and performed a detailed analysis of B cell responses in systemic and local lymphoid tissues following prime-boost vaccination with pLAIV and pISV. We show that the nonhuman primate model recapitulates the serologic observations from clinical studies. Further, we found that pLAIVs induced robust germinal center B cell responses in the mediastinal lymph node. Subsequent boosting with pISV in pLAIV-primed animals resulted in detection of B cells in the axillary lymph nodes, spleen, and peripheral blood. We demonstrate that intranasally administered pLAIV elicits a highly localized germinal center B cell response in the mediastinal lymph node that is rapidly recalled following pISV boost into germinal center reactions at numerous distant immune sites.

526 Non-Cytotoxic Ultrathin Antimicrobial Hydrogel Dressings Containing Ionic and Metallic Silver

Preventing wound infections is a major unresolved healthcare challenge. More than 2.1 million U.S. patients seek treatment for burns annually. These wounds, when infected, do not progress through the normal healing process. To address this problem, we have developed an ultrathin microfilm dressing that delivers a non-cytotoxic, yet effective dose of antimicrobial silver ions to the wound bed. Microfilms were fabricated as polyvinyl alcohol hydrogel sheets coated with a polymeric nanofilm containing 0.1 mg/in2 of ionic and metallic silver. In contrast, commercial antimicrobial dressings Aquacel® Ag, Acticoat® and Silverlon® contain 1.2, 16.1 and 54.6 mg/in2 of silver, respectively. Cytotoxicity of microfilms was compared to that of Aquacel Ag and Silverlon using MTT cytotoxicity assay. Release of silver from these dressings over 3 days in a simulated wound fluid was also characterized. An ISO 22196-compliant study was used to measure in-vitro antimicrobial activity. A porcine wound healing study evaluated local tissue response and wound healing characteristics following repeated application of microfilm to partial-thickness wounds over 14 days. Furthermore, porcine wound colonization studies were conducted to evaluate antimicrobial activity of microfilm in wounds contaminated with 103 CFU P. aeruginosa over 5 days post-surgery. Microfilms were non-cytotoxic whereas Aquacel Ag and Silverlon were cytotoxic. This was consistent with up to 10x lower release of silver ions from microfilms. In vitro, microfilms reduced 5 Log10 units of bacterial loads, including MRSA and VRE, on their surface for >3 days. The porcine wound healing study concluded that materials of microfilms had no significant effect on epithelial growth, granulation tissue formation, critical cellular responses, or acute inflammation following multiple applications until complete wound closure. Contaminated porcine wounds treated with Telfa pads (control) had robust P. aeruginosa colonization of >105 CFU/wound, while those treated with microfilm or Acticoat had significantly less (>2 Log10 lower) bacterial burden. The difference between the CFU in microfilm and Acticoat groups was insignificant. Our antimicrobial microfilm dressing is a pioneering advance because it is effective in clearing microbial burden in wounds without causing cytotoxicity, which is in stark contrast to conventional antimicrobial dressings. Microfilms are applied to wound with each bandage change followed by longer intervals as the wounds heal. Microfilm conform intimately to the wound bed where it reduces bacterial load thus allowing normal wound healing. Microfilms slough off as wounds heal or rinsed off with saline at bandage changes.

Long-Term Implantability of Resorbable Carboxymethyl Cellulose/Chitosan Microspheres in a Rabbit Renal Arterial Embolization Model.

To determine the physiologic response to resorbable carboxymethyl cellulose/chitosan (CMC/CN) microspheres in a long-term rabbit model, including the clinical response, gross pathology, and histopathology. Six rabbits were embolized with CMC/CN microspheres (300-500µm) in one kidney via an inferior renal artery branch. Angiography was performed immediately before and after embolization and prior to killing at 6months (180 ± 7days, n = 3) and 12months (365 ± 10days, n = 3). A complete necropsy was performed on each animal with dissection of the kidneys and harvesting of additional tissues as per ISO-10993-part 6 and ISO-10993-part 11 guidelines. All tissues were processed and stained for pathological analysis. The caudal third of target kidneys was successfully embolized with CMC/CN microspheres. Over the course of the study, there were neither notable clinical signs in either embolization group nor significant changes in the tissue/body weight ratio between 6- and 12-month time points. Gross examination revealed that all embolized kidneys had morphologic features consistent with infarction resulted from microsphere delivery. The percentage of infarction decreased from 9.1% ± 5.7% at 6months to 1.9% ± 0.4% at 12months. Microscopically, infarcted areas demonstrated evidence of chronic injury and repair, including loss of renal parenchyma with replacement fibrosis, tubular regeneration, and minimal to mild lymphoplasmacytic inflammation without any active changes such as necrosis or neutrophilic inflammation. No systemic toxicity was observed in the animals 6 and 12months after CMC/CN microspheres delivery. The local tissue response was mild.

Human Langerhans Cells with Pro-inflammatory Features Relocate within Psoriasis Lesions.

Psoriasis is a common skin disease that presents with well-demarcated patches of inflammation. Recurrent disease in fixed areas of the skin indicates a localized disease memory that is preserved in resolved lesions. In line with such concept, the involvement of tissue-resident immune cells in psoriasis pathology is increasingly appreciated. Langerhans cells (LCs) are perfectly placed to steer resident T cells and local tissue responses in psoriasis. Here, we present an overview of the current knowledge of LCs in human psoriasis, including findings that highlight pro-inflammatory features of LCs in psoriasis lesions. We also review the literature on conflicting data regarding LC localization and functionality in psoriasis. Our review highlights that further studies are needed to elucidate the molecular mechanisms that drive LCs functionality in inflammatory diseases.

Micromechanics of brain white matter tissue: A fiber-reinforced hyperelastic model using embedded element technique

A transverse-plane hyperelastic micromechanical model of brain white matter tissue was developed using the embedded element technique (EET). The model consisted of a histology-informed probabilistic distribution of axonal fibers embedded within an extracellular matrix, both described using the generalized Ogden hyperelastic material model. A correcting method, based on the strain energy density function, was formulated to resolve the stiffness redundancy problem of the EET in large deformation regime. The model was then used to predict the homogenized tissue behavior and the associated localized responses of the axonal fibers under quasi-static, transverse, large deformations. Results indicated that with a sufficiently large representative volume element (RVE) and fine mesh, the statistically randomized microstructure implemented in the RVE exhibits directional independency in transverse plane, and the model predictions for the overall and local tissue responses, characterized by the normalized strain energy density and Cauchy and von Mises stresses, are independent from the modeling parameters. Comparison of the responses of the probabilistic model with that of a simple uniform RVE revealed that only the first one is capable of representing the localized behavior of the tissue constituents. The validity test of the model predictions for the corona radiata against experimental data from the literature indicated a very close agreement. In comparison with the conventional direct meshing method, the model provided almost the same results after correcting the stiffness redundancy, however, with much less computational cost and facilitated geometrical modeling, meshing, and boundary conditions imposing. It was concluded that the EET can be used effectively for detailed probabilistic micromechanical modeling of the white matter in order to provide more accurate predictions for the axonal responses, which are of great importance when simulating the brain trauma or tumor growth.

Structure and Function Relationships in Diseases of the Small Airways.

It is well known that particulate matter suspended in the earth's atmosphere generated by tobacco smoke, automobile exhaust, industrial processes, and forest fires has been identified as a major risk factor for chronic lung disease. Particulate matter can be divided into large, intermediate, and fine particulates. When inhaled, large particulates develop sufficient momentum to leave the flowing stream of inhaled air and deposit by impaction in the nose, mouth, nasopharynx, larynx, trachea, and central bronchi. Intermediate-sized particulates that develop less momentum deposit in the smaller bronchi and larger bronchioles, and the finest particulates that develop the least momentum make it to the distal gas-exchanging tissue, where gas moves solely by diffusion. On the basis of Einstein's classic work on Brownian motion that showed particles suspended in a gas diffuse much more slowly than the gas in which they are suspended, we postulate that the small airways that accommodate the shift from bulk airflow to diffusion become the major site for deposition of fine particles, resulting in a host immune response. Much remains to be learned about the interaction between the deposition of fine particulates and the host immune and tissue responses; the purpose of this review is to examine the hypothesis that the smallest conducting airways and proximal gas-exchanging tissue are the primary sites for the deposition of the finest particulates inhaled into the lungs.

Five Hundred Fifty-five Retrieved Metal-on-metal Hip Replacements of a Single Design Show a Wide Range of Wear, Surface Features, and Histopathologic Reactions.

In 2010, a widely used metal-on-metal hip implant design was voluntarily recalled by the manufacturer because of higher than anticipated failure rates at 5 years. Although there was a large published range of revision rates, numerous studies had reported a higher risk of revision for excessive wear and associated adverse tissue reactions when compared with other metal-on-metal total hips. The reasons for this were suggested by some to be related to cup design features. From retrievals of ASR metal-on-metal implants and tissue samples obtained at revision surgery, we asked the following questions: (1) What were the common and uncommon surface features? (2) What were the common and uncommon linear and volumetric wear characteristics? (3) Were there common taper corrosion characteristics? (4) What aseptic lymphocytic vasculitis-associated lesion (ALVAL) features were present in the tissues? Five hundred fifty-five ASRs, including 23 resurfacings, were studied at one academic research center. Features of wear (eg, light and moderate scratching), damage (eg, deposits, gouges), and bone attachment on the porous coating were semiquantitatively ranked from 0 (none) to 3 (> 75%) based on the amount of a feature in each region of interest by the same experienced observer throughout the study. Visible features of head taper corrosion were ranked (Goldberg score) from 1 (none) to 4 (severe) by the same observer using a previously published scoring method. An experienced tribologist measured component wear depth using a coordinate measuring machine and quantified wear volume using previously validated methods. All available tissues were sampled and examined for features of ALVAL and scored from 0 to 10 by a single observer using a method they previously developed and published. A score from 0 to 4 is considered low, 5 to 8 is considered moderate, and 9 or 10 is considered high with regard to the risk of metal hypersensitivity features in the tissues. The most common bearing surface features were light and moderate scratches and removal or postremoval damage. Discoloration and deposits were commonly observed on femoral heads (55% [305 of 553]) and less commonly on cups (30% [165 of 546]). There was no evidence of impingement or dislocation damage. There was typically a small amount of bone attachment in at least one of eight designated regions of interest (84% [460 of 546]); extensive or no bone attachment was uncommon. Edge wear was highly prevalent. The maximum wear of 469 cups (88%) occurred near the edge, whereas the maximum wear of 508 femoral heads (94%) occurred between the pole and 45° from the pole. The median combined head-cup wear volume was 14 mm (range, 1-636 mm). One hundred sixty-nine pairs (32%) had a combined wear volume of < 10 mm, 42 pairs (8%) had volumetric wear of > 100 mm, and 319 pairs (60%) had wear volume between 10 and 100 mm³. Seventy-four percent of tapers (390 of 530) received a Goldberg score of 4, 22% (116 of 530) a score of 3, < 5% (24 of 530) a score of 2, and none received a score of 1. The most frequent ALVAL score was 5 out of 10 (35 of 144 hips [24%]) and ranged from 2 (one hip) to 10 (nine hips); 92 of 144 (64%) had a moderate score, 17 of 144 (12%) had a high score, and 35 (24%) had a low score. Although edge wear was prevalent, in most cases, this was not associated with high wear. The increased diameter and decreased coverage angle of the ASR design may have resulted in the observed high incidence of edge wear while perhaps decreasing the risk for impingement and dislocation. The role of bearing wear in the revisions of metal-on-metal implants is controversial, because it is known that there is a large range of in vivo wear rates even within the same implant type and that patient variability affects local tissue responses to wear debris. The observations from our study of 555 retrieved ASR implant sets indicate that there was a wide range of wear including a subset with very high wear. The results suggested that the failure of the ASR and ASR XL was multifactorial, and the failure of different subgroups such as those with low wear may be the result of mechanisms other than reaction to wear debris.

An Immunopathological Evaluation of the Porcine Cholecyst Matrix as a Muscle Repair Graft in a Male Rat Abdominal Wall Defect Model.

With the increasing use of animal-based biomaterials for regenerative medical applications, the need for their safety assessment is paramount. A porcine cholecyst-derived scaffold (CDS), intended as a muscle repair graft, prepared by a nondetergent/enzymatic method was engrafted in a rat abdominal wall defect model. Host tissue-scaffold interface samples were collected 2, 8, and 16 weeks postimplantation and evaluated by histopathology, immunohistochemistry, and electron microscopy. The nature of the tissue reaction was compared with those induced by a jejunum-derived scaffold (JDS) prepared by the same method and a commercial-grade small intestinal submucosa (CSIS) scaffold. A study of the immunopathological response in major lymphoid tissues and immunophenotyping for M1 and M2 macrophages was performed at the host tissue-scaffold interface. Further, "irritancy scores" for CDS and JDS were determined using CSIS as the reference material. Both CDS and JDS appeared to be potential biomaterials for muscle grafts, but the former stimulated a skeletal muscle tissue remodeling response predominated by M2 macrophages. The data support the notion that biomaterials with similar biocompatibility, based on local tissue response on implantation, may cause differential immunogenicity. Additionally, CDS compared to JDS and CSIS was found to be less immunotoxic.

Myocardial Inflammation Predicts Remodeling and Neuroinflammation After Myocardial Infarction

BackgroundThe local inflammatory tissue response after acute myocardial infarction (MI) determines subsequent healing. Systemic interaction may induce neuroinflammation as a precursor to neurodegeneration. ObjectivesThis study sought to assess the influence of MI on cardiac and brain inflammation using noninvasive positron emission tomography (PET) of the heart-brain axis. MethodsAfter coronary artery ligation or sham surgery, mice (n = 49) underwent serial whole-body PET imaging of the mitochondrial translocator protein (TSPO) as a marker of activated macrophages and microglia. Patients after acute MI (n = 3) were also compared to healthy controls (n = 9). ResultsInfarct mice exhibited elevated myocardial TSPO signal at 1 week versus sham (percent injected dose per gram: 8.0 ± 1.6 vs. 4.8 ± 0.9; p < 0.001), localized to activated CD68+ inflammatory cells in the infarct. Early TSPO signal predicted subsequent left ventricular remodeling at 8 weeks (rpartial = −0.687; p = 0.001). In parallel, brain TSPO signal was elevated at 1 week (1.7 ± 0.2 vs. 1.4 ± 0.2 for sham; p = 0.017), localized to activated microglia. After interval decline at 4 weeks, progressive heart failure precipitated a second wave of neuroinflammation (1.8 ± 0.2; p = 0.005). TSPO was concurrently up-regulated in remote cardiomyocytes at 8 weeks (8.8 ± 1.7, p < 0.001) without inflammatory cell infiltration, suggesting mitochondrial impairment. Angiotensin-converting enzyme inhibitor treatment lowered acute inflammation in the heart (p = 0.003) and brain (p = 0.06) and improved late cardiac function (p = 0.05). Patients also demonstrated elevation of cardiac TSPO signal in the infarct territory, paralleled by neuroinflammation versus controls. ConclusionsThe brain is susceptible to acute MI and chronic heart failure. Immune activation may interconnect heart and brain dysfunction, a finding that provides a foundation for strategies to improve heart and brain outcomes.

Identification and characterization of the major pseudocoelomic proteins of the giant kidney worm, Dioctophyme renale

BackgroundThe giant kidney worm, Dioctophyme renale, is a debilitating and potentially lethal parasite that inhabits and destroys, typically host’s right kidney, and may also be found in ectopic sites. It is circumglobally distributed, mainly in dogs, and is increasingly regarded as a threat to other domestic animals and humans. There is little information on the parasite’s true incidence, or immune responses to it, and none on its biochemistry and molecular biology.ResultsWe characterised the soluble proteins of body wall, intestine, gonads and pseudocelomic fluid (PCF) of adult parasites. Two proteins, P17 and P44, dominate the PCF of both male and females. P17 is of 16,622 Da by mass spectrometry, and accounts for the intense red colour of the adult parasites. It may function to carry or scavenge oxygen and be related to the ‘nemoglobins’ found in other nematode clades. P44 is of 44,460 Da and was found to associate with fatty acids by thin layer chromatography. Using environment-sensitive fluorescent lipid probes, P44 proved to be a hydrophobic ligand-binding protein with a binding site that is highly apolar, and competitive displacement experiments showed that P44 binds fatty acids. It may therefore have a role in distributing lipids within the parasites and, if also secreted, might influence local inflammatory and tissue responses. N-terminal and internal peptide amino-acid sequences of P44 indicate a relationship with a cysteine- and histidine-rich protein of unknown function from Trichinella spiralis.ConclusionsThe dominant proteins of D. renale PCF are, like those of large ascaridids, likely to be involved in lipid and oxygen handling, although there is evidence of strong divergence between the two groups.

In Vitro Biocompatibility, Radiopacity, and Physical Property Tests of Nano-Fe₃O₄ Incorporated Poly-l-lactide Bone Screws.

The aim of this study was to fabricate biodegradable poly-l-lactic acid (PLLA) bone screws containing iron oxide (Fe3O4) nanoparticles, which are radiopaque and 3D-printable. The PLLA composites were fabricated by loading 20%, 30%, and 40% Fe3O4 nanoparticles into the PLLA. The physical properties, including elastic modulus, thermal properties, and biocompatibility of the composites were tested. The 20% nano-Fe3O4/PLLA composite was used as the material for fabricating the 3D-printed bone screws. The mechanical performance of the nano-Fe3O4/PLLA bone screws was evaluated by anti-bending and anti-torque strength tests. The tissue response and radiopacity of the nano-Fe3O4/PLLA bone screws were assessed by histologic and CT imaging studies using an animal model. The addition of nano-Fe3O4 increased the crystallization of the PLLA composites. Furthermore, the 20% nano-Fe3O4/PLLA composite exhibited the highest thermal stability compared to the other Fe3O4 proportions. The 3D-printed bone screws using the 20% nano-Fe3O4/PLLA composite provided excellent local tissue response. In addition, the radiopacity of the 20% nano-Fe3O4/PLLA screw was significantly better compared with the neat PLLA screw.

Adverse Local Tissue Responses to Failed Temporomandibular Joint Implants

The purpose of this study was to determine whether failed alloplastic temporomandibular joint replacement (TMJR) devices can elicit the aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) reaction seen in some patients with metal-on-metal hip arthroplasties. This study involved analysis of paraffin-embedded sections of peri-implant tissue from failed TMJ implant cases obtained from 3 independent sources. Hematoxylin and eosin staining, conventional and polarized light microscopy, back-scattered electron imaging, and energy-dispersive x-ray analysis were used. Immunohistochemical methods were used to identify T and B lymphocytes and macrophages. The total TMJR device specimens showed primary macrophage and lymphocytic responses similar to responses reported previously for failed total hip implants, including ALVAL. No chronic or acute inflammation was apparent in the failed hemiarthroplasty TMJR cases. In this limited preliminary study, the local tissue responses to the failed TMJR implants showed similar primary macrophage and lymphocyte responses to previously reported failed metal-on-metal and metal-on-polyethylene orthopedic total joint replacement devices. No such local inflammatory responses were seen with the failed TMJR hemiarthroplasty devices.

A COMPARATIVE EXAMINATION OF QUANTITATIVE AND SPECIFIC STRUCTURE OF YEAST FUNGI OF THE CANDIDA GENUS CONSIDERING PATIENTS WITH CHRONIC GENERALIZED PERIODONTITIS OF VARYING SEVERITY

Background: Periodontitis is a major reason of tooth loss in people of middle and older age groups that requires more attention of researchers to the problems of etiology, pathogenesis, diagnosis, and therapy of the disease. Currently, the interaction of the microbial content of dental plaque and local tissue response is considered to be the main reason for the development of inflammatory periodontal diseases. At the same time, Russian and foreign researchers mark a high frequency of so-called torpid or resistant to antimicrobial therapy forms of periodontitis.Aim: To study the specific and quantitative composition of a fungi of the Candida genus in the contents of periodontal pockets of patients with varying severity of chronic generalized periodontitis.Materials and methods: A non-randomized non-controlled retrospective research was conducted. We studied the main clinical and radiological indicators of periodontal status and its contamination with fungi of the Candida genus in people with mild, moderate, or severe chronic periodontitis.Results: Research participants enrolled in four groups: 1–3 groups included patients with chronic generalized light, medium and heavy periodontitis; group 4 included twenty-five subjects with no signs of periodontal disease. Among the examined patients from the control group, the frequency allocation of Candida from the contents of the gingival sulcus was 9.09%, patients with periodontal pathology of periodontal pockets ― 64.91%. High contamination with fungal flora (more than 6.0 CFU / mL) was observed in all patients with chronic periodontitis. Total allocated 40 strains of Candida with a predominance of Candida albicans.Conclusions: The results of microbiological research confirmed an increase of contamination of periodontal with fungi of the Candida genus in patients with periodontitis if compared with healthy people. The degree of the contamination with fungi of the Candida genus decreased with increasing severity of periodontitis. It can be assumed that the presence of Candida fungi in the periodontal pocket can be considered as a possible component of the etiology of chronic periodontitis and as a factor aggravating it.

Effects of Local Antibiotic Delivery from Porous Space Maintainers on Infection Clearance and Induction of an Osteogenic Membrane in an Infected Bone Defect.

Reconstruction of large bone defects can be complicated by the presence of both infection and local antibiotic administration. This can be addressed through a two-stage reconstructive approach, called the Masquelet technique, that involves the generation of an induced osteogenic membrane over a temporary poly(methyl methacrylate) (PMMA) space maintainer, followed by definitive reconstruction after the induced membrane is formed. Given that infection and antibiotic delivery each have independent effects on local tissue response, the objective of this study is to evaluate the interaction between local clindamycin release and bacterial contamination with regards to infection prevention and the restoration of pro-osteogenic gene expression in the induced membrane. Porous PMMA space maintainers with or without clindamycin were implanted in an 8 mm rat femoral defect model with or without Staphylococcus aureus inoculation for 28 days in a full-factorial study design (four groups, n = 8/group). Culture results demonstrated that 8/8 animals in the inoculated/no antibiotic group were infected at 4 weeks, which was significantly reduced to 1/8 animals in the inoculated/antibiotic group. Quantitative polymerase chain reaction analysis demonstrated that clindamycin treatment restores inflammatory cytokine and growth factor expression to the same levels as the no inoculation/no antibiotic group, demonstrating that clindamycin can ameliorate the negative effects of bacterial inoculation and does not itself negatively impact the expression of important cytokines. Main effect analysis shows that bacterial inoculation and clindamycin treatment have independent and interacting effects on the gene expression profile of the induced membrane, further highlighting that antibiotics play an important role in the regeneration of infected defects apart from their antimicrobial properties.

The SUMO-targeted ubiquitin ligase, Dgrn, is essential for Drosophila innate immunity.

The ability of metazoans to combat pathogenic infection involves both systemic and local responses to the invading pathogens. Ubiquitin and SUMO pathways molecularly regulate the response to infection, immune signaling and gene expression. Here, we report that Degringolade (Dgrn, CG10981), a SUMO-targeted ubiquitin ligase connecting the two pathways, is essential for the innate immunity response in Drosophila. dgrnDK null and heterozygous mutant adult flies are severely immune-compromised and succumb rapidly to both pathogenic bacteria and fungi infections. The sensitivity to infection stems from the inability to produce multiple anti-microbial peptides, and transcriptional analyses suggest that the overexpression of Dgrn enhances the transcriptional output of the NF-&kgreen;B related Toll and immune deficiency (IMD)-pathways. Moreover, expression of Dgrn alleviated the inhibitory impact of the cytoplasmic NF-&kgreen;B inhibitor Cactus and the nuclear co-repressor Groucho/TLE (Gro). Additionally, we found that Dgrn is required for the local regenerative response of the mid-gut following infection. Upon oral infection, dgrn mutant flies fail to activate the Delta-Notch pathway in stem cells and enteroblasts, and are unable to regenerate and replace the damaged and dying enterocytes. Interestingly, the ubiquitin-specific protease CG8334 (dUSP32/dUSP11) antagonizes Dgrn activity in the gut, and halving the dose of CG8334 restores Delta-Notch signaling and rescues the lethality observed in dgrn mutants. Collectively, our data suggest that Dgrn is essential for both systemic and local tissue response to infection.

Tsetse fly tolerance to T. brucei infection: transcriptome analysis of trypanosome-associated changes in the tsetse fly salivary gland.

BackgroundFor their transmission, African trypanosomes rely on their blood feeding insect vector, the tsetse fly (Glossina sp.). The ingested Trypanosoma brucei parasites have to overcome a series of barriers in the tsetse fly alimentary tract to finally develop into the infective metacyclic forms in the salivary glands that are transmitted to a mammalian host by the tsetse bite. The parasite population in the salivary gland is dense with a significant number of trypanosomes tightly attached to the epithelial cells. Our current knowledge on the impact of the infection on the salivary gland functioning is very limited. Therefore, this study aimed to gain a deeper insight into the global gene expression changes in the salivary glands of Glossina morsitans morsitans in response to an infection with the T. brucei parasite. A detailed whole transcriptome comparison of midgut-infected tsetse with and without a mature salivary gland infection was performed to study the impact of a trypanosome infection on different aspects of the salivary gland functioning and the mechanisms that are induced in this tissue to tolerate the infection i.e. to control the negative impact of the parasite presence. Moreover, a transcriptome comparison with age-matched uninfected flies was done to see whether gene expression in the salivary glands is already affected by a trypanosome infection in the tsetse midgut.ResultsBy a RNA-sequencing (RNA-seq) approach we compared the whole transcriptomes of flies with a T. brucei salivary gland/midgut infection versus flies with only a midgut infection or versus non-infected flies, all with the same age and feeding history. More than 7500 salivary gland transcripts were detected from which a core group of 1214 differentially expressed genes (768 up- and 446 down-regulated) were shared between the two transcriptional comparisons. Gene Ontology enrichment analysis and detailed gene expression comparisons showed a diverse impact at the gene transcript level. Increased expression was observed for transcripts encoding for proteins involved in immunity (like several genes of the Imd-signaling pathway, serine proteases, serpins and thioester-containing proteins), detoxification of reactive species, cell death, cytoskeleton organization, cell junction and repair. Decreased expression was observed for transcripts encoding the major secreted proteins such as 5′-nucleotidases, adenosine deaminases and the nucleic acid binding proteins Tsals. Moreover, expression of some gene categories in the salivary glands were found to be already affected by a trypanosome midgut infection, before the parasite reaches the salivary glands.ConclusionsThis study reveals that the T. brucei population in the tsetse salivary gland has a negative impact on its functioning and on the integrity of the gland epithelium. Our RNA-seq data suggest induction of a strong local tissue response in order to control the epithelial cell damage, the ROS intoxication of the cellular environment and the parasite infection, resulting in the fly tolerance to the infection. The modified expression of some gene categories in the tsetse salivary glands by a trypanosome infection at the midgut level indicate a putative anticipatory response in the salivary glands, before the parasite reaches this tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3283-0) contains supplementary material, which is available to authorized users.

Abstract 13832: GPR39 is a High Affinity Vascular Smooth Muscle Receptor for P450 Eicosanoids

Epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic acids (HETEs) are endogenous vasoactive P450 eicosanoids that play important roles in cardiovascular physiology and disease, but their mechanisms of action are not fully understood. We developed a clickable photo-crosslinking probe (EET-P) based on EETs regio-isomer 14,15-EET, which allowed us to purify and enrich 14,15-EET binding proteins in mouse heart vascular smooth muscle cells (VSMCs). Proteomic analysis generated a list of proteins bound by EET-P that included the orphan G-protein coupled receptor 39 (GPR39). Consistent with the molecular weight of GPR39, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis revealed a prominent ~50 kD protein crosslinked to EET-P, and pretreatment with either 14,15-EET or its antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) completely displaced EET-P binding. Heterologous expression of GPR39 but not a control plasmid in HEK293 cells conferred responsiveness to both 14,15-EET and 15-HETE, as assayed by extracellular-regulated kinase (ERK) activation. Immuno-histochemical analysis localized GPR39 expression in both mouse heart micro-vessels and primary cultured VSMCs. Live-cell imaging of VSMC intracellular calcium ([Ca 2+ ]i) revealed a dual and antagonistic regulatory mechanism in which 15-HETE induces and 14,15-EET suppresses VSMC calcium transients. Short hairpin RNA (shRNA) knockdown of GPR39 in VSMCs was sufficient to abolish effects of both 15-HETE and 14,15-EET on VSMC calcium. Our results identify GPR39 as a high-affinity, lipid-sensing GPCR antagonistically regulated by two endogenous P450 eicosanoids with opposing actions on VSMC calcium, and offer a unique window into multi-ligand GPCRs and their role in integrating the multitude of hormonal signals to regulate local tissue function and response to disease state.

Complications from carcinoid syndrome: review of the current evidence.

Patients with well-differentiated neuroendocrine tumours may develop carcinoid syndrome (CS), which is characterised by flushing, abdominal cramps, diarrhoea, and bronchospasms. In this scenario, long-term secretion of vasoactive substances—serotonin, tachynins, and others, may induce fibrogenic responses in local or distant tissues, leading to complications such as carcinoid heart disease (CHD), mesenteric and/or retroperitoneal fibrosis. Rare cases of lung/pleural fibrosis and scleroderma have also been described. Despite it not being well described yet, current evidence suggests the pathogenesis of such fibrogenic complications relies on signalling through 5-HT2B and TGF-β1. Medical management is still very limited and lacks prospective and randomised studies for definitive recommendations. Surgical procedures remain the best definitive treatment option for CHD and abdominal fibrosis. Recently, cognitive impairment has also been described as a potential consequence of CS. This review critically discusses the literature concerning the epidemiology, pathogenesis, clinical features, diagnosis, and treatment options for CS-related long-term complications.

Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix

Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective “readouts” of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

Cell-Mediated Pathologies in Traumatic Orthopaedic Injuries

Physical trauma is one of the most common mechanisms leading to orthopaedic injury. The trauma and associated inflammatory response initiates the process of tissue regeneration and repair, which includes the recruitment and induction of multipotential cells to participate in the process. Too much inflammation can overwhelm this response to cause scarring. Although stem cells have been extensively studied as therapy for regenerating functional tissues, concerns of ethics, availability, and ease of clinical utility call for investigation into an alternative source of multipotential cells for potential therapeutic use. Here, we describe trauma-derived mesenchymal progenitor cells (MPCs). These cells are morphologically and functionally similar to bone marrow-derived mesenchymal stems cells. MPCs, which are not present in large numbers in untraumatized tissue, but are abundant in injured muscle tissue and after isolation have the potential for clinical application in regenerative medicine. MPCs appear to be activated by injury, enhancing their capability of differentiating into multiple cell lines, generating trophic factors, and producing functional tissue. This cell lineage is now thought to be involved in the pathologic process of heterotopic ossification, whereby inflammation and scarring dominates the local tissue response to injury. The utility of MPCs as a therapeutic arm for regenerative medicine can be further realized when the microenvironment commands that trigger these cells to activate are elucidated. This in turn can create patient-specific resource of nongenetically modified induced multipotent cells for regenerative therapy after orthopaedic injury.