Cardiomyocytes of newborn mice proliferate after injury or exposure to growth factors. However, these responses are diminished after postnatal day-6 (P6), representing a barrier to building new ...cardiac muscle in adults. We have previously shown that exogenous thyroid hormone (T3) stimulates cardiomyocyte proliferation in P2 cardiomyocytes, by activating insulin-like growth factor-1 receptor (IGF-1R)-mediated ERK1/2 signaling. But whether exogenous T3 functions as a mitogen in post-P6 murine hearts is not known. Here, we show that exogenous T3 increases the cardiomyocyte endowment of P8 hearts, but the proliferative response is confined to cardiomyocytes of the left ventricular (LV) apex. Exogenous T3 stimulates proliferative ERK1/2 signaling in apical cardiomyocytes, but not in those of the LV base, which is inhibited by expression of the nuclear phospho-ERK1/2-specific dual-specificity phosphatase, DUSP5. Developmentally, between P7 and P14, DUSP5 expression increases in the myocardium from the LV base to its apex; after this period, it is uniformly expressed throughout the LV. In young adult hearts, exogenous T3 increases cardiomyocyte numbers after DUSP5 depletion, which might be useful for eliciting cardiac regeneration.
In this review, we discuss the state of our knowledge as it relates to embryonic brain vascular patterning in model systems zebrafish and mouse. We focus on the origins of endothelial cell and the ...distinguishing features of brain endothelial cells compared to non-brain endothelial cells, which is revealed by single cell RNA-sequencing methodologies. We also discuss the cross talk between brain endothelial cells and neural stem cells, and their effect on each other. In terms of mechanisms, we focus exclusively on Wnt signaling and the recent developments associated with this signaling network in brain vascular patterning, and the benefits and challenges associated with strategies for targeting the brain vasculature. We end the review with a discussion on the emerging areas of meningeal lymphatics, endothelial cilia biology and novel cerebrovascular structures identified in vertebrates.
The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to ...impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.
Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase ...followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.
The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) ...establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection.
The 9-month-long chemotherapy of tuberculosis often results in poor compliance and emergence of drug-resistant strains. So, improved therapeutic strategy is urgently needed. Immunotherapy could be ...beneficial for the effective management of the disease. Previously we showed the protective efficacy of Mycobacterium indicus pranii (MIP) when given as prophylactic vaccine in animal models of tuberculosis.
We sought to investigate whether MIP can be used as an adjunct to the chemotherapy in guinea pig models of tuberculosis. Efficacy of MIP was evaluated when given subcutaneously or by aerosol.
MIP-therapy as an adjunct to the chemotherapy was found to be effective in accelerating bacterial killing and improving organ pathology. MIP-immunotherapy resulted in higher numbers of activated antigen-presenting cells and lymphocytes in the infected lungs and also modulated the granulomatous response. Early increase in protective Th1 immune response was observed in the immunotherapy group. Following subsequent doses of MIP, decrease in the inflammatory response and increase in the immunosuppressive response was observed, which resulted in the improvement of lung pathology.
MIP immunotherapy is a valuable adjunct to chemotherapy for tuberculosis. Aerosol route of immunotherapy can play a crucial role for inducing immediate local immune response in the lung.
Introduction: Dual specific phosphatases (DUSPs) are mitogen-activated protein kinase (MAPK) regulators, which also serve as drug targets for treating various vascular diseases. Previously, we have ...presented mechanistic characterizations of DUSP5 and its interaction with pERK, proposing a dual active site. Methods: Herein, we characterize the interactions between the DUSP5 phosphatase domain and the pT-E-pY activation loop of ERK2, with specific active site assignments. We also report the full NMR chemical shift assignments of DUSP5 that now enable chemical shift perturbation and dynamics studies. Results and Discussion: Both phosphates of the pT-E-pY tripeptide are dephosphorylated, based on 31 P NMR; but, steady state kinetic studies of the tripeptide both as a substrate and as an inhibitor indicate a preference for binding and dephosphorylation of the phospho-tyrosine before the phospho-threonine. Catalytic efficiency (k cat /K m ) is 3.7 M −1 S −1 for T-E-pY vs 1.3 M −1 S −1 for pT-E-Y, although the diphosphorylated peptide (pT-E-pY) is a better substrate than both, with k cat /K m = 18.2 M −1 S −1 . Steady state inhibition studies with the p NPP substrate yields K is values for the peptide inhibitors of: 15.82 mM (pT-E-Y), 4.932 mM (T-E-pY), 1.672 mM (pT-E-pY). Steady state inhibition studies with pNPP substrate and with vanadate or phosphate inhibitors indicated competitive inhibition with Kis values of 0.0006122 mM (sodium vanadate) and 17.32 mM (sodium phosphate), similar to other Protein Tyrosine Phosphatases with an active site cysteine nucleophile that go through a five-coordinate high energy transition state or intermediate. Molecular dynamics (MD) studies confirm preferential binding of the diphosphorylated peptide, but with preference for binding the pY over the pT reside in the catalytic site proximal to the Cys263 nucleophile. Based on MD, the monophosphorylated peptide binds tighter if phosphorylated on the Tyr vs the Thr. And, if the starting pose of the docked diphosphorylated peptide has pT in the catalytic site, it will adjust to have the pY in the catalytic site, suggesting a dynamic shifting of the peptide orientation. 2D 1 H- 15 N HSQC chemical shift perturbation studies confirm that DUSP5 with tripeptide bound is in a dynamic state, with extensive exchange broadening observed—especially of catalytic site residues. The availability of NMR chemical shift assignments enables additional future studies of DUSP5 binding to the ERK2 diphosphorylated activation loop. Summary: These studies indicate a preference for pY before pT binding, but with ability to bind and dephosphorylate both residues, and with a dynamic active site pocket that accommodates multiple tripeptide orientations.
Primary cilia from the brain microvascular endothelial cells (ECs) are specialized cell-surface organelles involved in mediating sensory perception, cell signaling, and vascular stability. ...Immunofluorescence (IF) analysis of human primary brain microvascular ECs reveals two cilia per cell. To confirm the
observation of the two-cilia phenotype in human primary brain ECs, ECs isolated from mouse brain were cultured and stained for cilium. Indeed, brain ECs from a ciliopathic mouse (polycystic kidney disease or
) also possess more than one cilium. Primary cilium emerges from the mother centriole. Centriole analysis by IF suggests that in brain ECs, markers for the mother and daughter centrioles stain both cilia, suggesting that the second cilium in brain ECs arises from the daughter centriole. Further quantification of cilia size in brain ECs revealed that cilia arising from the mother centriole are bigger in size compared with cilia from the daughter centriole. Cell cycle analyses using immunoblotting and flow cytometry suggest that the ciliary proteins ARL13B and IFT88 involved in brain EC ciliogenesis are highly expressed only in the G0/G1 and S phases of the cell cycle. The IF analyses of cells arrested at different cell cycle stages indicate that the two-cilia phenotype is highly specific to the G0/G1 phase. Our findings suggest that in addition to the mother centriole, the daughter centriole also plays a role in ciliogenesis in primary cultured ECs.
Visceral white adipose tissues (WAT) regulate systemic lipid metabolism and inflammation. Dysfunctional WAT drive chronic inflammation and facilitate atherosclerosis. Adipose tissue-associated ...macrophages (ATM) are the predominant immune cells in WAT, but their heterogeneity and phenotypes are poorly defined during atherogenesis. The scavenger receptor CD36 mediates ATM crosstalk with other adipose tissue cells, driving chronic inflammation. Here, we combined the single-cell RNA sequencing technique with cell metabolic and functional assays on major WAT ATM subpopulations using a diet-induced atherosclerosis mouse model ( Apoe -null). We also examined the role of CD36 using Apoe / Cd36 double-null mice. Based on transcriptomics data and differential gene expression analysis, we identified a previously undefined group of ATM displaying low viability and high lipid metabolism and labeled them as “unhealthy macrophages”. Their phenotypes suggest a subpopulation of ATM under lipid stress. We also identified lipid-associated macrophages (LAM), which were previously described in obesity. Interestingly, LAM increased 8.4-fold in Apoe / Cd36 double-null mice on an atherogenic diet, but not in Apoe -null mice. The increase in LAM was accompanied by more ATM lipid uptake, reduced adipocyte hypertrophy, and less inflammation. In conclusion, CD36 mediates a delicate balance between lipid metabolism and inflammation in visceral adipose tissues. Under atherogenic conditions, CD36 deficiency reduces inflammation and increases lipid metabolism in WAT by promoting LAM accumulation.
Since 2000, a resurgence of syphilis has been noted in many developed and developing countries, especially among men who have sex with men (MSM). Incidence and prevalence of syphilis in pregnant ...women have been reduced drastically by mandatory screening in early pregnancy. Insufficient data in other populations especially from developing countries limit targeted public health interventions. This study aimed to describe the clinical and epidemiological profile of serologically confirmed syphilis cases among the non-pregnant high-risk group reporting to a tertiary care center in Southern India. A retrospective study was carried out in a tertiary care center in Southern India for 6 years from 2015 to 2020. A total of 265 serologically confirmed syphilis patients were included. A statistically significant increase in positivity from 0.52 to 2.1% was observed in this study (2015 to 2020). Among risk factors, high-risk behavior with multiple heterosexual partners was the commonest (51.3%), followed by marital partners who tested positive (9.4%) and MSM (7.5%). The majority of the patients were diagnosed at the latent stage (79%), followed by secondary syphilis (10%) and tertiary syphilis (8%). A quarter of patients (23%) were coinfected with HIV. Serological non-responsiveness was more common among HIV infected (47 vs. 24%). Sixteen had neurosyphilis and six had ocular involvement. HIV co-infection complicated 50% (8/16) of neurosyphilis patients. Syphilis is still prevalent, especially in high-risk groups including those are attending STI clinics. Further prospective multicentric studies are needed to identify and implement public health measures.
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