Abstract
Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 ...antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.
Communication between cancer cells and the surrounding stromal cells of the tumor microenvironment (TME) plays a key role in promoting metastasis, which is the major cause of cancer death. Small ...membrane-bound particles called extracellular vesicles (EVs) are released from both cancer and stromal cells and have a key role in mediating this communication through transport of cargo such as various RNA species (mRNA, miRNA, lncRNA), proteins, and lipids. Tumor-secreted EVs have been observed to induce a pro-tumorigenic phenotype in non-malignant cells of the stroma, including fibroblasts, endothelial cells, and local immune cells. These cancer-associated cells then drive metastasis by mechanisms such as increasing the invasiveness of cancer cells, facilitating angiogenesis, and promoting the formation of the pre-metastatic niche. This review will cover the role of EV-mediated signaling in the TME during metastasis and highlight the therapeutic potential of targeting these pathways to develop biomarkers and novel treatment strategies.
The purpose of this study was to determine the mitochondrial content, and the oxidative and nitrosative stress of the placenta in women with gestational diabetes mellitus (GDM).
Full-term placentas ...from GDM and healthy pregnancies were collected following informed consent. The lipid peroxidation (TBARS) and oxidized protein (carbonyls) levels were determined by spectrophotometry, and 3-nitrotyrosine (3-NT), subunit IV of cytochrome oxidase (COX4), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and actin were determined by western blot, whereas ATPase activity was performed by determining the adenosine triphosphate (ATP) consumption using a High-performance liquid chromatography (HPLC) system.
TBARS and carbonyls levels were lower in the placentas of women with GDM compared with the normal placentas (p < 0.001 and p < 0.05, respectively). Also, 3-NT/actin and AMPK/actin ratios were higher in GDM placentas than in the normal placentas (p = 0.03 and p = 0.012, respectively). Whereas COX4/actin ratio and ATPase activity were similar between GDM placentas and those controls.
These data suggest that placentas with GDM are more protected against oxidative damage, but are more susceptible to nitrosative damage as compared to normal placentas. Moreover, the increased expression levels of AMPK in GDM placentas suggest that AMPK might have a role in maintaining the mitochondrial biogenesis at normal levels.
HGRL28072011 . Registered 28 July 2011.
Circulating biological markers, such as miRNAs, hold the greatest possibilities to complement tissue biopsy and clinical diagnostic tests. The objective of this study was to evaluate the relative ...abundance of three circulating miRNAs in serum from 17 HPV16‐positive patients with early cervical lesions known as Low‐Grade Squamous Intraepithelial Lesions (LSILs). The expression of circulating microRNAs miR‐15b, miR‐34a and miR‐218 in patients with LSILs was compared to 23 HPV‐negative individuals showing normal cervical epithelium (healthy women) and 23 Squamous Cell Carcinoma (SCC) samples. The expression levels of miR‐15b remained unchanged while those of miRNAs 34a and 218 were relatively high in serum obtained from LSIL patients in comparison with healthy women (results were statistically significant with a p of < 0.01 or < 0.001). According to previous findings, miR‐15b was overexpressed and miRNAs 34a and 218 were underexpressed in serum from SCC patients. Additionally, the mRNA expression levels of some selected gene targets were determined Cyclin D1 (CCND1), Cyclin E1 (CCNE1), B‐cell lymphoma 2 (Bcl‐2) and MutS homolog 2 (MSH‐2). All serum results correlated with tissue samples from the same patients. We propose that circulating microRNAs can be valuable as molecular markers for the early follow‐up of cervical carcinogenesis risk.
Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also known as IPS-1, MAVS, or VISA), the activation of the transcription factors NF-κB and IRF3, which ...collaborate to induce an antiviral type I interferon (IFN) response. FADD and RIP1, known as mediators of death-receptor signaling, are implicated in this antiviral pathway; however, the link between death-receptor and antiviral signaling is not known. Here we showed that TRADD, a crucial adaptor of tumor necrosis factor receptor (TNFRI), was important in RIG-like helicase (RLH)-mediated signal transduction. TRADD is recruited to Cardif and orchestrated complex formation with the E3 ubiquitin ligase TRAF3 and TANK and with FADD and RIP1, leading to the activation of IRF3 and NF-κB. Loss of TRADD prevented Cardif-dependent activation of IFN-β, reduced the production of IFN-β in response to RNA viruses, and enhanced vesicular stomatitis virus replication. Thus, TRADD is not only an essential component of proinflammatory TNFRI signaling, but is also required for RLH-Cardif-dependent antiviral immune responses.
Introduction/Aims
We studied a patient with a congenital myasthenic syndrome (CMS) caused by a dominant mutation in the synaptotagmin 2 gene (SYT2) and compared the clinical features of this patient ...with those of a previously described patient with a recessive mutation in the same gene.
Methods
We performed electrodiagnostic (EDX) studies, genetic studies, muscle biopsy, microelectrode recordings and electron microscopy (EM).
Results
Both patients presented with muscle weakness and bulbar deficits, which were worse in the recessive form. EDX studies showed presynaptic failure, which was more prominent in the recessive form. Microelectrode studies in the dominant form showed a marked reduction of the quantal content, which increased linearly with higher frequencies of nerve stimulation. The MEPP frequencies were normal at rest but increased markedly with higher frequencies of nerve stimulation. The EM demonstrated overdeveloped postsynaptic folding, and abundant endosomes, multivesicular bodies and degenerative lamellar bodies inside small nerve terminals.
Discussion
The recessive form of CMS caused by a SYT2 mutation showed far more severe clinical manifestations than the dominant form. The pathogenesis of the dominant form likely involves a dominant‐negative effect due to disruption of the dual function of synaptotagmin as a Ca2+‐sensor and modulator of synaptic vesicle exocytosis.
A. Pes cavus in a patient with a heterozygous mutation in the CB2 domain of synaptotagmin 2 (SYT2). B. Schematic view of SYT2 showing the identified mutation. C. Repetitive stimulation demonstrating presynaptic failure of neuromuscular transmission. D. Distorted neuromuscular junction showing intermingled extensions of the nerve terminal (black asterisks) and Schwann cell (white asterisks), endosomes (white arrowheads), coated pits (black arrows), and a lamellar body (LB). Markers are: 5 mV and 5 ms in C, and 0.6 μm in D.
Zika virus (ZIKV) can be vertically transmitted during pregnancy resulting in a range of adverse pregnancy outcomes. The decidua is commonly found to be infected by ZIKV, yet the acute immune ...response to infection remains understudied
. We hypothesized that
African-lineage ZIKV infection induces a pro-inflammatory response in the decidua. To test this hypothesis, we evaluated the decidua in pregnant rhesus macaques within the first two weeks following infection with an African-lineage ZIKV and compared our findings to gestationally aged-matched controls. Decidual leukocytes were phenotypically evaluated using spectral flow cytometry, and cytokines and chemokines were measured in tissue homogenates from the decidua, placenta, and fetal membranes. The results of this study did not support our hypothesis. Although ZIKV RNA was detected in the decidual tissue samples from all ZIKV infected dams, phenotypic changes in decidual leukocytes and differences in cytokine profiles suggest that the decidua undergoes mild anti-inflammatory changes in response to that infection. Our findings emphasize the immunological state of the gravid uterus as a relatively immune privileged site that prioritizes tolerance of the fetus over mounting a pro-inflammatory response to clear infection.
A successful pregnancy requires many physiological adaptations from the mother, including the establishment of tolerance toward the semiallogeneic fetus. Innate lymphoid cells (ILCs) have arisen as ...important players in immune regulation and tissue homeostasis at mucosal and barrier surfaces. Dimensionality reduction and transcriptomic analysis revealed the presence of two novel CD56
decidual ILCs that express low T-bet and divergent Eomes levels. Transcriptional correlation with recently identified first trimester decidual dNKs suggests that these novel decidual ILCs might be present throughout pregnancy. Functional testing with permutation analysis revealed production of multiple factors by individual cells, with a preference for IFNγ and VEGF. Overall, our data suggests continuity of a unique decidual innate lymphocytes across pregnancy with a polyfunctional functional profile conducive for pregnancy.
NBSGW mice are highly immunodeficient and carry a hypomorphic mutation in the
gene, providing a host environment that supports robust human hematopoietic expansion without pre-conditioning. These ...mice thus provide a model to investigate human hematopoietic engraftment in the absence of conditioning-associated damage. We compared transplantation of human CD34
HSPCs purified from three different sources: umbilical cord blood, adult bone marrow, and adult G-CSF mobilized peripheral blood. HSPCs from mobilized peripheral blood were significantly more efficient (as a function of starting HSPC dose) than either cord blood or bone marrow HSPCs at generating high levels of human chimerism in the murine blood and bone marrow by 12 weeks post-transplantation. While T cells do not develop in this model due to thymic atrophy, all three HSPC sources generated a human compartment that included B lymphocytic, myeloid, and granulocytic lineages. However, the proportions of these lineages varied significantly according to HSPC source. Mobilized blood HSPCs produced a strikingly higher proportion of granulocyte lineage cells (~35% as compared to ~5%), whereas bone marrow HSPC output was dominated by B lymphocytic cells, and cord blood HSPC output was enriched for myeloid lineages. Following transplantation, all three HSPC sources showed a shift in the CD34
subset towards CD45RA
progenitors along with a complete loss of the CD45RA
CD49f
long-term HSC subpopulation, suggesting this model promotes mainly short-term HSC activity. Mice transplanted with cord blood HSPCs maintained a diversified human immune compartment for at least 36 weeks after the primary transplant, although mice given adult bone marrow HSPCs had lost diversity and contained only myeloid cells by this time point. Finally, to assess the impact of non-HSPCs on transplantation outcome, we also tested mice transplanted with total or T cell-depleted adult bone marrow mononuclear cells. Total bone marrow mononuclear cell transplants produced significantly lower human chimerism compared to purified HSPCs, and T-depletion rescued B cell levels but not other lineages. Together these results reveal marked differences in engraftment efficiency and lineage commitment according to HSPC source and suggest that T cells and other non-HSPC populations affect lineage output even in the absence of conditioning-associated inflammation.
The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, ...the degree, timing, and subset-specific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques (
) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy.
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