Minimal Cellular Infiltration Research Articles

Macrophage membrane-functionalized nanofibrous mats and their immunomodulatory effects on macrophage polarization

Immunomodulation is an important phenomenon in the normal mammalian host response toward an injury, and plays a critical role in tissue regeneration and regenerative medicine. Different phenotypes of macrophages show an array of activation states compassing pro-inflammatory to pro-alleviating cells, which are the critical players to modulate immune response and tissue regeneration. In this study, macrophage membranes of different phenotypes (macrophages (M0), classically activated macrophages (M1) and alternatively activated macrophages (M2)) were coated onto poly-ε-caprolactone (PCL) nanofibers to acquire exterior surface proteins and similar functions of the natural membranes. In vitro results unveiled that these nanofibers, especially the M2-PCL nanofibers, can suppress the activities of inflammatory markers such as TNF-α and IL-1β, and stimulate anti-inflammatory markers such as Arg-1, IL-10 and TGF-β. In a C57BL/6 mouse model, the macrophage membrane-coated nanofibers, especially the M2-PCL nanofibers, displayed minimal cellular infiltration and low collagen deposition, increased anti-inflammatory CD206 and decreased inflammatory CD86 levels. The M2-PCL nanofibers most effectively neutralized inflammatory chemokines, regulated the expression of inflammation-associated genes as well as anti-inflammatory genes, and showed strong immunomodulatory effects than the PCL, M0-PCL and M1-PCL nanofibers. Statement of SignificanceDifferent types of macrophage membrane-functionalized PCL nanofibers were successfully prepared and well characterized. They inherited the surface proteins imitating the source macrophages, and played an important role in limiting cellular infiltration and collagen deposition. These different macrophages and their membrane-coated nanofibers (M0-PCL, M1-PCL and M2-PCL) behaved like their respective source cells. The M2 mimicking M2-PCL nanofibers effectively polarized macrophages to M2 phenotype and decreased the expression of inflammation-associated chemokines and promoted the anti-inflammation in vitro and in vivo, which is critical for tissue regeneration. The mice implanted with the bio-mimicking M2-PCL nanofibers effectively inhibited toll like receptors signaling induced NF-kB and IRF-5 and their target genes such as Edn-1, IL-6, iNOS, TNF-α, etc. compared to the PCL, and M0-PCL and M1-PCL macrophage membrane-coated nanofibers.

Histological and Physiological Studies of the Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Bleomycin Induced Lung Fibrosis in Adult Albino Rats.

Background:Lung fibrosis is considered as an end stage for many lung diseases including lung inflammatory disease, autoimmune diseases and malignancy. There are limited therapeutic options with bad prognostic outcome. The aim of this study was to explore the effect of mesenchymal stem cells (MSCs) derived from bone marrow on Bleomycin (BLM) induced lung fibrosis in albino rats.Methods:30 adult female albino rats were distributed randomly into 4 groups; negative control group, Bleomycin induced lung fibrosis group, lung fibrosis treated with bone marrow-MSCs (BM-MSCs) and lung fibrosis treated with cell free media. Lung fibrosis was induced with a single dose of intratracheal instillation of BLM. BM-MSCs or cell free media were injected intravenously 28 days after induction and rats were sacrificed after another 28 days for assessment. Minute respiratory volume (MRV), forced vital capacity (FVC) and forced expiratory volume 1 (FEV1) were recorded using spirometer (Power lab data acquisition system). Histological assessment was performed by light microscopic examination of H&E, and Masson’s trichrome stained sections and was further supported by morphometric studies. In addition, electron microscopic examination to assess ultra-structural changes was done. Confocal Laser microscopy and PCR were used as tools to ensure MSCs homing in the lung.Results:Induction of lung fibrosis was confirmed by histological examination, which revealed disorganized lung architecture, thickened inter-alveolar septa due excessive collagen deposition together with inflammatory cellular infiltration. Moreover, pneumocytes depicted variable degenerative changes. Reduction in MRV, FVC and FEV1 were recorded. BM-MSCs treatment showed marked structural improvement with minimal cellular infiltration and collagen deposition and hence restored lung architecture, together with lung functions.Conclusion:MSCs are promising potential therapy for lung fibrosis that could restore the normal structure and function of BLM induced lung fibrosis.Electronic supplementary materialThe online version of this article (10.1007/s13770-020-00294-0) contains supplementary material, which is available to authorized users.

Attenuating ischemia/reperfusion injury in rat cardiac transplantation by intracoronary infusion with siRNA cocktail solution.

Tumor necrosis factor-α (TNF-α), caspase-8, and complement component 5a receptor (C5aR) are known to play a crucial role in the myocardial ischemia/reperfusion (I/R) injury in cardiac transplantation. We hypothesized that the intracoronary infusion of TNF-α, caspase-8, and C5aR small interfering RNAs (siRNA) would protect cardiac allograft function and improve graft survival from I/R injury-induced organ failure. I/R injury of cardiac allograft was induced by syngeneic rat cardiac transplantation, in which the transplanted hearts were infused with saline or different amounts of siRNA cocktail solution targeting TNF-α, caspase-8, and C5aR via coronary arteries, and subsequently subjected to 18 h of preservation at 4°C in histidine–tryptophan–ketoglutarate (HTK) solution. The effects of siRNA cocktail solution on prolonged cold I/R injury were determined by assessing graft survival, histopathological changes, myeloperoxidase (MPO) activity, and malondialdehyde (MDA) concentration. The perfused siRNA cocktail solution successfully knocked down the expression of TNF-α, caspase-8, and C5aR in vitro and in vivo. Approximately 91.7% of control hearts that underwent 18 h of cold ischemia ceased their function after transplantation; however, 87.5% of cardiac allografts from the highest dose siRNA cocktail solution-pretreated hearts survived >14 days and exhibited minimal histological changes, with minimal cellular infiltration, interstitial edema, and inflammation and maximal reduced MPO activity and MDA concentration in the cardiac allograft. We demonstrated the feasibility and efficiency of infusion of TNF-α, caspase-8, and C5aR siRNA via the intracoronary route as a promising strategy for gene silencing against I/R injury in cardiac transplantation.

Dog-specific hemorrhagic changes induced by liposomal formulations, in the liver and the gallbladder

Although several liposomal drugs, including liposomal doxorubicin, have been approved, the etiology of the pathological responses caused by their physicochemical properties remains unknown. Herein, we investigated the pathological changes in the liver and the gallbladder of dogs following a single injection of liposomal doxorubicin (1 or 2.5 mg/kg) or an empty liposomal formulation (i.e., liposomal formulation without doxorubicin, ca. 21 mg/kg as lipid content). Injection of liposomal doxorubicin or the empty liposomal formulation induced hemorrhagic changes in the liver and the gallbladder. These changes were accompanied by minimal cellular infiltration with no obvious changes in the blood vessels. As there were no differences in the incidence and severity of hemorrhage between the groups administered comparable amounts of total lipid, the physicochemical properties of the liposomal formulation rather than an active pharmacological ingredient, doxorubicin, were associated with the hemorrhagic changes. Furthermore, decreased cytoplasmic granules with low electron density in mast cells beneath the endothelium of the hepatic vein were observed in the liver of dogs treated with liposomal doxorubicin or empty liposomal formulation. Injection of compound 48/80, a histamine releaser induced comparable hemorrhage in dogs, implying that hemorrhage caused by injection of liposomal doxorubicin or the empty liposomal formulation could be attributed to the histamine released from mast cells. The absence of similar hemorrhagic lesions in other species commonly used in toxicology studies (i.e., rats and monkeys), as well as humans, is due to the lack of mast cells beneath the endothelium of the hepatic vein in these species.

Prolonged Cold Ischemia Induces Necroptotic Cell Death in Ischemia-Reperfusion Injury and Contributes to Primary Graft Dysfunction after Lung Transplantation.

Primary graft dysfunction (PGD) is a major cause of morbidity and mortality after lung transplantation. Ischemia-reperfusion injury (IRI) is a key event that contributes to PGD, though complex interactions affect donor lungs status, such as preceding brain death (BD), hemorrhagic shock (HS), and pre-engraftment lung management, the latter recognized as important risk factors for PGD. We hypothesized that a multi-hit isogenic mouse model of lung transplantation is more closely linked to PGD than IRI alone. Left lung transplants were performed between inbred C57BL/6 mice. A one-hit model of IRI was established by inducing cold ischemia (CI) of the donor lungs at 0°C for 1, 72, or 96 hours before engraftment. Multi-hit models were established by inducing 24 hours of HS and/or 3 hours of BD before 24 hours of CI. The recipients were killed at 24 hours after transplant and lung graft samples were analyzed. In the one-hit model of IRI, up to 72-hour CI time resulted in minimal cellular infiltration near small arteries after 24-hour reperfusion. Extension of CI time to 96 hours led to increased cellular infiltration and necroptotic pathway activation, without evidence of apoptosis, after 24-hour reperfusion. In a multi-hit model of PGD, "HS + BD + IRI" demonstrated increased lung injury, cellular infiltration, and activation of necroptotic and apoptotic pathways compared with IRI alone. Treatment with an inhibitor of receptor-interacting protein kinase 1 kinase, necrostatin-1, resulted in a significant decrease of downstream necroptotic pathway activation in both single- and multi-hit models of IRI. Thus, activation of necroptosis is a central event in IRI after prolonged CI, though it may not be sufficient to cause PGD alone. Pathological evaluation of donor lungs after CI-induced IRI, in conjunction with pre-engraftment donor lung factors in our multi-hit model, demonstrated early evidence of lung injury consistent with PGD. Our findings support the premise that pre-existing donor lung status is more important than CI time alone for inflammatory pathway activation in PGD, which may have important clinical implications for donor lung retrieval.

Response of the inner ear to lipopolysaccharide introduced directly into scala media

In an attempt to develop an animal model of immune mediated Meniere's disease, we have injected lipopolysaccharide (LPS) directly into scala media of guinea pigs and monitored functional and morphological changes over a period of 6 weeks. Depending on the concentration of LPS, changes ranged from moderate-to-severe hearing loss and endolymphatic hydrops with minimal cellular infiltrate or fibrosis, to dense cellular infiltration that filled the scalae. Interestingly, higher concentrations of LPS not only induced severe cellular infiltration, hydrops, and hearing loss, but also a substantial enlargement of the endolymphatic duct and sac. Moreover, LPS injections into perilymph failed to induce hydrops, yet still resulted in cellular infiltration and fibrosis in the cochlea. This suggests that chronic hydrops resulting from an immune challenge of the cochlea may not be due to blockage of the endolymphatic duct and sac, restricting fluid absorption. Furthermore, injecting antigen into endolymph may produce chronic immune-mediated hydrops, and provide a more promising animal model of Meniere's, although animals did not display signs of vestibular dysfunction, and the hearing loss was relatively severe.

Research Highlights

Ablation of Transcription Factor IRF4 Promotes Transplant Acceptance by Driving Allogenic CD4+ T Cell Dysfunction Jie Wu, Hedong Zhang, Xiamomin Shi, et al. Immunity. 2017;47(6):1114–1128.e6. The transcriptional programs regulating allogenic T-cell responses have not been adequately documented thus far. Here, the authors used a knockout mouse model to demonstrate the importance of interferon regulatory factor 4 (IRF4) in controlling graft rejection. IRF4 is a key transcriptional factor running T-cell responses during and after transplantation. The authors performed several experiments on IRF4 deficient (IRF4−/−) as well as wild type mice by transplanting them with mismatched heart allografts. First, they demonstrated that graft-infiltrating T cells expressed a consistently elevated mean fluorescence intensity of IRF4. Notably, they were able to show that IRF4−/− mice did not reject their allografts. Moreover, all IRF−/− mice survived the observation period of 100 days with intact myocytes and only minimal cellular infiltrates into the graft. The authors then went on transferring either 2 million wild type CD4+ or CD8+ T cells or 20 million IRF4−/− CD4+ or CD8+ T cells into IRF4−/− mice and found that the adaptive transfer of unmanipulated T cells facilitated rejection, whereas the transfer of IRF−/− T cells did not. Furthermore, IRF4−/− mice failed to show increased T cells in their spleen after transplantation, indicating that expansion of alloreactive T cells in IRF4−/− was impaired. Mechanistically, the authors demonstrated the critical role of PD-1 expression by alloantigen-specific T cells for the long-term acceptance of cardiac allografts in IRF−/− mice. Finally, the authors showed that trametinib, a MEK1/2 inhibitor most potently reduced IRF4 expression in activated T cells, inhibited Th1 and Th17 cell differentiation and prolonged heart allograft survival. This intriguing data show the critical role of IRF4 expression on T cells in alloimmunity and indicate the translational potential of trametinib to in improving transplant outcomes. Cutting Edge: Allograft Rejection Is Associated With Weak T-Cell Responses to Many Different Graft Leukocyte-Derived Peptides Burrack AL, Malhotra D, Dileepan T, Osum KC, Swanson LA, Fife BT, Jenkins MK. J Immunol. 2018; 200: 477–482. Allografts are rejected by T lymphocytes that recognize foreign MHC class I and II antigens. The robustness of this T-cell response, however, is not understood. Previous concepts have attributed the rejection process largely on the frequency of T cells that recognize either allogeneic MHC molecules alone or MHC plus-bound peptides. The authors explored the specificity of directly alloreactive T cells, using a peptide-MHC II tetramer-based approach. Tail skin from H2-DM- and CD74-deficient mice were grafted onto the flanks of recipient laboratory-bred strain house mouse. These grafts were rejected after 10 to 12 days. Spleen, lymph node, or blood cells were harvested and stained 7 to 14 days after transplantation. The total number of tetramer-positive cells in the enriched fraction from each mouse was determined, using AccuCheck counting beads. Differences in T-cell expansion and differentiations after transplantation between and across groups were established. CD4+ cells were identified as viable lymphocyte-sized single cells that expressed CD3 or CD90.2. Graft-responsive CD4 T cells expanded an average of 28-fold after transplantation. Mice transplanted with B6 skin also demonstrated a clonal T-cell expansion, indicating that this phenomenon was not limited to autoimmunity-prone NOD mice. In the transplanted mice, tetramer binding sites were examined and sites increased fivefold in NOD mice that had been transplanted with B6 skin. These results demonstrate that directly alloreactive T cells recognize an allogeneic MHC II molecule and a graft leukocyte peptide in the same way that microbe-reactive T cells recognize a syngeneic MHC II molecule and a microbial peptide. The data suggest that the potency of direct alloreactivity is not due to a high frequency of T cells that recognize MHC molecules alone, but rather the consequence of an additional weak activation of T cells, specific for hundreds of different graft leukocyte peptide-allogeneic MHC complexes. These data have important conceptual relevance in understanding basic mechanisms that drive alloimmunity and inflammation.

Protective Role of N-Acetylcysteine on Isoprenaline-Induced Myocardial Injury: Histological, Immunohistochemical and Morphometric Study.

Several researchers studied the protective effect of the N-acetylcysteine (NAC) when it was given before the induction of myocardial infarction (MI). Other researchers studied such protective effect when it was before done and after done of the MI. The missing data are the comparison between the protective effect of NAC before myocardial injury with its protective effect both before and after myocardial injury. The aim of the study was to compare the cardioprotective effect of NAC on the isoprenaline-induced myocardial injury before the isoprenaline (ISP) injection with its protective effect both before and after the ISP injection. This study was applied over both short and long time periods. A total of 90 male adult Wistar albino rats were used in the study. The rats were divided into four groups: control group, ISP-treated group, NAC-pretreated group and NAC-pre-& posttreated group. Based on the duration of the experiment, the second and third groups were further subdivided into a and b groups. Histological, immunohistochemical and histomorphometric analysis were used. The myocytes in the ISP-treated groups were fragmented, disrupted with karyolysis. The blood vessels were dilated, congested and associated with blood extravasation, interstitial edema and cellular inflammatory infiltration. Much improvement was observed in the NAC-pretreated group. Focal degeneration was detected in the muscle fibers. The capillaries were normal. Minimal blood extravasation and cellular infiltration were seen. The cardiac muscle fibers in the NAC-pre-& posttreated group were regularly arranged. The mean collagen fiber area percent of the ISP-treated groups was significantly higher by 8.3-folds and 10.1-folds as compared with that of the control group and was also higher by 5.5-folds and 6.8-folds as compared with that of the NAC-pre-&posttreated groups. The α-SMA area percent in the ISP-treated groups was significantly higher by 12.2-folds and 23.9-folds as compared with that of the control group and was higher by 7.5-folds and 15-folds as compared with that of the NAC-pre-& posttreated groups. The mean PCNA area percent of the ISP-treated groups was significantly higher by 126.2 and 164.8% as compared with that of the control group and was higher by 106.3 and 141.5% as compared with that of NAC-pre-& posttreated groups. ISP had deleterious effects on the heart. Administration of NAC before ISP injection could largely reduce the ISP-induced short- and long-term alterations. The protection was maximum with the use of NAC before the ISP injection and continued after the injection for 12days.

Relationship of clusterin with renal inflammation and fibrosis after the recovery phase of ischemia-reperfusion injury.

BackgroundLong-term outcomes after acute kidney injury (AKI) include incremental loss of function and progression towards chronic kidney disease (CKD); however, the pathogenesis of AKI to CKD remains largely unknown. Clusterin (CLU) is a chaperone-like protein that reduces ischemia-reperfusion injury (IRI) and enhances tissue repair after IRI in the kidney. This study investigated the role of CLU in the transition of IRI to renal fibrosis.MethodsIRI was induced in the left kidneys of wild type (WT) C57BL/6J (B6) versus CLU knockout (KO) B6 mice by clamping the renal pedicles for 28 min at the body temperature of 32 °C. Tissue damage was examined by histology, infiltrate phenotypes by flow cytometry analysis, and fibrosis-related gene expression by PCR array.ResultsReduction of kidney weight was induced by IRI, but was not affected by CLU KO. Both WT and KO kidneys had similar function with minimal cellular infiltration and fibrosis at day 14 of reperfusion. After 30 days, KO kidneys had greater loss in function than WT, indicated by the higher levels of both serum creatinine and BUN in KO mice, and exhibited more cellular infiltration (CD8 cells and macrophages), more tubular damage and more severe tissue fibrosis (glomerulopathy, interstitial fibrosis and vascular fibrosis). PCR array showed the association of CLU deficiency with up-regulation of CCL12, Col3a1, MMP9 and TIMP1 and down-regulation of EGF in these kidneys.ConclusionOur data suggest that CLU deficiency worsens renal inflammation and tissue fibrosis after IRI in the kidney, which may be mediated through multiple pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12882-016-0348-x) contains supplementary material, which is available to authorized users.

Widespread Myocardial Delivery of Heart-Derived Stem Cells by Nonocclusive Triple-Vessel Intracoronary Infusion in Porcine Ischemic Cardiomyopathy: Superior Attenuation of Adverse Remodeling Documented by Magnetic Resonance Imaging and Histology.

Single-vessel, intracoronary infusion of stem cells under stop-flow conditions has proven safe but achieves only limited myocardial coverage. Continuous flow intracoronary delivery to one or more coronary vessels may achieve broader coverage for treating cardiomyopathy, but has not been investigated. Using nonocclusive coronary guiding catheters, we infused allogeneic cardiosphere-derived cells (CDCs) either in a single vessel or sequentially in all three coronary arteries in porcine ischemic cardiomyopathy and used magnetic resonance imaging (MRI) to assess structural and physiological outcomes. Vehicle-infused animals served as controls. Single-vessel stop-flow and continuous-flow intracoronary infusion revealed equivalent effects on scar size and function. Sequential infusion into each of the three major coronary vessels under stop-flow or continuous-flow conditions revealed equal efficacy, but less elevation of necrotic biomarkers with continuous-flow delivery. In addition, multi-vessel delivery resulted in enhanced global and regional tissue function compared to a triple-vessel placebo-treated group. The functional benefits after global cell infusion were accompanied histologically by minimal inflammatory cellular infiltration, attenuated regional fibrosis and enhanced vessel density in the heart. Sequential multi-vessel non-occlusive delivery of CDCs is safe and provides enhanced preservation of left ventricular function and structure. The current findings provide preclinical validation of the delivery method currently undergoing clinical testing in the Dilated cardiomYopathy iNtervention With Allogeneic MyocardIally-regenerative Cells (DYNAMIC) trial of CDCs in heart failure patients.

The effect of coenzyme Q10 on age-related changes of the distal colon mucosa of male albino rats

Introduction Aging is associated with a number of diverse effects on the colon leading to deterioration in its functions. The reactive oxygen species have been suggested as an important factor in aging. Coenzyme Q10 (CoQ10) is a vitamin-like substance, endogenously synthesized inside the cell. It serves as an antioxidant. CoQ10 decreases gradually with aging. Aim of Work The aim of this study was to evaluate the role of CoQ10 in age-related histological changes of the distal colon of male albino rats. Materials and methods A total of 20 male albino rats were used in this study. The rats were divided into three groups: group I, group II, and group III. Group I comprised 10 rats aged 4±1 months, which served as the control group. Group II (the aged group) comprised five rats aged 18±1 months. Group III comprised five rats of similar age as that of group II and received CoQ10 daily through the intraperitoneal route at a dose of 5 mg/kg body weight/day for 8 weeks. Specimens of the distal colon were excised and paraffin sections were prepared for histological and immunohistochemical staining. Results In aged rats (group II), the mucosa showed discontinuity and loss of surface epithelium with increased intraepithelial lymphocytes. There was significant decrease in the number of PAS-positive (periodic acid Schiff) goblet cells, with abnormal proliferation within the crypt. Intense immunopositive stain for tumor necrosis factor-α was detected in the mucosa. Extensive mononuclear cellular infiltration was eminent within the lamina propria. Significant increase in collagen fibers was detected in the lamina propria of the distal colon. In group III, the mucosa appeared intact with minimal cellular infiltration. PAS-positive goblet cells were nearly similar to those of controls. Immunopositive reaction for tumor necrosis factor-α was significantly decreased compared with group II. Significant decrease in collagen fiber content was seen in the lamina propria. Conclusion The administration of CoQ10 improved age-related histological changes that occurred in the distal colon of rats.

CpG-depleted adeno-associated virus vectors evade immune detection

Due to their efficient transduction potential, adeno-associated virus (AAV) vectors are leading candidates for gene therapy in skeletal muscle diseases. However, immune responses toward the vector or transgene product have been observed in preclinical and clinical studies. TLR9 has been implicated in promoting AAV-directed immune responses, but vectors have not been developed to circumvent this barrier. To assess the requirement of TLR9 in promoting immunity toward AAV-associated antigens following skeletal muscle gene transfer in mice, we compared immunological responses in WT and Tlr9-deficient mice that received an AAV vector with an immunogenic capsid, AAVrh32.33. In Tlr9-deficient mice, IFN-γ T cell responses toward capsid and transgene antigen were suppressed, resulting in minimal cellular infiltrate and stable transgene expression in target muscles. These findings suggest that AAV-directed immune responses may be circumvented by depleting the ligand for TLR9 (CpG sequences) from the vector genome. Indeed, we found that CpG-depleted AAVrh32.33 vectors could establish persistent transgene expression, evade immunity, and minimize infiltration of effector cells. Thus, CpG-depleted AAV vectors could improve outcome of clinical trials of gene therapy for skeletal muscle disease.

Cryptococcus neoformans growth and protection from innate immunity are dependent on expression of a virulence-associated DEAD-box protein, Vad1.

The fungus Cryptococcus neoformans has emerged as a major cause of meningoencephalitis worldwide. Host response to the fungus involves both innate and adaptive immunity, but fungal genes that modulate these processes are poorly understood. Previous studies demonstrated attenuated virulence of a mutant of a virulence-associated DEAD-box protein (VAD1) in mice, despite normal growth at host temperatures, suggesting modulation of the immune response. In the present study, the Δvad1 mutant demonstrated progressive clearance from lung and was unable to induce pathological lesions or to cause extrapulmonary disease, despite retaining its ability to grow in mouse serum and a J774.16 macrophage cell line. Pulmonary clearance occurred with a minimal cellular infiltrate, marked by reduced CD4 cells, CD11b(+) Ly6C(high) monocytes, and F4/80(+) macrophages, but the mutant strain retained recruitment of CD8 cells, compared to infections with wild-type fungi. Adaptive cytokine responses were reduced, including Th1, Th2, and Th17 cytokines; however, early gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) responses were retained while nonprotective interleukin 4 (IL-4) and IL-5 were diminished. Furthermore, the Δvad1 mutant was controlled in lungs despite CD4/CD8 cell depletion. These data, along with improved phagocytosis by macrophages and increases in early/innate IL-1α, IFN-γ, and chemokines elicited in the lungs within 3 days of infection with the Δvad1 mutant, indicate that VAD1 expression reduces innate recognition of C. neoformans, rendering the yeast resistant to elimination by the innate mechanisms of host defense. Thus, our studies define a novel role of the cryptococcal Vad1 protein as a central regulator of cryptococcal virulence and illustrate that Vad1 promotes microbe resistance to innate host defenses.

Myocardial migration by fibroblast progenitor cells is blood pressure dependent in a model of angII myocardial fibrosis

Activation of the renin-angiotensin system (RAS) is thought to promote myocardial fibrosis. However, it is unclear whether this physiological fibrotic response results from chronic hemodynamic stress or from direct cellular signaling. Male C57B/6 mice were randomly assigned to receive angiotensin II (AngII) (2.0 μg kg(-1) min(-1)), AngII+hydralazine (6.9 μg kg(-1) min(-1)) or saline (control) via osmotic pumps for 7 days. Blood pressure was measured via noninvasive plethysmography. Hearts were harvested and processed for analysis. Cellular infiltration and collagen deposition were analyzed using histological staining. Molecular mediators were assessed using quantitative RT-PCR. As previously described, animals that received AngII developed hypertension and multifocal cellular infiltration by SMA(+)/CD133(+) fibroblast progenitors followed by collagen deposition. The coadministration of hydralazine with AngII completely inhibited the hypertensive effects of AngII (P0.01) and resulted in minimal cellular infiltration and minimal collagen deposition. These findings were in the context of persistent RAS activation, which was evidenced by elevation in serum aldosterone levels in animals that received AngII or AngII+hydralazine compared with animals that received saline. At the molecular level, infusion of AngII resulted in the significant upregulation of profibrotic factors (connective tissue growth factor-7.8±0.7 fold), proinflammatory mediators (TNFα-4.6±0.8 fold; IL-1β-6.4±2.6 fold) and chemokines (CCL2-3.8±1.0 fold; CXCL12-3.2±0.4 fold), which were inhibited when hydralazine was also infused. We provide evidence that myocardial infiltration by fibroblast progenitor cells secondary to AngII and the resultant fibrosis can be prevented by the addition of hydralazine. Furthermore, the beneficial effects of hydralazine were observed while maintaining RAS activation, suggesting that the mechanism of fibrosis is blood pressure dependent.

Rock1 Inhibitor Abrogates Chronic Rejection in Rat Cardiac Model System

Background: We have shown previously that the abrogation of acute and chronic rejection of rat cardiac allografts occurs through the down-regulation of RhoA pathway and involves the changes in RhoA kinase (ROCK)-dependent actin cytoskeleton and T cell motility. Here we studied the ability of the Y-27632, a highly selective inhibitor of Rho-associated protein kinase p160ROCK (ROCK1), to abrogate chronic rejection of the allograft and influence T cell infiltration. Methods: Heterotopic cardiac transplants were performed between donor Wistar Furth (WF) and ACI recipient rats. Controls received sub-therapeutic dose of cyclosporine (CsA, 10 mg/kg) for 3 days or 7 days therapeutic dose of cyclosporine. The experimental groups of ACI recipient received one preoperative dose of the Y-27632 inhibitor (2 mg/kg, gavage feed) in conjunction with the sub-therapeutic dose of CsA for 3 days or inhibitor alone for 7 days. The cardiac grafts were harvested at 100 days of post-transplantation for histological and immunohistochemical assessment of chronic rejection, vascular sclerosis, and infiltration by different T cell subtypes. Results: Cardiac allografts from recipients exposed to Y-27632 inhibitor in conjunction with sub-therapeutic dose of CsA showed drastically reduced vascular sclerosis, minimal myocardial total cellular infiltration, and were selectively infiltrated with Foxp3+ T regulatory (Treg) cells. Conclusions: Our novel finding that a single dose of the ROCK1 inhibitor Y-27632 attenuates chronic rejection in rat cardiac model system by promoting development of Treg cells warrants its potential as a novel therapeutic agent specific for the inhibition of chronic rejection.

An Elastomeric Patch Electrospun from a Blended Solution of Dermal Extracellular Matrix and Biodegradable Polyurethane for Rat Abdominal Wall Repair

A biodegradable elastomeric scaffold was created by electrospinning a mixed solution of poly(ester urethane)urea (PEUU) and porcine dermal extracellular matrix (dECM) digest, with PEUU included to provide elasticity, flexibility, and mechanical support and dECM used to enhance bioactivity and biocompatibility. Micrographs and differential scanning calorimetry demonstrated partial miscibility between PEUU and dECM. With greater dECM content, scaffolds were found to possess lower breaking strains and suture retention strength, although initial modulus was greater with higher dECM concentrations. The hybrid scaffolds containing 0% to 50% dECM had tensile strengths of 5 to 7 MPa, breaking strains of 138% to 611%, initial moduli of 3 to 11 Mpa, and suture retention strengths of 35 to 59 MPa. When hydrated, scaffolds were found to contract markedly with 50% dECM content. When used in a rat full-thickness abdominal wall replacement model, no herniation, infection, or tissue adhesion was observed after 4 and 8 weeks with a scaffold containing 25% dECM or a control 100% PEUU scaffold. Scaffolds incorporating dECM were significantly thicker at the time of explant, with greater numbers of associated smooth muscle actin-positive staining cells than in the control, but minimal cellular infiltration and remodeling of the scaffold were detected regardless of dECM addition. The processing of dECM and PEUU from a mixed solution thus provided a scaffold with evidence of better bioactivity and with mechanical properties not achievable with digested dECM alone.

Myopathy and Neuropathy with Pipestem Capillaries and Vascular Activated Complement Deposition

Necrotizing myopathy with pipestem capillaries defines an immune-mediated disorder of skeletal muscle characterized by minimal cellular infiltration, muscle fiber necrosis, and microangiopathy with complement membrane attack complex (MAC; C5b9) deposition. Thickened, hyalinized (“pipestem”) vessels, ultrastructurally embedded in amorphous material, and capillary loss are further features.1 The rare patients reported showed proximal weakness and high creatine kinase,1,–,4 frequently associated with myalgia. They presented with other symptoms suggesting autoimmune disorders (including cerebral vasculitis) or cancer and an overall unfavorable prognosis,1,2 despite the response of muscle symptoms to immunosuppressive treatments. A pathophysiologic concept to link these multiorgan symptoms is not known, possibly because only one sample outside muscle has so far been taken for comparison of histopathologic features. That skin probe showed “mild fibrosis and lymphocytic perivascular cuffs in superficial dermis.”2 A 57-year-old normotensive, nondiabetic man presented with rapid onset edema of the distal legs, dysesthesia, loss of ankle reflex, and bilateral foot drop (Medical Research Council grade 0). Proximal and upper limb strength and reflexes were intact. Neurophysiologic examination demonstrated loss of compound muscle action potentials (CMAPs) of both peroneal nerves and the left tibial nerve, reduced CMAP amplitude of the right tibial nerve, and loss of sensory nerve action potentials of both sural nerves. Neurography of the right median and ulnar nerves showed normal results. Needle myography showed abnormal spontaneous activity and only a few recordable muscle unit potentials in the tibialis anterior …

Comparison of collagen scaffold tubes for possible esophagus organ tissue engineering applications: In-situ omental implantation study in an ovine model

BACKGROUND: Esophagus tissue engineering requires optimal scaffolds that can support the implanted cells and integrate with the implanted tissue. METHODS: Four collagen scaffolds were investigated in-situ in an ovine model after 8 weeks of omental implantation to assess their suitability with regards to integration and cellular infiltration for esophagus organ tissue engineering: (a) Acellular bovine pericardial collagen (BPC), (b) Acellular cross-linked (HDMI hexamethylene diisocyanate) porcine dermal collagen (PDC), (c) Porcine porous collagen (PPC) and (d) Bovine tendon dual-layered (type-1) collagen (DLC). RESULTS: BPC demonstrated minimal integration with minimal cellular infiltration. PDC was inert to both integration and cellular infiltration. PPC enabled integration to omental tissue; however, demonstrated minimal cellular infiltration. DLC exhibited both omental integration and cellular infiltration. CONCLUSION: DLC has close to ideal properties for application in esophagus tissue engineering. BPC and PPC could have limited application in esophagus tissue engineering whereas PDC has been found to be unsuitable.

Desmoplasia: not always a bad thing

Desmoplasia describes a histological pattern characterized by a hyalinized stroma and a minimal cellular infiltrate. In non-cutaneous neoplasms, this pattern of stromal response is classically associated with malignancy, whereas in cutaneous pathology, desmoplasia is observed in malignant as well as benign neoplasms. Given this, the obvious question is whether desmoplasia associated with a benign neoplasm is any different from that associated with malignant tumours. Is the stromal response a mere epiphenomenon, or does it actually contribute to the biological behaviour of the neoplasm? What happens at the tumour-host interface? Which molecules are involved in mediating the desmoplastic reaction pattern? This review is an attempt to answer these questions. Examples of benign and malignant cutaneous neoplasms associated with the desmoplastic reaction pattern will be included.

Heavy metal exposure reverses genetic resistance to Chlamydia-induced arthritis

IntroductionWe have previously observed that Brown Norway (BN) rats display a relative resistance to experimental Chlamydia-induced arthritis. In the present study, we examine an environmental toxin, mercuric chloride (HgCl2), as a modulator of this innate resistance to arthritis.MethodsTo assess the effect of the heavy metal exposure, one group of rats received two subcutaneous injections of HgCl2 (1 mg/kg) 48 hours apart. Seven days later, the animals received the intra-articular injection of synoviocyte-packaged Chlamydia.ResultsHistopathology revealed that BN rats receiving only Chlamydia had a minimal cellular infiltration in the joint, which was predominantly mononuclear in character. In contrast, mercury-exposed rats had a marked exacerbation of the histopathological severity of the arthritis, and the infiltration was predominantly neutrophilic. Mercury exposure was also associated with marked enhancement in IgE levels and an alteration in IgG2a/IgG1 ratio, reflecting a Th2 shift. The local cytokine profile in the joint was markedly altered after mercury exposure, with a suppression of tumour necrosis factor-alpha and interferon-gamma but an enhancement of vascular endothelial growth factor. This was associated with decreased host clearance capacity reflected in enhanced bacterial load in both the spleen and the joint and was accompanied by enhanced detection of microbial antigens in the synovial tissues by immunohistological staining.ConclusionsGenetically defined cytokine production in the joint defines the severity of reactive arthritis by dictating the local clearance of the pathogen. This interplay can be altered dramatically by heavy metal exposure, which results in suppression of protective cytokines in the microenvironment of the joint.