Characterizing temporal and spatial recruitment of systemically administered RPE65-programmed bone marrow-derived cells to the retina in a mouse model of age-related macular degeneration.

PURPOSE: Previously, we reported that the intravenous injection of bone marrow-derived cells (BMDC) infected with lentivirus expressing the human RPE65 gene resulted in the programming of BMDC to promote visual recovery in a mouse model of age-related macular degeneration (AMD). The aim of this study was to characterize the spatial and temporal recruitment of these programmed BMDC to the retinal pigment epithelial (RPE) layer. METHODS: C57BL/6J female mice received a subretinal injection of AAV1-SOD2 ribozyme to knock down (KD) superoxide dismutase 2 (SOD2) and induce AMD-like pathology. BMDC were isolated from GFP+ mice and infected with a lentivirus expressing RPE65. One month after SOD2 KD, fifty thousand GFP+ RPE65-BMDC were injected in the mouse tail vein. Animals were terminated at different time points up to 60 min following cell administration, and localization of GFP+ cells was determined by fluorescence microscopy of neural retina and RPE flat mounts and tissue sections. RESULTS: GFP+ RPE65- BMDC were observed in SOD2 KD neural retina and RPE as early as 1 min following administration. With increasing time, the number of cells in the neural retina decreased, while those in the RPE increased. While the number of cells in peripheral and central retina remained similar at each time point, the number of BMDC recruited to the central RPE increased in a time-dependent manner up to a maximum by 60 min post administration. Immunohistochemistry of cross-sections of the RPE layer confirmed the incorporation of donor GFP+ BMDC into the RPE layer and that these GFP+ human RPE65 expressing cells co-localized with murine RPE65. No GFP+ cells were observed in the neural retina or RPE layer of normal uninjured control eyes. CONCLUSIONS: Our study shows that systemically administered GFP+ RPE65-BMDC can reach the retina within minutes and that the majority of these BMDC are recruited to the injured RPE layer by 60 min post injection.

Castration-induced prostate epithelial cell apoptosis results from targeted oxidative stress attack of M1142 -macrophages.

Prostate development and function are regulated by androgens. Epithelial cell apoptosis in response to androgen deprivation is caspase-9-dependent and peaks at Day 3 after castration. However, isolated epithelial cells survive in the absence of androgens. Znf142 showed an on-off expression pattern in intraepithelial CD68-positive macrophages, with the on-phase at Day 3 after castration. Rats treated with gadolinium chloride to deplete macrophages showed a significant drop in apoptosis, suggesting a causal relationship between macrophages and epithelial cell apoptosis. Intraepithelial M1-polarization was also limited to Day 3, and the inducible nitric oxide synthase (iNOS) knockout mice showed significantly less apoptosis than wild-type controls. The epithelial cells showed focal DNA double-strand breaks (DSB), 8-oxoguanine, and protein tyrosine-nitrosylation, fingerprints of exposure to peroxinitrite. Cultured epithelial cells induced M1-polarization and showed focal DSB and underwent apoptosis. The same phenomena were reproduced in LNCaP cells cocultured with Raw 264.7 macrophages. In conclusion, the M1 142 -macrophage (named after Znf142) attack causes activation of the intrinsic apoptosis pathway in epithelial cells after castration.

Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration.

BACKGROUND: Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis (LAP) is involved and contribute to prevent inflammation. METHODS: Flow cytometry, RT-PCR and immunohistochemistry were used to characterize the macrophage subpopulation residing in the epithelial layer of the rat ventral prostate (VP) after castration. Stereology was employed to determine variations in the number of ED1 and ED2. Mice were treated with either chloroquine or L-asparagine to block autophagy. RESULTS: M1 (iNOS-positive) and M2 macrophages (MRC1+ and ARG1+) were not found in the epithelium at day 5 after castration. The percentage of CD68+ (ED1) and CD163+ (ED2) phenotypes increased after castration but only CD68+ cells were present in the epithelium. RT-PCR showed increased content of the autophagy markers Bcl1 and LC3 after castration. In addition, immunohistochemistry showed the presence of LC3+ and ATG5+ cells in the epithelium. Double immunohistochemistry showed these cells to be CD68+ /LC3+ , compatible with the LAP phenotype. LC3+ cells accumulate significantly after castration. Chloroquine and L-asparagine administration caused inflammation of the glands at day 5 after castration. CONCLUSIONS: CD68+ macrophages phagocytose apoptotic cell corpses and activate the LAP pathway, thereby contributing to the preservation of a non-inflammed microenvironment. Marked inflammation was detected when autophagy blockers were administered to castrated animals.

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes.

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin-induced DM1 and ligature-induced PD models. Increased IL-23 (80-fold) and Mmp8 expression (25-fold) was found in DM1. Ligature resulted in an IL-1ß/IL-6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non-diabetics. PD in DM1 involved IL-1ß (but not IL-6) and IL-23/IL-17, reduced IL-6 and IL-10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20-fold higher counts of neutrophils and macrophages. IL-23 and Mmp8 expression are hallmarks of DM1. In association with the IL-1/IL-6 (Th1) response in PD, one found a secondary IL-17 (Th17) pathway in non-diabetic rats. Low IL-6/TNF-α suggest that the Th1 response was compromised in DM1, while IL-17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD-associated IL-1/IL-6 (Th1), IL-10, and Reck expression are associated with the acute-to-chronic inflammation transition, which is lost in DM1. In conclusion, IL-23/IL-17 are associated with the PD progression in DM1.

Progress in kidney transplantation: The role for systems immunology.

The development of systems biology represents an immense breakthrough in our ability to perform translational research and deliver personalized and precision medicine. A multidisciplinary approach in combination with use of novel techniques allows for the extraction and analysis of vast quantities of data even from the volume and source limited samples that can be obtained from human subjects. Continued advances in microfluidics, scalability and affordability of sequencing technologies, and development of data analysis tools have made the application of a multi-omics, or systems, approach more accessible for use outside of specialized centers. The study of alloimmune and protective immune responses after solid organ transplant offers innumerable opportunities for a multi-omics approach, however, transplant immunology labs are only just beginning to adopt the systems methodology. In this review, we focus on advances in biological techniques and how they are improving our understanding of the immune system and its interactions, highlighting potential applications in transplant immunology. First, we describe the techniques that are available, with emphasis on major advances that allow for increased scalability. Then, we review initial applications in the field of transplantation with a focus on topics that are nearing clinical integration. Finally, we examine major barriers to adapting these methods and discuss potential future developments.

Reduced blood pressure in sickle cell disease is associated with decreased angiotensin converting enzyme (ACE) activity and is not modulated by ACE inhibition.

BACKGROUND: Sickle cell disease (SCD) incurs vaso-occlusive episodes and organ damage, including nephropathy. Despite displaying characteristics of vascular dysfunction, SCD patients tend to present relatively lower systemic blood pressure (BP), via an unknown mechanism. We investigated associations between BP and renin-angiotensin-system (RAS) components in SCD and determined whether an inhibitor of angiotensin converting enzyme (ACE; often used to slow SCD glomerulopathy) further modulates BP and RAS components in a murine model of SCD. METHODS: BP was compared in human subjects and mice with/without SCD. Plasma angiotensin II, ACE and renin were measured by immunoassay. BP was reevaluated after treating mice with enalapril (25 mg/kg, 5x/week) for 5 weeks; plasma and organs were stored for angiotensin II and ACE activity measurement, and quantitative real-time PCR. RESULTS: Diastolic BP and systolic BP were significantly lower in patients and mice with SCD, respectively, compared to controls. Reduced BP was associated with increased plasma renin and markers of kidney damage (mice) in SCD, as well as significantly decreased plasma ACE concentrations and ACE enzyme activity. As expected, enalapril administration lowered BP, plasma angiotensin II and organ ACE activity in control mice. In contrast, enalapril did not further reduce BP or organ ACE activity in SCD mice; however, plasma angiotensin II and renin levels were found to be significantly higher in enalapril-treated SCD mice than those of treated control mice. CONCLUSION: Relative hypotension was confirmed in a murine model of SCD, in association with decreased ACE concentrations in both human and murine disease. Given that ACE inhibition has an accepted role in decreasing BP, further studies should investigate mechanisms by which ACE depletion, via both Ang II-dependent and alternative pathways, could contribute to reduce BP in SCD and understand how ACE inhibition confers Ang II-independent benefits on kidney function in SCD.

Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells.

The vascular endothelial growth factor receptors (VEGFRs) can shape the neovascular phenotype of vascular endothelial cells when translocated to the nucleus, however the spatial and temporal changes in the intracellular distribution and translocation of VEGFRs to the nucleus and the organelles involved in this process is unclear. This study reports the effect of exogenous VEGF on translocation of VEGFRs and organelles in micro- and macrovascular endothelial cells. We showed that VEGF is responsible for: a rapid and substantial nuclear translocation of VEGFRs; VEGFR1 and VEGFR2 exhibit distinct spatial, temporal and structural translocation characteristics both in vitro and in vivo and this determines the nuclear VEGFR1:VEGFR2 ratio which differs between microvascular and macrovascular cells; VEGFR2 nuclear translocation is associated with the endosomal pathway transporting the receptor from Golgi in microvascular endothelial cells; and an increase in the volume of intracellular organelles. In conclusion, the nuclear translocation of VEGFRs is both receptor and vessel (macro versus micro) dependent and the endosomal pathway plays a key role in the translocation of VEGFRs to the nucleus and the subsequent export to the lysosomal system. Modulating VEGF-mediated VEGFR1 and VEGFR2 intracellular transmigration pathways may offer an alternative for the development of new anti-angiogenic therapies.

The origin of prostate gland-secreted IgA and IgG.

The prostate secretes immunoglobulin (Ig) A (IgA) and IgG; however, how immunoglobulins reach the secretion, where the plasma cells are located, whether immunoglobulins are antigen-specific and where activation of the adaptive response occurs are still unknown. Immune cells, including CD45RA+ cells, were scattered in the stroma and not organized mucosae-associated lymphoid-tissue. IgA (but not IgG) immunostaining identified stromal plasma cells and epithelial cells in non-immunized rats. Injected tetramethylrhodamine-IgA transcytosed the epithelium along with polymeric immunoglobulin receptor. Oral immunization with ovalbumin/mesopourous SBA-15 silica adjuvant resulted in more stromal CD45RA+/IgA+ cells, increased content of ovalbumin-specific IgA and IgG, and the appearance of intraepithelial CD45RA+/IgG+ cells. An increased number of dendritic cells that cooperate in other sites with transient immunocompetent lymphocytes, and the higher levels of interleukin-1ß, interferon-γ and transforming growth factor-ß, explain the levels of specific antibodies. Nasal immunization produced similar results except for the increase in dendritic cells. This immunomodulatory strategy seems useful to boost immunity against genitourinary infections and, perhaps, cancer.

Changes in MMPs and inflammatory cells in experimental gingivitis.

In periodontal disease, extensive disorganization of the extracellular matrix promotes the loss of adhesion between the teeth and periodontium. A previous study suggested a reduction in the area occupied by collagen in the gingiva, during the first week of periodontal disease induction, however, the remaining fibers were more compact and thicker. Therefore, it was decided to investigate which of the MMP-2, -9, -14 and RECK, an MMP inhibitor, were involved in these modifications taking place in early gingivitis induced by ligature. The results of gene expression analysis indicated no changes for RECK. MMP-14 showed a reduction at 7 days of inflammation, and there was an immediate increase in MMP-2 gene expression and enzymatic activity, apparently by the stimulation of resident cells such as fibroblasts. A peak of MMP-9 expression 5 days after ligature followed after the peak of enzymatic activity found two days earlier. This pattern was consistent with the kinetics of macrophage and neutrophil recruitment. Immunohistochemistry suggested that MMP-9 was produced by both resident and inflammatory cells. Based on this evidence, it is suggested that extracellular matrix remodeling is related to MMP-2 and -9 production and activation. This allowed us to conclude that the host inflammatory response represents a significant factor for the advance of periodontal diseases.

Lipocalina associada à gelatinase de neutrófilos (NGAL) e calprotectina no tecido laminar de equinos após obstrução jejunal, tratados ou não com hidrocortisona / Neutrophil gelatinase associated lipocalin (NGAL) and calprotectin in equine laminar tissue after jejunal obstruction, treated or not with hidrocortisone

A laminite é uma doença podal grave que acomete os equídeos, sendo responsável por intenso sofrimento. Neste estudo foram pesquisadas a presença de calprotectina por meio da imunoistoquímica, e de lipocalina associada à gelatinase de neutrófilos (NGAL), por zimografia, no tecido laminar do casco de equinos após obstrução intestinal. Os animais foram divididos em quatro grupos: Grupo controle (Gc), contendo sete animais normais, sem procedimento cirúrgico; Grupo Instrumentado (Gi), contendo cinco animais, os quais passaram por todo o procedimento cirúrgico sem sofrerem obstrução intestinal; Grupo Não Tratado (Gnt), contendo quatro equinos submetidos a obstrução intestinal do jejuno por distensão de balão intraluminal, sem tratamento; e Grupo Tratado (Gt), contendo quatro equinos submetidos a obstrução intestinal, e tratados preventivamente com hidrocortisona. Houve imunomarcação de calprotectina em todos os grupos experimentais, com aumento nos equinos do grupo distendido em relação ao Gc. Com relação ao NGAL, houve aumento também do Gnt e do Gi em relação ao Gc. O Gt não diferiu dos demais. Conclui-se que a distensão do intestino delgado pode promover acúmulos de leucócitos nos cascos de equinos e que o NGAL é um método viável para se detectar infiltração neutrofílica em equinos. Novos estudos deverão ser realizados para se verificar possível benefício anti-inflamatório da hidrocortisona no casco de equinos com obstrução intestinal.

Laminitis is a severe hoof condition in horses that may cause intense suffering. In this study, leukocyte infiltration in hoof laminar tissue was investigated in horses subject to intestinal obstruction using immunohistochemistry to detect calprotectin, and zymography to detect neutrophil gelatinase associated lipocalin (NGAL). There were four groups: the Control Group (Gc), with seven horses, without surgical procedures; the Sham-operated Group (Gi), with five horses that were subjected to surgical procedure without intestinal obstruction; the No Treat group (Gnt), with four horses subjected to intestinal obstruction (jejunal distention using an intraluminal balloon) without treatment; and Treated group (Gt), with four horses subjected to intestinal obstruction and treated with hydrocortisone. Positive calprotectin imunostaining was detected in all experimental groups, with increase cell counts in horses of the distended group compared with the control group. NGAL expression was increased in Gd compared with Gc e Gi. The Gt did not differ from the others. In conclusion, small intestine distension can promote leukocyte infiltration in equine hoof laminar tissue, and NGAL zymography was considered a useful method for leukocyte tissue detection in horses. New studies will be conducted to verify the possible beneficial anti-inflammatory effects of hydrocortisone in hoof of horses with intestinal obstruction.