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Author(s):  
Taylor L. T. Wherry ◽  
Rohana Dassanayake ◽  
Eduardo Casas ◽  
Shankumar Mooyottu ◽  
John P. Bannantine ◽  
...  

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of ruminant enteritis, targets intestinal macrophages. During infection, macrophages contribute to mucosal inflammation and development of granulomas in the small intestine which worsens as disease progression occurs. Vitamin D3 is an immunomodulatory steroid hormone with beneficial roles in host-pathogen interactions. Few studies have investigated immunologic roles of 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in cattle, particularly cattle infected with MAP. This study examined the effects of exogenous vitamin D3 on immune responses of monocyte derived macrophages (MDMs) isolated from dairy cattle naturally infected with MAP. MDMs were pre-treated with ± 100 ng/ml 25(OH)D3 or ± 4 ng/ml 1,25(OH)2D3, then incubated 24 hrs with live MAP in the presence of their respective pre-treatment concentrations. Following treatment with either vitamin D3 analog, phagocytosis of MAP by MDMs was significantly greater in clinically infected animals, with a greater amount of live and dead bacteria. Clinical cows had significantly less CD40 surface expression on MDMs compared to subclinical cows and noninfected controls. 1,25(OH)2D3 also significantly increased nitrite production in MAP infected cows. 1,25(OH)2D3 treatment played a key role in upregulating secretion of pro-inflammatory cytokines IL-1β and IL-12 while downregulating IL-10, IL-6, and IFN-γ. 1,25(OH)2D3 also negatively regulated transcripts of CYP24A1, CYP27B1, DEFB7, NOS2, and IL10. Results from this study demonstrate that vitamin D3 compounds, but mainly 1,25(OH)2D3, modulate both pro- and anti-inflammatory immune responses in dairy cattle infected with MAP, impacting the bacterial viability within the macrophage.


Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 100
Author(s):  
Raveendra R. Kulkarni ◽  
Carissa Gaghan ◽  
Javid Mohammed

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.


2022 ◽  
Author(s):  
Eduard Otto Roos ◽  
William Mwangi ◽  
Wilhelm Gerner ◽  
Ryan Waters ◽  
John A Hammond

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-colour, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve, TAV, TCM, TEM, TTE and γδ-TCR cells. This panel will improve our ability to examine T cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimised for other bovid species as many of these reagents are likely to cross react.


2022 ◽  
Vol 9 (1) ◽  
pp. 27
Author(s):  
Giovanni Cimmino ◽  
Stefano Conte ◽  
Mariarosaria Morello ◽  
Grazia Pellegrino ◽  
Laura Marra ◽  
...  

Background: Thrombosis with cardiovascular involvement is a crucial complication in COVID-19 infection. COVID-19 infects the host by the angiotensin converting enzyme-2 receptor (ACE2r), which is expressed in endothelial cells too. Thus, COVID-related thrombotic events might be due to endothelial dysfunction. IL-6 is one of the main cytokines involved in the COVID-19 inflammatory storm. Some evidence indicates that Vitamin D (VitD) has a protective role in COVID-19 patients, but the molecular mechanisms involved are still debated. Thus, we investigated the effect of VitD on Tissue Factor and adhesion molecules (CAMs) in IL-6-stimulated endothelial cells (HUVEC). Moreover, we evaluated levels of the ACE2r gene and proteins. Finally, we studied the modulation of NF-kB and STAT3 pathways. Methods: HUVEC cultivated in VitD-enriched medium were stimulated with IL-6 (0.5 ng/mL). The TF gene (RT-PCR), protein (Western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. Similarly, CAMs soluble values (ELISA) and ACE2r (RT-PCR and Western blot) levels were assessed. NF-kB and STAT3 modulation (Western blot) were also investigated. Results: VitD significantly reduced TF expression at both gene and protein levels as well as TF-procoagulant activity in IL-6-treated HUVEC. Similar effects were observed for CAMs and ACE2r expression. IL-6 modulates these effects by regulating NF-κB and STAT3 pathways. Conclusions: IL-6 induces endothelial dysfunction with TF and CAMs expression via upregulation of ACE2r. VitD prevented these IL-6 deleterious effects. Thus, it might be speculated that this is one of the hypothetical mechanism(s) by which VitD exerts its beneficial effects in COVID-19 infection.


2022 ◽  
Vol 23 (2) ◽  
pp. 848
Author(s):  
Rodrigo P. Silva-Aguiar ◽  
Diogo B. Peruchetti ◽  
Lucas S. Florentino ◽  
Christina M. Takiya ◽  
María-Paz Marzolo ◽  
...  

Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as “albumin-induced albumin endocytosis”. Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.


2022 ◽  
Author(s):  
Orest Tsymbalyuk ◽  
Volodymyr Gerzanich ◽  
Marc Simard ◽  
Chozha Rathinam

Abstract Pathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), remains completely unknown. Here, we report that DC- differentiation, maintenance and functions are altered at both early and late phases of TBI. Our studies identify that; 1. frequencies and absolute numbers of DCs in the spleen and BM are altered at both acute and late phases of TBI; 2. surface expression of key molecules involved in antigen presentation of DCs were affected both at early and late phases of TBI; 3. distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; 4. early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors, were deregulated after TBI; and 5. intracellular ROS levels were reduced in DC progenitors and differentiated DCs at both early and late phases of TBI. Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs at both early and late phases of TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries.


2022 ◽  
Vol 13 ◽  
Author(s):  
Woosuk Chung ◽  
Dian-Shi Wang ◽  
Shahin Khodaei ◽  
Arsene Pinguelo ◽  
Beverley A. Orser

Background: Perioperative neurocognitive disorders (PNDs) occur commonly in older patients after anesthesia and surgery. Treating astrocytes with general anesthetic drugs stimulates the release of soluble factors that increase the cell-surface expression and function of GABAA receptors in neurons. Such crosstalk may contribute to PNDs; however, the receptor targets in astrocytes for anesthetic drugs have not been identified. GABAA receptors, which are the major targets of general anesthetic drugs in neurons, are also expressed in astrocytes, raising the possibility that these drugs act on GABAA receptors in astrocytes to trigger the release of soluble factors. To date, no study has directly examined the sensitivity of GABAA receptors in astrocytes to general anesthetic drugs that are frequently used in clinical practice. Thus, the goal of this study was to determine whether the function of GABAA receptors in astrocytes was modulated by the intravenous anesthetic etomidate and the inhaled anesthetic sevoflurane.Methods: Whole-cell voltage-clamp recordings were performed in astrocytes in the stratum radiatum of the CA1 region of hippocampal slices isolated from C57BL/6 male mice. Astrocytes were identified by their morphologic and electrophysiologic properties. Focal puff application of GABA (300 μM) was applied with a Picospritzer system to evoke GABA responses. Currents were studied before and during the application of the non-competitive GABAA receptor antagonist picrotoxin (0.5 mM), or etomidate (100 μM) or sevoflurane (532 μM).Results: GABA consistently evoked inward currents that were inhibited by picrotoxin. Etomidate increased the amplitude of the peak current by 35.0 ± 24.4% and prolonged the decay time by 27.2 ± 24.3% (n = 7, P < 0.05). Sevoflurane prolonged current decay by 28.3 ± 23.1% (n = 7, P < 0.05) but did not alter the peak amplitude. Etomidate and sevoflurane increased charge transfer (area) by 71.2 ± 45.9% and 51.8 ± 48.9% (n = 7, P < 0.05), respectively.Conclusion: The function of astrocytic GABAA receptors in the hippocampus was increased by etomidate and sevoflurane. Future studies will determine whether these general anesthetic drugs act on astrocytic GABAA receptors to stimulate the release of soluble factors that may contribute to PNDs.


2022 ◽  
pp. petgeo2021-029
Author(s):  
Diveena Danabalan ◽  
Jon G. Gluyas ◽  
Colin G. Macpherson ◽  
Thomas H. Abraham-James ◽  
Josh J. Bluett ◽  
...  

Commercial helium systems have been found to date as a serendipitous by-product of petroleum exploration. There are nevertheless significant differences in the source and migration properties of helium compared with petroleum. An understanding of these differences enables prospects for helium gas accumulations to be identified in regions where petroleum exploration would not be tenable. Here we show how the basic petroleum exploration playbook (source, primary migration from the source rock, secondary longer distance migration, trapping) can be modified to identify helium plays. Plays are the areas occupied by a prospective reservoir and overlying seal associated with a mature helium source. This is the first step in identifying the detail of helium prospects (discrete pools of trapped helium). We show how these principles, adapted for helium, can be applied using the Rukwa Basin in the Tanzanian section of the East African Rift as a case study. Thermal hiatus caused by rifting of the continental basement has resulted in a surface expression of deep crustal gas release in the form of high-nitrogen gas seeps containing up to 10% 4He. We calculate the total likely regional source rock helium generative capacity, identify the role of the Rungwe volcanic province in releasing the accumulated crustal helium, and show the spatial control of helium concentration dilution by the associated volcanic CO2. Nitrogen, both dissolved and as a free gas phase, plays a key role in the primary and secondary migration of crustal helium and its accumulation into what might become a commercially viable gas pool. This too is examined. We identify and discuss evidence that structures and seals suitable for trapping hydrocarbon and CO2 gases will likely also be efficient for helium accumulation on the timescale of the Rukwa basin activity.The Rukwa Basin prospective recoverable P50 resources of helium have been independently estimated to be about 138 billion standard cubic feet (2.78 x 109 m3 at STP). If this volume is confirmed it would represent about 25% of the current global helium reserve. Two exploration wells Tai 1 and Tai 2 completed by August 2021 have proved the presence of seal and reservoir horizons with the reservoirs containing significant helium shows.This article is part of the Energy Geoscience Series available at https://www.lyellcollection.org/cc/energy-geoscience-series


2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Charlotte A. James ◽  
Yuexin Xu ◽  
Melissa S. Aguilar ◽  
Lichen Jing ◽  
Erik D. Layton ◽  
...  

AbstractT cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.


Antibodies ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 4
Author(s):  
Ashleigh J. Nicaise ◽  
Amye McDonald ◽  
Erin Rushing Sears ◽  
Trell Sturgis ◽  
Barbara L. F. Kaplan

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD’s effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD’s EAE disease attenuation.


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