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With a wide variety of prostate supplements on the market today, there’s rising interest in natural ways to promote prostate health. Prostate supplements are used to treat symptoms including painful urination, painful ejaculation and pelvic discomfort.
Prostate supplements can be taken as vitamins, minerals, tea, smoothies, and other different forms. These supplements offer various benefits and have proven to be beneficial and necessary.
Several things can have an impact on the prostate’s health. Men can develop many prostate-related conditions, including prostatitis and benign prostatic hyperplasia (BPH). An interest in prostate supplements has increased significantly to encourage awareness around male health.
These products are said to enhance prostate health and reduce many prostate-related matters. The prostate is one of the many prostate supplements that have proven to fight against several prostate-related conditions while reducing any chance of active cancer cells.
These supplements have shown an impressive effect on the development of age-related macular degeneration and heart disease, and encourage strong bones. Buy Prostadine to eliminate any risks of gambling with your health.
Supplements for Prostate health typically contain a mix of vitamins, herbal extracts,minerals, and other compounds. These supplements have shown evidence to reduce BPH symptoms and improve urinary flow while reducing the volume of residual urinary post-void. Common supplement ingredients include:
One of the most common ingredients in prostate supplements is the saw palmetto berry. Many of the chemicals found in saw palmetto are known to offer health advantages. The berry of the plant is where most of the nutrients come from. These berries prevent the formation of a hormone that contributes to prostate growth.
Beta-sitosterol is a plant sterol and is used in many prostate supplements. Clinical trials indicate that it might reduce urinary symptoms of BPH and improve urinary flow rates.
Extracts of flower pollen are rich in several minerals and vitamins. These include sterols, enzymes, amino acids, flavonoids, carotenoids, lipids and more. These components provide both immediate and long-term health advantages that will be noticeable in strengthening the immune system and reducing enlarged prostate glands.
Pygeum Africanum, an extract from the African plum tree, is utilised for its anti-inflammatory properties and its ability to improve urinary symptoms related to BPH.
Extracts from the root of the stinging nettle plant are often incorporated because of their anti-inflammatory and analgesic properties. They are mainly used for symptomatic relief in cases of BPH.
Pumpkin seeds offer a high dose of zinc which helps with prostate health. Phytosterols can also be found in pumpkin seeds. The strong elements of Phytosterols fight hormones that build prostate cells and maintain a healthy prostate.
Vitamin D is one of the key ingredients in producing any form of medication related to prostate health. It is known to eliminate any growth relating to cancer cells in the prostate.
Zinc is an important mineral in assisting male fertility. A healthy prostate is an indication of high concentrations of zinc and prostate health conditions are often associated with a lack of zinc.
Vitamin E plays a key role in protecting the body’s cells from any health concerns. It helps reduce oxidative stress, which can potentially lead to the progression of various chronic diseases, including cancer.
It’s important to be knowledgeable of the drawbacks that can be experienced before taking medication regularly. Prostate supplements often offer a safety disclaimer. However, every medication has possible side effects that need to be carefully observed.
It’s important to note that supplements aren’t strictly monitored by the Food and Drug Administration (FDA) compared to medication. Users might face potential harm from incorrect dosages or the presence of potentially harmful ingredients.
Supplements vary according to each individual. However, there have been common side effects such as headaches, dizziness, and sometimes even gastrointestinal problems.
These side effects could worsen if they’re taken with other medications. Any decision to start on prostate supplements should involve a discussion with healthcare providers considering a user’s medical history and concurrent medications.
Prostate supplements can improve prostate health, but they shouldn’t be considered an exclusive solution. For many individuals, lifestyle changes can significantly improve prostate health. Maintaining a healthy diet, regular exercise and moderating the intake of fluids can help manage symptoms of BPH.
Medical treatments for prostate issues are continually developing and improving. Taking supplements regularly will improve your prostate and overall health. However, should you experience severe cases of prostate health issues, please consider prescription medication, minimally invasive therapies and surgeries.
Dietary supplements and vitamins are a type of complementary and alternative medicine (CAM) treatment that can be used instead of or in addition to conventional medical procedures. To guarantee a healthy prostate, CAM also contains certain meals, herbs, minerals, and vitamins.
While prostate supplements promise a natural and non-invasive solution to manage prostate issues, their actual efficacy and safety need consideration. Although they can contribute towards better prostate health, they’re not a substitute for comprehensive medical care or healthy living practices.
It’s wise to always stay informed about prostate health and get information from credible healthcare providers. Prostate supplements are accessible to alleviate mild symptoms, but not to form part of a cure for serious health concerns.


The feature FX Stack > KeyFramer (or other FX) > Add to the FX Bin is quite useful, except that the list of my saved Keyframer FX added to the FX Bin is now a very long , cluttered list. I know I can Export them to save them in a separate folder until such time as I want to Import them again , and I know I can delete individual FX in the Bin that I no longer need , but is there a way to Add an FX to the Bin for only a specific project , but then when I open a new project the same FX will not be saved in the FX Bin of the new project ? I don't necessarily need these saved FX in the FX Bin to be in every project , you know ? It's because there are certainly some FX I want to save to the FX Bin to re-use later, but I'm trying to cut down on the clutter in the list of saved FX in the Bin.
Another Feature/Improvement request:
When I want to save an FX by Exporting it , it is annoying that the name of the FX in the Export window is "Untitled" and must be renamed , even through in the FX Bin I've already named it. So for example , let's say I have a KeyFramer FX that I've added to the FX Bin named SC_05_Slow_Pan_v2 ... I have that FX currently in the FX Stack and now I want to Export it. When I click on Export the name shows as Untitled , which means I have to retype in SC_05_Slow_Pan_v2 . This seems like it should be unnecessary, since the FX already has a name assigned in the FX Bin so Exporting the FX it should have the same name (with the option to rename it if desired).
By the way, where are these individual FX saved in the FX Bin stored ? Is it possible to delete them all at the same time , instead of deleting them one at a time ?
This section is dedicated to the feature & improvement requests (be sure what you are asking does not exist yet in TVPaint Animation )
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What Is a Medical Credit Card?
In today’s world, medical expenses can be a burden for many individuals and families. To help manage these costs, medical credit cards have emerged as a convenient financing option. In this article, we will explore the concept of a medical credit card, how it works, and its benefits for patients seeking healthcare services.
Chapter 1: Understanding Medical Credit Cards
What is a Medical Credit Card?
Learn about the definition and purpose of a medical credit card. Understand how it differs from traditional credit cards and personal loans.
How Do Medical Credit Cards Work?
Discover the mechanics of medical credit cards, including the application process, credit limits, and repayment options. Gain insights into the interest rates and fees associated with these cards.
Chapter 2: Benefits of Medical Credit Cards
Access to Immediate Medical Care
Highlight how medical credit cards provide patients with the ability to access necessary medical treatments and procedures without delay. Emphasize the importance of timely healthcare services.
Flexible Payment Options
Discuss the flexibility that medical credit cards offer in terms of repayment plans. Explain how patients can choose affordable monthly installments that fit their budget.
Special Financing and Promotional Offers
Explore the various special financing options and promotional offers that medical credit cards may provide. Showcase how these features can help patients save on interest and manage their healthcare expenses effectively.
Chapter 3: Considerations and Tips for Using Medical Credit Cards
Carefully Review Terms and Conditions
Advise readers to carefully review the terms and conditions of medical credit cards before applying. Highlight the importance of understanding interest rates, repayment periods, and any potential penalties or fees.
Verify Acceptance by Healthcare Providers
Encourage patients to ensure that their healthcare providers accept medical credit cards as a payment method. Provide tips for verifying acceptance in advance to avoid any inconvenience.
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Educate readers on responsible credit card usage practices, such as making timely payments and keeping track of expenses. Promote the importance of maintaining a good credit score.
Chapter 4: Alternatives to Medical Credit Cards
Health Savings Accounts (HSAs) and Flexible Spending Accounts (FSAs)
Introduce HSAs and FSAs as alternatives to medical credit cards. Explain how these accounts allow individuals to set aside pre-tax funds for medical expenses.
Payment Plans with Healthcare Providers
Highlight the option of negotiating payment plans directly with healthcare providers. Explain how this approach may offer more flexibility and potentially lower costs.
Medical credit cards can be a valuable financial tool for individuals seeking healthcare services. By understanding how these cards work, exploring their benefits, and considering alternative options, patients can make informed decisions about managing their medical expenses. Remember to review terms and conditions, verify acceptance by healthcare providers, and use credit responsibly. With careful consideration and responsible usage, a medical credit card can help individuals access necessary medical care while effectively managing their finances.

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Cats are amazing creatures, and they can be wonderful pets. Being a cat owner is an enjoyable venture in life. Keep reading to learn more about cat care.
If you plan on getting a cat for a pet, try looking into a local shelter. They have tons of amazing cats who desperately need a home. Adopting a cat this way helps to save their life and cut down on unwanted animal births.
Be certain your cat has regular vet visits. They should go at least once yearly for a check-up, and more often if they need any shots. Make vet appointments immediately if your cat has a medical condition or has become injured.
Prevent your cat from getting zapped by coating exposed wires with bitter apple. Most office stores sell a tube that you can stick all of your wires and cords through if the spray doesn’t work. The tube from a used-up roll of paper towels makes an excellent shield for loose cords. Whenever you aren’t using any thin, electronic cords, you should store them away.
The correct location is important when picking a spot for a cat’s litter box. Never put the box near your cat’s food, and pick a quiet place away from areas that are heavily used. Make sure it’s properly ventilated to keep smell down. This will keep both you and your cat happy.
Male felines tend to have a build-up of crystals that present in the urine, so you need to avoid this with proper nutrition. If they have to pass the crystals it can cause the cat a lot of pain and paying a vet to help with this can cost you a lot of money. Choose a cat food low in magnesium. This means you should pay attention to labels on food. Fish products can have more magnesium when compared to poultry.
Brush your cat often. This can help spread their fur’s natural oils around and stimulate blood flow to their skin. It is also a good way to reduce shedding. Loose hair causes hairballs, a common problem for cats. The hair enters the pet’s stomach and they later cough it up.
Acclimate your cat to a cat carrier. You cannot punish a cat the same way you would a dog. Cats learn well when they feel encouraged. Place a beloved blanket or item inside the carrier and let it stay open near the cat’s usual haunts. After some time, your cat will respond better to the carrier. This takes the hassle out of traveling with your cat in its carrier.
To help avoid a finicky cat, feed your cat different brands of cat food. If you raise your kitten with the same food for their entire life, this can become the only type of food they will willingly eat.
Plan on spending the money for good cat food that contains the nutrients that your cat needs. Check out the ingredients, and make sure it includes protein, such as meat, as the first item in the list. If the food uses fillers like corn and other things that are not protein, that’s not a sign of the good nutrition a cat needs. This type of food should be avoided. You have a carnivorous cat, and a diet consisting of animal proteins is essential.
Moist, canned food can be a very good option for your cat. Dry food is less expensive, but there are added benefits to be had in canned cat food. Canned food is rich in protein and fat and also has a higher water content. It’s also much softer on older teeth if your cat is getting up there in years. Speak with your vet, however generally speaking, canned cat food is better for your cat.
As you know already, it can be very rewarding to be a cat owner. But they should be treated as the special species they are. They behave differently and have different nutritional needs. Read through this article a couple of times to soak up all of the info it contains. Your cat will thank you!

domain: pet


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ORIGINAL RESEARCH article
Sec. NK and Innate Lymphoid Cell Biology
Volume 10 - 2019 | https://doi.org/10.3389/fimmu.2019.02469
Pregnancy-Induced Alterations in NK Cell Phenotype and Function
- 1Department of Medicine, Stanford University, Palo Alto, CA, United States
- 2Department of Stanford Immunology Program, Stanford University, Palo Alto, CA, United States
- 3Department of Statistics, Stanford University, Palo Alto, CA, United States
- 4Department of Pediatrics, Stanford University, Palo Alto, CA, United States
- 5Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
- 6Department of Obstetrics and Gynecology, Stanford Prevention Research Center, Stanford University School of Medicine, Stanford University, Palo Alto, CA, United States
- 7Department of Microbiology and Immunology, Stanford University, Palo Alto, CA, United States
- 8Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, United States
- 9Chan Zuckerberg Biohub, San Francisco, CA, United States
Pregnant women are particularly susceptible to complications of influenza A virus infection, which may result from pregnancy-induced changes in the function of immune cells, including natural killer (NK) cells. To better understand NK cell function during pregnancy, we assessed the ability of the two main subsets of NK cells, CD56dim, and CD56bright NK cells, to respond to influenza-virus infected cells and tumor cells. During pregnancy, CD56dim and CD56bright NK cells displayed enhanced functional responses to both infected and tumor cells, with increased expression of degranulation markers and elevated frequency of NK cells producing IFN-γ. To better understand the mechanisms driving this enhanced function, we profiled CD56dim and CD56bright NK cells from pregnant and non-pregnant women using mass cytometry. NK cells from pregnant women displayed significantly increased expression of several functional and activation markers such as CD38 on both subsets and NKp46 on CD56dim NK cells. NK cells also displayed diminished expression of the chemokine receptor CXCR3 during pregnancy. Overall, these data demonstrate that functional and phenotypic shifts occur in NK cells during pregnancy that can influence the magnitude of the immune response to both infections and tumors.
During pregnancy, the immune system has to finely balance its activity in order to tolerate the semi-allogeneic fetus, while maintaining the ability to fight microbial challenges (1–4). These immune alterations may be at least partially responsible for the increased susceptibility of pregnant women to complications from influenza virus infection (5–9). Recent studies have demonstrated enhanced responses to influenza virus by several innate immune cell subsets during pregnancy, including monocytes, plasmacytoid dendritic cells and natural killer (NK) cells (2, 10–14). It remains unclear whether such changes could contribute to the enhanced pathogenesis of influenza virus during pregnancy because the role of NK cells in the pathogenesis of influenza virus remains controversial. Several mouse studies have shown that NK cell depletion or the use of mice deficient in NK cells improved the outcome of influenza infection (15, 16), suggesting that NK cell activity may be pathogenic in the setting of influenza infection. On the contrary, NK cells reduced influenza virus burden and promoted clearance of the virus in mice deficient in NKp46, a major NK cell receptor thought to play a role in influenza recognition (17), suggesting that NK cells may contribute to protection from influenza. Controversy remains as another mouse strain deficient in NKp46 expression is resistant to viral infection (18). In humans, NK cells were found in abundance in the lungs of fatally infected patients with the 2009 H1N1 pandemic strain of influenza virus (19). This NK cell recruitment correlated with severity of lung inflammation and poor patient outcome, but the causality in the relation between infiltration of NK cells and viral clearance and pathogenesis is unproven.
NK cells mediate their response to influenza and other pathogens using an array of germline receptors. Inhibitory receptors serve to protect healthy cells from NK cells and include the killer-cell immunoglobulin-like receptors (KIRs) and the heterodimer NKG2A-CD94. NK cell activating receptors signal ‘altered self' and include NKp46, NKp30, NKp44, NKG2C, and NKG2D, among others. Together, the activating and inhibitory receptors define the degree of NK cell maturation and responsiveness to stimuli (20, 21). In response to virus-infected or cancerous cells, NK cells can kill cells via release of cytolytic molecules or through engagement of death receptors. They can also produce cytokines, such as IFN-γ, which limit viral replication and tumor proliferation (21). CD56dim and CD56bright NK cells are two major NK cell subsets identified in the peripheral blood that tend to differ in their responsiveness. CD56dim NK cells are more cytotoxic and CD56bright are better at secreting cytokines (22, 23). Due to their robust cytotoxic capabilities and immune regulatory potential, NK cell activation is tightly regulated to limit tissue damage at the site of infection. Here, we sought to better understand how NK cell activity is regulated during pregnancy and gain insight into the unusual susceptibility of pregnant women to complications from influenza virus and other infections. We used mass cytometry and ex vivo influenza infection to profile the expression of NK cell activating and inhibitory receptors during this critical period of development.
Materials and Methods
Pregnant women in their second and third trimester and control non-pregnant women were enrolled in two cohorts in separate years. In the discovery cohort, twenty-one healthy pregnant women were recruited between October 2013 and March 2014 from the Obstetrics Clinic at Lucile Packard Children's Hospital at Stanford University. Twenty-one non-pregnant (control) women were recruited for Stanford influenza vaccine studies (NCT numbers: NCT03020537, NCT03022422, and NCT02141581). In the validation cohort, 32 non-pregnant (control) women were recruited for Stanford vaccine studies (NCT numbers: NCT01827462 and NCT03022422) and 21 healthy pregnant women were recruited between October 2012 and March 2013 from the Obstetrics Clinic at Lucile Packard Children's Hospital at Stanford. Venous blood was collected from all participants at baseline; pregnant women also provided a sample at 6 weeks post-partum. Exclusion criteria included concomitant illnesses, immunosuppressive medications, or receipt of blood products within the previous year. Pregnant women were also excluded for known fetal abnormalities and morbid obesity (pre-pregnancy body mass index >40). This study was performed in accordance with the Declaration of Helsinki and approved by the Stanford University Institutional Review Board (IRB-25182); written informed consent was obtained from all participants. Blood from anonymous healthy donors at the Stanford blood bank center was obtained for confirmatory functional assays.
PBMC Isolation, Cryopreservation, and Cell Purification for Functional Assays
PBMCs from healthy donors were isolated from whole blood by Ficoll-Paque (GE Healthcare) and cryopreserved in 90% fetal bovine serum (Thermo Scientific)/10% dimethyl sulfoxide (Sigma-Aldrich). Cryopreserved PBMCs were thawed and washed with complete RP10 media [RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Life Technologies)] and 50 U/mL benzonase (EMD Millipore). NK cells and/or monocytes were sorted using Sony sorter SH800 (Sony) with the following antibodies: CD3-Allophycocyanine (clone OKT3; BioLegend), CD14-Brilliant Violet 421 (clone HCD14; BioLegend), CD19-Alexa Fluor 488 (clone HIB19; Biolegend), and CD56-Phycoerythrin Cyanine 7 (clone NCAM; BioLegend).
NK Cell: Infected Monocyte Co-culture
A/California/7/2009 influenza (pH1N1) wild-type influenza A virus obtained from Kanta Subbarao at the National Institutes of Health was propagated in embryonated chicken eggs. Monocytes were washed and re-suspended in serum-free RPMI media at 1 × 105 per 100 μL and infected at a multiplicity of infection (MOI) of 3 for 1 h at 37°C with 5% carbon dioxide. One-hour post-infection, viral inoculum was removed and cells were resuspended in 100 μL of complete RP10. Autologous NK cells were then exposed to pH1N1-infected monocytes at a effector:target (E:T) ratio 1:1. After a further 2-h incubation, 2 μM monensin, 3 μg/mL brefeldin A (eBiosciences), and anti-CD107a-allophycocyanin-H7 (BD Pharmingen) were added to the co-culture for 4 h, followed by cell staining for flow cytometry analysis.
K562 Cell Assay
Following purification, NK cells were exposed to K562 tumor cells (ATCC) at an effector:target (E:T) ratio of 1:1. Immediately following co-incubation, 2 μM monensin, 3 μg/mL brefeldin A, and anti-CD107a-allophycocyanin-H7 were added to the co-culture for 4 h, followed by cell staining for flow cytometry analysis.
Cell Staining and Flow-Cytometry Analysis
Cells were stained with LIVE/DEAD fixable Aqua Stain (Life Technologies), followed by surface staining and then fixed and permeabilized with FACS Lyse and FACS Perm II (BD Pharmingen) according to the manufacturer's instructions. Cells were stained with anti-CD3-PE or -APC, anti-CD16-PerCPCy5.5 (clone 3G8; BioLegend), anti-IFNγ-FITC or V450 (clone B27; BD Biosciences), anti-CD56-PEcy7, or anti-CD14-APC or -APC-H7 and fixed using 1% paraformaldehyde. Uncompensated data were collected using a three-laser MACSQuant® Analyser (Miltenyi). Analysis and compensation were performed using FlowJo flow-cytometric analysis software, version 9.9.4 (Tree Star).
Antibody Labeling for CyTOF
Purified antibodies (lacking carrier proteins) were labeled 100 μg at a time according to instructions provided by DVS Sciences with heavy metal-preloaded maleimide-coupled MAXPAR chelating polymers and as previously described (24, 25). Qdot antibodies purchased from Invitrogen were used for Cd112 and were not conjugated. In115, Gd155, and Gd157 were ordered from Trace Sciences and conjugated with exactly as with metals purchased from DVS Sciences. Following labeling, antibodies were diluted in PBS to a concentration between 0.1 and 0.3 mg/mL. Each antibody clone and lot was titrated to optimal staining concentrations using cell lines and primary human samples. The gating shown in Figures S2, S4, S6, S8 displays one individual as an example. Gates were set based on both positive and negative controls known to express markers, and all stains were validated by comparison to conventional flow cytometry, as described in our prior studies (20, 26). Cell subsets known to not express markers were used as negative controls in many cases (for instance, B cells do not express many NK cell markers). For some stains such as NKG2C, as new antibody conjugations and panels were used for the second cohort, the gating strategy modified if better ability to distinguish populations was possible. Gating was not used as part of the GLM analysis.
PBMC Staining for CyTOF Acquisition
Cryopreserved PBMCs from non-pregnant and pregnant women in discovery and validation cohort were thawed and cells were transferred to 96-well deep-well-plates, resuspended in 25 μM cisplatin (Enzo Life Sciences) for 1 min and quenched with 100% serum. Cells were stained for 30 min, fixed (BD FACS Lyse), permeabilized (BD FACS Perm II), and stained with intracellular antibodies for 45 min on ice. Staining panels are described in Tables S3, S4. All antibodies were conjugated using MaxPar X8 labeling kits (DVS Sciences). Cells were suspended overnight in iridium intercalator (DVS Sciences) in 2% paraformaldehyde in phosphate-buffered saline (PBS) and washed 1 × in PBS and 2 × in H2O immediately before acquisition on a CyTOF-1 (Fluidigm).
Modeling of Predictors of Pregnancy in Mass Cytometry Data
To identify markers that were consistently changed during pregnancy, we used a generalized linear model (GLM) with bootstrap resampling to account for the donor-specific heterogeneity. We implemented the GLM approach and other regression models in an open source R package CytoGLMM (27) available here: https://christofseiler.github.io/CytoGLMM/.
Linear discriminant analyses were implemented in R using the package MASS (28, 29). Statistical analyses for functional experiments were performed using GraphPad Prism, version 6.0d (GraphPad Software). A Mann-Whitney U-test was used to compare control to pregnant women and a Wilcoxon signed-rank was used to compare the paired data in women between pregnancy and the post-partum period.
Mass cytometry data supporting this publication is available at ImmPort (https://www.immport.org) under study accession SDY1537.
NK Cell Immune Response to Influenza Virus During Pregnancy
To investigate how pregnancy alters NK cell phenotype and function, we recruited two cohorts of pregnant and non-pregnant (control) women in subsequent years (Tables S1, S2). We assessed NK cell antiviral function during pregnancy by flow cytometry after exposing sorted NK cells to autologous infected monocytes [Figure 1A; (12, 30)]. We observed that the frequency of CD56dim NK cells expressing CD107a, a marker of cytolytic activity, and IFN-γ was significantly greater in pregnant women than in controls or in post-partum women (Figures 1B,C and Figure S1A). Similarly, the frequency of CD56bright NK cells expressing CD107a and IFN-γ was also significantly greater during pregnancy than in controls and post-partum (Figures 1D,E and Figure S1A). Bulk NK cells from pregnant women displayed enhanced killing of influenza-infected monocytes (Figure 1F and Figure S1B). These data demonstrate that the two major NK cell subsets have enhanced responses to influenza-virus infected cells during pregnancy.
Figure 1. CD56dim and CD56bright NK cell immune response to influenza infected and tumor cells during pregnancy. (A) PBMCs from controls (N = 10), pregnant women (N = 10), and post-partum women (N = 10) in discovery cohort were isolated from blood samples. Monocytes and total NK cells were sorted and monocytes were infected with the H1N1 influenza virus strain. NK cells were either exposed to H1N1-infected monocytes or to K562 tumor cells for 7 or 4 h, respectively. (B–I) CD56dim and CD56bright NK cell immune response was then determined by flow cytometry. The frequency of (B) CD107a- and (C) IFN-γ-expressing CD56dim NK cells in response to influenza-infected monocytes is represented. The frequency of (D) CD107a- and (E) IFN-γ-expressing CD56bright NK cells in response to influenza-infected monocytes is represented. (F) The frequency of dead or dying monocytes based on staining with viability dye in NK cell co-culture. The frequency of CD107a (G) and IFN-γ-production (H) by CD56dim NK cells in response to K562 cells is represented. The frequency of CD107a (I) and IFN-γ-production (J) by CD56bright NK cells in response to K562 cells. (K) The frequency of dead or dying K562 tumor cells based on staining with viability dye in NK cell co-culture. *P < 0.05, **P < 0.01, and ***P < 0.001 (Mann–Whitney U-Tests to compare controls vs. pregnant; Wilcoxon matched-paired test to compare pregnant vs. post-partum).
NK Cell Immune Response to Cancer Cells During Pregnancy
During pregnancy, monocytes respond more robustly to influenza virus (11) which could activate NK cells through inflammatory cytokine production, potentially explaining enhanced NK cell responses. We hypothesized that if NK cell function was intrinsically elevated during pregnancy, we should observe enhanced anti-tumor responses as well. We therefore exposed sorted total NK cells from controls and pregnant women to the K562 tumor cell line (Figure 1A), which represents a homogenous, identical target for NK cells from controls and pregnant women. CD56dim NK cells from pregnant women had 1.6-fold greater expression of CD107a than CD56dim NK cells from non-pregnant women in response to K562 cells (Figure 1G and Figure S1C), though IFN-γ responses were not significantly different (Figure 1H and Figure S1C). CD56bright NK cells also displayed enhanced degranulation (Figure 1I and Figure S1C) but no difference in IFN-γ production (Figure 1J and Figure S1C). This increased degranulation by both NK cell subsets from pregnant women resulted in enhanced killing of K562 cells by bulk NK cells (Figure 1K and Figure S1D). These data indicate that NK cells have an intrinsically enhanced ability to kill both infected and tumor targets during pregnancy.
Deep Profiling of CD56dim and CD56bright NK Cells During Pregnancy in the Discovery Cohort
To understand potential drivers of this enhanced NK cell function during pregnancy, we profiled the expression patterns of inhibitory and activating surface receptors on NK cells in control non-pregnant women, pregnant women, and post-partum women (including the 10 individuals per group tested in Figure 1). PBMCs in both cohorts were evaluated by mass cytometry as outlined in Figure 2A and Tables S3, S4. NK cells were identified as CD3−CD19−CD20−CD14−CD56dim/brightCD16+/− cells (Figure S2A). The frequency of NK cells did not significantly differ between pregnant and control women, nor in pregnant vs. post-partum women in either cohort (Figures S2B,C). To identify NK cell markers predictive of pregnancy, we used a Generalized Linear Model (GLM) with bootstrap resampling to account for correlations between cells and inter-individual variability [Figure 2A; (27)].
Figure 2. Deep profiling of CD56dim and CD56bright NK cells in non-pregnant and pregnant women from discovery cohort. (A) PBMCs from controls (N = 21), pregnant women (N = 21), and post-partum women (N = 21) in discovery cohort were isolated from blood samples. PBMCs were isolated and stained for mass cytometry. Data obtained were analyzed using a linear regression model, GLM. (B,C) Markers predictive of CD56dim NK cells in control vs. pregnant women in discovery cohort were assessed by GLM with bootstrap resampling. The markers are listed on the y-axis and the x-axis represents the log-odds that the marker expression levels predict the outcome (control on the left vs. pregnancy on the right). Summary data are depicted, showing the 95% confidence interval. Markers in which the bar does not cross zero are significantly predictive of one state vs. the other. (D) Represents the linear discriminant analysis (LDA) for CD56dim NK cells between non-pregnant, pregnant, and post-partum samples from discovery cohort. (E,F) Markers predictive of CD56bright NK cells in control vs. pregnant women in discovery cohort were assessed by GLM with bootstrap resampling. The markers are listed on the y-axis and the x-axis represents the log-odds that the marker expression levels predict the outcome (control on the left vs. pregnancy on the right). Summary data are depicted, showing the 95% confidence interval. Markers in which the bar does not cross zero are significantly predictive of one state vs. the other. (G) Represents the linear discriminant analysis (LDA) for CD56bright NK cells between non-pregnant, pregnant, and post-partum samples from discovery cohort.
Expression of several markers such as CD38, NKp46, PD-1, and CD27 were predictive of pregnancy on CD56dim NK cells, while NKp30 was more likely to predict control (Figure 2B). When comparing the same women during pregnancy and post-partum, CD38, NKp46, NKG2C, NKG2D, and NKp44 were predictive of pregnancy on CD56dim NK cells (Figure 2C). Manual gating confirmed elevated expression of CD38 and NKp46 on CD56dim NK cells during pregnancy (Figures S3, S4). To further define the markers that distinguish pregnancy, a linear discriminant analysis (LDA) was performed, revealing that CD38 and NKp46 best separate the CD56dim NK cell population of pregnant women from that of control and post-partum women (Figure 2D). Together, our data indicate that there are differences in NK receptor expression patterns during pregnancy, and that CD38 and NKp46 expression are major drivers of these pregnancy-related changes.
As CD56bright NK cells differ from CD56dim NK cells in their maturation and receptor expression patterns, we analyzed them separately. CD38 and NKp46 expression levels are also predictive of pregnancy on CD56bright NK cells, as is the inhibitory receptor NKG2A, which is highly expressed on CD56bright NK cells (Figure 2E and Figures S5, S6). Expression of the chemokine receptor, CXCR3, and activating receptor, NKp44, were associated with non-pregnant state. Similar differences were seen when comparing pregnant and post-partum samples (Figure 2F and Figures S5, S6). LDA reveals that CD38, NKp46, NKG2A, and NKG2D best separate CD56bright NK cells of pregnant women from that of control and post-partum women (Figure 2G). Together, these data suggest that during pregnancy, both CD56dim and CD56bright NK cell subsets have the potential for greater activation through an increased expression of CD38 and NKp46.
Deep Profiling of CD56dim and CD56bright NK Cells in the Validation Cohort
We performed a deeper profiling of NK cells in the validation cohort, using an antibody panel including an increased number of specific NK cell receptors such as KIRs (Figure 2A and Table S4). Similar to the discovery cohort, CD38 and NKp46 are predictive of pregnancy on CD56dim NK cells compared to controls (Figure 3A). CD56dim NK cells from pregnant women also display an increased expression of NKG2C, LILRB1, and KIR2DL3 compared to control, while NKG2D and CD11b expression predicted control. CD38, NKG2A, and CD244 expression are also predictive of pregnancy when compared with post-partum conditions, while several markers including KIRs predicted the post-partum state (Figure 3B). NKp46 predicted the post-partum state among CD56dim NK cells in the validation cohort (Figure 3B). Manual gating confirmed the results of the GLM for this cohort (Figures S7, S8). LDA performed on these data showed that CD38 and NKG2A best explained the separation between CD56dim NK cells from pregnant women with controls and post-partum in the validation cohort (Figure 3C).
Figure 3. Deep profiling of CD56dim and CD56bright NK cells in non-pregnant and pregnant women from validation cohort. (A,B) Markers predictive of CD56dim NK cells in control vs. pregnant women in validation cohort were assessed by GLM with bootstrap resampling. The markers are listed on the y-axis and the x-axis represents the log-odds that the marker expression levels predict the outcome (control on the left vs. pregnancy on the right). Summary data are depicted, showing the 95% confidence interval. Markers in which the bar does not cross zero are significantly predictive of one state vs. the other. (C) Represents the linear discriminant analysis (LDA) for CD56dim NK cells between non-pregnant, pregnant and post-partum samples from validation cohort. (D,E) Markers predictive of CD56bright NK cells in control vs. pregnant women in validation cohort were assessed by GLM with bootstrap resampling. The markers are listed on the y-axis and the x-axis represents the log-odds that the marker expression levels predict the outcome (control on the left vs. pregnancy on the right). Summary data are depicted, showing the 95% confidence interval. Markers in which the bar does not cross zero are significantly predictive of one state vs. the other. (F) Represents the linear discriminant analysis (LDA) for CD56bright NK cells between non-pregnant, pregnant and post-partum samples from validation cohort.
For CD56bright NK cells, CD11b, CD38, LILRB1, CD25, KIR2DL3, NKG2A, and NKG2C are predictive of pregnancy, while several markers predict the post-partum state (Figures 3D,E). These data were confirmed by manual gating (Figures S9, S10). LDA separation showed that CD38, NKp30, CD94, and CD244 most contribute to the separation of CD56bright NK cells from pregnant women compared to controls and post-partum (Figure 3F). Several markers differed in their predictions between the discovery and validation cohorts. For instance, NKG2C was predictive of pregnancy in comparison to control among both CD56dim and CD56bright NK cells in the validation cohort, but not in the discovery cohort. This raises the possibility that there are differences in CMV status between cohorts driving the effect. Unfortunately, CMV serologies were not available; however, there were no significant differences in the frequency of “adaptive” NKG2C+CD57+ NK cells between the control, pregnant, or post-partum women in either cohort, making it less likely that differences in CMV status were driving the differences in NK cell phenotype (Figure S11). Overall, the most consistent finding in pregnancy is the increased expression of CD38 on both CD56dim and CD56bright NK cells. There is significant variation in the expression patterns of activating and inhibitory NK cell receptors during pregnancy, but pregnancy is associated with a higher activation status and enhanced CD38 expression.
Co-expression of CD38 and NKp46
As the most consistently observed difference was enhanced expression of CD38 and NKp46 on CD56dim NK cells during pregnancy, we examined the frequency of CD56dim NK cells co-expressing these markers (Figure 4). CD38 and NKp46 were co-expressed on a greater frequency of NK cells both the discovery (Figures 4A,B) and validation cohorts during pregnancy (Figures 4C,D). In the discovery cohort, the frequency of CD38highNKp46+ NK cells returned to levels found in controls during the post-partum period, but in the validation cohort, the frequency of CD38highNKp46+ NK cells remained high in the post-partum period. There was no significant association between the frequency of CD38highNKp46+ NK cells and “adaptive” NKG2C+CD57+ NK cells (Figure S11).
Figure 4. NKp46 and CD38 co-expression in NK cells from controls, pregnant and post-partum women in the discovery and validation cohort. (A) Representative mass cytometry plots showing the co-expression of NKp46 and CD38 in peripheral NK cells from a control, pregnant or post-partum women in the discovery cohort. (B) Frequency of CD38+NKp46+ NK cells from controls, pregnant and post-partum individuals in the discovery cohort. ***P < 0.001 (Mann–Whitney U-Tests to compare controls vs. pregnant; Wilcoxon matched-paired test to compare pregnant vs. post-partum). (C) Representative mass cytometry plots showing the co-expression of NKp46 and CD38 in peripheral NK cells from a control, pregnant, or post-partum women in the validation cohort. (D) Frequency of CD38+NKp46+ NK cells from controls, pregnant and post-partum individuals in the validation cohort. *P < 0.05 (Mann–Whitney U-Tests to compare controls vs. pregnant; Wilcoxon matched-paired test to compare pregnant vs. post-partum).
During pregnancy, the maternal immune system is engaged in a fine balance: tolerance is required to preserve the fetus while defenses must be maintained to protect mother and baby from microbial challenges. NK cells play a critical role in this balance as their job is to patrol the body for “altered self” (31). NK cell activity had been thought to be suppressed during pregnancy to protect the fetus, but recent studies have suggested a more nuanced view (2). NK cells from pregnant women display diminished responses to stimulation with cytokines and phorbol-myristate acetate and ionomycin, yet NK cell responses to influenza-infected cells are enhanced (12–14, 32). Here we show that both CD56dim and CD56bright NK cell subsets have enhanced responses to both the influenza virus and to cancer cells, indicating a cell-intrinsic enhancement in their response to threats. Profiling CD56dim and CD56bright NK cells from pregnant and non-pregnant women showed that during pregnancy, both subsets are characterized by increased expression of the activation marker, CD38. CD38 is expressed on a large proportion of NK cells even in non-pregnant individuals and is significantly increased in cell surface density during pregnancy. CD56dim NK cells also demonstrate increased expression of the activating receptor NKp46 during pregnancy (though it is even higher in the post-partum period in one study); this receptor may play a role in recognition of influenza-infected cells (33, 34). These observations indicate that NK cells have an enhanced expression of receptors that mark NK cell activation and contribute to the response to influenza virus and cancer cells.
Pregnant women are significantly more likely to suffer adverse consequences from influenza infection than are the general population. During the 1918 influenza pandemic, the case fatality rate for influenza infection was estimated to be 27–75% among pregnant women but only 2–3% among the general population (35). Even with improved supportive care, the case-fatality rate among pregnant women was twice that of the general population during the 2009 pandemic (36). Thus, an understanding of the mechanisms driving this enhanced susceptibility to influenza infection during pregnancy represents an important challenge for the scientific community. During influenza virus infection, the recruitment of peripheral NK cells into the lungs represents one of the first lines of defense following influenza infection (37). Though isolated NK cells stimulated with cytokines or chemicals have suppressed responses during pregnancy, our data here confirm earlier findings that NK cell responses to autologous influenza-infected cells are enhanced during pregnancy (12). This enhanced responsiveness could be deleterious to lung integrity and drive pathogenesis. Consistent with this idea, Kim et al. demonstrated that pregnant mice infected by influenza virus have increased lung inflammation and damage compared to non-pregnant mice (38). Further, Littauer et al. suggested that innate immune responses play a role in the initiation of pregnancy complications such as preterm birth and stillbirth following influenza virus infection (5). Finally, the idea that enhanced NK cell responses could be detrimental in pregnant women is consistent with observations that hyperinflammatory responses are a driving force behind severe influenza disease in humans (39–41).
To deepen our understanding of the effect of pregnancy on NK cell responses, we turned to mass cytometry to profile the expression of NK cell surface receptors. We were surprised to discover that both CD56dim and CD56bright NK cell subsets had a consistent and significant increase in CD38 expression during pregnancy compared to non-pregnant and post-partum samples. While CD38 is commonly viewed as an activation marker on T cells, it is more highly expressed on NK cells and has several important functions. First, CD38 confers lymphocytes with the ability to adhere to endothelial cells through its binding to CD31, a necessary step in extravasation. CD38 also functions as an ectoenzyme, converting extracellular NAD+ to cADPR through its cyclase activity or cADPR to Adenosyl-di-phosphate ribose through its hydrolase activity (42). These molecules in turn can diffuse into the cell and promote its activation by driving intracellular calcium increase, phosphorylation of signaling molecules, production of cytokines, and vesicular transport (43). CD38 crosslinking can enhance the cytotoxic activity of cytokine-activated NK cells (44–46) and plays a role in immune synapse formation in T cells (47) and NK cells (Le Gars et al., unpublished data). Thus, this increased CD38 expression during pregnancy might explain the enhanced responses of NK cells to influenza and tumor cells. Interestingly, decidual NK cells express high levels of CD38 compared to peripheral NK cells, yet their origin is still unclear (48). It has been proposed that subsets of NK cells can migrate from the maternal blood to the decidua and acquire the unique features of decidual NK cells upon exposure to decidual environment (49, 50). Our data suggest that the overall environment during pregnancy could enhance CD38 expression. Several studies suggest that KIR2DL4 could play a significant role in regulating IFN-γ production by decidual NK cells (51–53). Further, an NK cell population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, is characterized by high expression of the receptors NKG2C and LILRB1 (54). This NK cell population has open chromatin around the enhancers of IFNG and VEGF genes, which leads to an increased production of IFN-γ and VEGF upon activation. This is consistent with our finding that NKG2C and LILRB1 expression is increased in our validation cohort, and could explain the increased activation of peripheral NK cells upon encounter with infected or tumor cells during pregnancy.
Another interesting finding is the consistent increased expression of NKp46 on CD56dim NK cells during pregnancy. Intriguingly, in the validation cohort, NKp46 levels were even higher on CD56dim NK cells during the post-partum period. NKp46 has been shown to contribute to NK cell influenza virus responses through binding of influenza hemagglutinin (34). Signaling mediated by NKp46 following influenza sensing leads to the production of IFN-γ (33, 55). Therefore, an increased expression of NKp46 during pregnancy could make NK cells more responsive to influenza virus. Further, more elevated expression of NKp46 facilitates the control of lung cancer in mice (56) and NKp46 alteration is associated with tumor progression in human gastric cancer (57). Thus, the increased expression of NKp46 on CD56dim NK cells, together with CD38, could explain the enhanced response to cancer cells during pregnancy. Two factors limited our ability to directly attribute the enhanced expression of CD38 and NKp46 to NK cell hyperresponsiveness during pregnancy. First, we did not have sufficient PBMC samples from pregnant women to perform blocking experiments. Second, even with enough material, CD38 and NKp46 are expressed on NK cells from non-pregnant women as well, albeit at lower levels, thus blocking would be expected to diminish responses in both pregnant and non-pregnant women.
Several markers differed in their expression pattern during pregnancy in only one cohort, and there was significant variation in the expression patterns of some markers between cohorts. For instance, NKG2D cells was predictive of pregnancy in the discovery cohort and predictive of post-partum/control in the validation cohort. This may reflect the substantial differences in NK cell phenotype between individuals. In earlier work we noted that NK cell receptor expression profiles, particularly those of activating receptors, differed dramatically between identical twins and based on maturation status, suggesting that these expression patterns are influenced by the environment (20, 26). This high variation between individuals may also explain our failure to observe consistent pregnancy-related changes in NKp30 or NKp44 expression. Other changes associated with pregnancy, including expression patterns of LILRB1, KIR3DL2, and KIR2DL5 were only evaluated in the validation cohort and warrant follow-up in future studies. Changes in NKG2C expression observed on CD56dim NK cells between the pregnant and control subjects could reflect differences in the CMV status between the cohorts in the cross-sectional analyses; unfortunately, CMV status is not known for the cohorts. An additional feature that was observed in the discovery cohort, but unfortunately not evaluated in the validation cohort, was decreased expression of CXCR3 on CD56bright NK cells during pregnancy. CXCR3, through the binding to its ligand IP-10, is an important receptor responsible for the recruitment of NK cells to the site of infection or inflammation. The CXCR3/IP-10 axis has been shown to enhance acute respiratory distress syndrome (ARDS) by the increased systemic presence of IP-10 (58). Thus, decreased level of CXCR3 on NK cells fails to explain their enhanced responses during pregnancy but could represent a mechanism of protection to avoid an excessive recruitment of CD56bright NK cells to the lung of influenza-infected pregnant women and to restrain lung damage.
Why does NK cell phenotype undergo such changes during pregnancy? The answer remains unclear. The presence of fetal antigens in maternal blood could explain the increased activation state of NK cells. Monocytes and dendritic cells exert a pro-inflammatory phenotype during pregnancy (2, 11, 14) and this could be in part due to parental antigens present in the fetus. In turn, monocytes and pDCs could produce several cytokines such as IL-15,−18, or IFN-type I to promote increased NK cell receptor expression and activate NK cells (59). Another possibility to explain the observed phenotypic changes of NK cells during pregnancy is hormonal variation. These fluctuations could promote transcriptomic and epigenetic modifications driving alteration of NK cell phenotype and response to influenza virus and tumor cells. However, several studies suggest that progesterone and estrogen dampen NK cell cytotoxic activities (60, 61). A deep analysis of the transcriptomic and epigenetic landscape of NK cells during pregnancy could lead to a better understanding of these NK cell changes.
There are several limitations of our study, including the fact that our mass cytometry panels differed between the two cohorts and remain limited to ~40 markers. Thus, we may have excluded other molecules involved in NK cell immune responses during pregnancy, including critical NK cell surface molecules such as DNAM-1, TIGIT, and Siglec-7. We also did not follow-up on other differences that were seen in only one cohort. Further, here we studied peripheral blood NK cells and were not able to sample lung resident NK cells or uterine NK cells. Finally, we had limited data reflecting the history of the pregnant and control women in terms of their prior vaccination status, prior influenza infection status, cigarette and drug use, and others. We cannot exclude that unmeasured factors could influence the NK cell phenotype and the quality of the NK cell responses to influenza and cancer cells.
Here, our goal was to refine current understanding of NK cell biology and activity in the context of pregnancy and influenza virus infection. Our work reveals enhanced activity of both CD56dim and CD56bright NK cell subsets to influenza-infected cells and tumor cells during pregnancy. These enhanced responses are associated with a more robust expression of CD38, a receptor that plays a role in activation and cytotoxicity, and NKp46, a receptor associated with a better response to influenza virus and certain cancers. Together, our data provide a more complete view of the immune changes mediated by pregnancy and enhances our understanding of the susceptibility of pregnant women to influenza virus.
Data Availability Statement
Mass cytometry data supporting this publication is available at ImmPort (https://www.immport.org) under study accession SDY1537.
The studies involving human participants were reviewed and approved by Stanford University Institutional Review Board. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
ML, AK, NB, and CB designed experiments. ML, AK, and NB, and CS analyzed the data. LM, SS-O, and PK collaborated and provided advice in the analysis of the data. MD, CD, GS, and NA coordinated and provided human samples. ML and CB wrote the manuscript. All authors contributed revisions and edits.
This was supported by an Elizabeth and Russell Siegelman Fellowship in Infectious Diseases from the Stanford Child Health Research Institute (CHRI) to AK, a Stanford CHRI post-doctoral fellowship to ML, the CHRI – Stanford Clinical and Translational Science Award grant number UL1 TR000093 (AK), a National Institutes of Health (NIH) Training Grant: Viral Infections in Children T32 AI78896-05 (AK), a Smith Family Stanford Graduate Fellowship (NB), Ruth L. Kirschstein NRSA 1F31HD089675 (NB), a Clinical Scientist Development Award #2013099 from the Doris Duke Charitable Foundation (CB), the McCormick Faculty Award (CB), Tasha and John Morgridge Endowed Faculty Scholar in Pediatric Translational Medicine from Stanford CHRI and Stanford University School of Medicine (CB), a NIH Director's New Innovator Award DP2AI112193 (CB), an Infrastructure and Opportunity Fund (CB) as part of the Stanford Human Immunology Project Consortium (HIPC) Grant U19AI090019 (MD), and an investigator award from the Chan Zuckerberg Biohub (CB). Clinical cohorts were supported by NIH U19AI057229 (MD) and an NIH/NCRR CTSA award UL1 RR025744 (H. Greenberg).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank our study volunteers for their participation, Sally Mackey for regulatory and data management, Sue Swope for consenting and conducting study visits, and the staff of the Stanford Vaccine Program for overall study coordination. This manuscript has been released as a Pre-Print at Biorxiv (62).
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2019.02469/full#supplementary-material
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54. Gamliel M, Goldman-Wohl D, Isaacson B, Gur C, Stein N, Yamin R, et al. Trained memory of human uterine NK cells enhances their function in subsequent pregnancies. Immunity. (2018) 48:951–62.e5. doi: 10.1016/j.immuni.2018.03.030
55. Glasner A, Isaacson B, Viukov S, Neuman T, Friedman N, Mandelboim M, et al. Increased NK cell immunity in a transgenic mouse model of NKp46 overexpression. Sci Rep. (2017) 7:13090. doi: 10.1038/s41598-017-12998-w
56. Shi L, Li K, Guo Y, Banerjee A, Wang Q, Lorenz UM, et al. Modulation of NKG2D, NKp46, and Ly49C/I facilitates natural killer cell-mediated control of lung cancer. Proc Natl Acad Sci USA. (2018) 115:11808–13. doi: 10.1073/pnas.1804931115
57. Han B, Mao F-Y, Zhao Y-L, Lv Y-P, Teng Y-S, Duan M, et al. Altered NKp30, NKp46, NKG2D, and DNAM-1 expression on circulating NK cells is associated with tumor progression in human gastric cancer. J Immunol Res. (2018) 2018:6248590. doi: 10.1155/2018/6248590
58. Ichikawa A, Kuba K, Morita M, Chida S, Tezuka H, Hara H, et al. CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin. Am J Respirat Critic Care Med. (2013) 187:65–77. doi: 10.1164/rccm.201203-0508OC
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Keywords: NK cells, pregnancy, influenza virus, cancer cells, NK repertoire
Citation: Le Gars M, Seiler C, Kay AW, Bayless NL, Starosvetsky E, Moore L, Shen-Orr SS, Aziz N, Khatri P, Dekker CL, Swan GE, Davis MM, Holmes S and Blish CA (2019) Pregnancy-Induced Alterations in NK Cell Phenotype and Function. Front. Immunol. 10:2469. doi: 10.3389/fimmu.2019.02469
Received: 23 May 2019; Accepted: 03 October 2019;
Published: 23 October 2019.
Edited by:Henrik Mei, Deutsches Rheuma-Forschungszentrum (DRFZ), Germany
Reviewed by:Kanutte Huse, Oslo University Hospital, Norway
Quirin Hammer, Karolinska Institute (KI), Sweden
Bertram Bengsch, Freiburg University Medical Center, Germany
Copyright © 2019 Le Gars, Seiler, Kay, Bayless, Starosvetsky, Moore, Shen-Orr, Aziz, Khatri, Dekker, Swan, Davis, Holmes and Blish. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

domain: medical


French Open round up: Nadal imperious in Paris as Djokovic battles through
Novak Djokovic and Rafael Nadal advanced to the fourth round of the French Open in sharply contrasting fashion on Friday, the Serbian reigning champion battling hard to avoid an upset after his long-term rival produced another exhibition of Roland Garros mastery.
Nadal annihilated Nikoloz Basilashvili as he targets a 10th title in Paris and 15th overall, but it was a very different affair for Djokovic, who twice had to come from behind to beat Diego Schwartzman in a five-set thriller.
David Goffin and Guillermo Garcia-Lopez both retired hurt and Grigor Dimitrov bowed out, but Milos Raonic and Dominic Thiem remain in the hunt for a quarter-final berth.
TWO BAGELS AND A BAGUETTE FOR RAFA
If Nadal is the King of Clay, Roland Garros must be his favourite palace. And the Spaniard held court in supremely commanding fashion on Friday, beating the unfortunate Basilashvili 6-0 6-1 6-0, the most comprehensive victory of his illustrious career at the grand slam venue in Paris.
Georgian Basilashvili could only hit five winners in the match, which lasted – by slam standards – a particularly brisk 94 minutes.
“The score is quite embarrassing but I have to accept it,” said Basilashvili, before departing the throne room, as Roberto Bautista Agut prepares to petition the sovereign in the fourth round, having seen off Jiri Vesely in straight sets.
75e victoire pour Rafa à Roland-Garros


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Saz feels bad as she is the only member of the group who is still a virgin so the others help her chat up the boy of her dreams, bookworm Joe. With Rob and Anna away for the night, Viva arranges for Saz and Joe to do the deed at their flat. In the meantime, Viva and Holli admit that none has gone all the way so, to rectify matters, they all invite boys over for the night. Unfortunately, Rob and Anna return early.
don @ minifie-1

domain: entertainment


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Ted Cruz States He Is a ‘Huge Follower’ In Bitcoin Mining
The Texas Bitcoin mining market is fortunate to have the support of Sen. Ted Cruz.
Cruz, who revealed his withdrawal from the Republican governmental race in February, was on Fox News Radio today promoting the advantages Bitcoin mining is giving the only star state.
Describing how Bitcoin can assist energy grids throughout batches of severe weather condition, Cruz mentioned “I am a huge follower in Bitcoin mining and cryptocurrency.”
” Bitcoin mining can improve the resiliency of the grid,” the senator continued, discussing how makers can quickly power down in “times of crisis,” ending up being “emergency situation tanks of power.”
Texas is going through a bout of harsh weather condition at the minute. Recording its 2nd most popular summertime on record, the state’s regional grid– which provides power to 25 million Texans, approximately 90% of the state– was required to carry out emergency situation steps.
These steps implied Bitcoin miners required to cut their energy intake, creating countless dollars in profits to some mining business due to how their power purchase arrangements with the energy function.
Cruz included that while Bitcoin mining itself produces a great deal of worth, in times of alarming weather, these makers can be shut down “in seconds.” He stated that this can “maximize energy for health centers and to warm individuals’s houses,” staunchly safeguarding the market.
He went on to tweet: “Texas is a center for tasks, development, and liberty,” concluding that the state is “a natural sanctuary for Bitcoin mining.”
” Senator Cruz is really on point with Bitcoin,” stated Dennis Porter, co-founder and CEO of the Satoshi Act Fund, a non-profit that supporters for favorable policymaking with concerns to Bitcoin mining.
Porter informed Decrypt that although he does not constantly concur with Cruz, nor do a number of his pro-Bitcoin circle left wing, they “like his position on Bitcoin.”
A Texas senator because 2013, Cruz has actually revealed he will be running for senatorial re-election next year.
Politicians from both sides of the U.S. aisle have actually been gradually heating up to Bitcoin and digital possessions.
A Number Of Republican governmental hopefuls have honestly backed some kind of cryptocurrency, whether through legal procedures or by calling out SEC chairman Gary Gensler. These consist of Florida Guv Ron DeSantis, business owner Vivek Ramaswamy, and Senator Tim Scott of South Carolina.
Democrats may be a bit hesitant when it pertains to honestly revealing assistance for crypto, however there are some noteworthy backers. One example is the Senator from New York City, Kirsten Gillibrand, who co-sponsored an expense with Republican Senator Cynthis Lummis that would develop specific crypto guidelines.
Porter described that he is “delighted to see the variety of pro-BTC governmental prospects due to the fact that it reveals simply just how much the area has actually grown.”
The policy supporter informed Decrypt that “wise political leaders have actually observed that Bitcoin is a winning concern,” concluding that these political leaders “can draw in a brand-new kind of citizen and donor by lining up with Bitcoin and guaranteeing the U.S.A. leads on this innovation.”
There are strong critics in Washington D.C., nevertheless. Significantly, Senator Elizabeth Warren leads the charge, in addition to Senator Brad Sherman from California, both Democrats. Republican Senator Roger Marshall has actually likewise been tagged as a member of the “Anti-Crypto Army,” calling it a “nationwide security risk.”


This Vietnam syndrome must come to an end in Afghanistan. It is reminiscent of a slot machine gambler who pours money into the machine, hoping to get the jackpot that never materializes, leaving the machine exasperated and broke. Neither Bush nor Obama learned the bitter lessons of Vietnam, and both poured money and resources into a failing enterprise with no end in sight.
Unless the Pakistani army planners see the disastrous errors of their ways and decide to roll back both the theory and practice of their jihadist enterprise, there will likely be more attacks like the one on Bacha Khan University. Blaming Afghanistan for the Pakistan army's sins and dereliction may be a good excuse, but it makes for a horrible solution to Pakistan's terrorism conundrum.
While relatively few Afghans are anxious to see the Taliban's return, many seem willing to believe their promises to govern differently than in the past. Incidents like the strike on Kunduz's Doctors Without Borders hospital by American gunships can also serve to channel anger against a Kabul regime reliant on foreign troops.
General Hamid Gul was the military equivalent of Osama bin Laden. He died with his boots on and blood of the innocents on his hands. One must never speak ill of the dead; it is the jihadist life and legacy of General Hamid Gul, however, which is impossible to ignore if further bloodshed and mayhem in Pakistan and the region is to be averted.
KABUL, Afghanistan (AP) -- The new leader of the Afghan Taliban vowed to continue his group's bloody, nearly 14-year insurgency in an audio message re...
The height of the Pakistani state's chutzpah is that it does not only harbor these terrorists for decades and unleash them on the neighbors and the world, but also that it wants to be given credit and a thank you note even when America or Allah takes them out. The fundamental question about Mullah Omar's death in Karachi is who in Pakistan knew about his presence there, when did they know it and what, if anything at all, did they do about it.
A second round of peace talks between Afghan government officials and Taliban representatives, expected to begin before the end of July, 2015, suggests that some parties to the fighting want to declare a ceasefire. But even in the short time since the first round on July 7, fighting has intensified.


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MORE foreign portfolio investment flowed into the Philippines in June, but the total was not enough to pull the year-to-date total from a negative position, data released by the Bangko Sentral ng Pilipinas (BSP) on Thursday showed.
A net inflow of $79.56 million in foreign portfolio investments — or hot money — entered the country in June, the biggest since it registered a net inflow of $301.33 million in January, the BSP numbers showed.
In May, the Bangko Sentral noted that there was a net outflow of $24.35 million compared with $450.87 million a year earlier.
Total inflows amounted to $2.01 billion in June, up 35.8 percent from $1.48 billion in May and by 11.4 percent from $1.80 billion in June 2016.
“This may be attributed to positive investor sentiment relative to the anticipated resolution of the conflict in Marawi City, accelerated net foreign buying, and approval by Congress of the first tax reform package,” central bank said.
The government is confident that the battle for Marawi will soon be over, with no less than President Rodrigo Duterte saying that the conflict will end in 10 to 15 days.
The first package of the Comprehensive Tax Reform Program, which was incorporated with 53 other tax-related bills under the proposed Tax Reform for Acceleration and Inclusion Act, was approved by the House of Representatives on May 31.
Total outflows last month reached $1.93 billion, up 28.3 percent from $1.5 billion in May “due to profit- taking and investor reaction to the United States Federal Reserve’s decision to increase interest rates,” the BSP said.
The same trend was noted on a yearly basis, as outflows rose by 42.5 percent from $1.48 billion, it added.
In the first half of the year, hot money registered a net outflow of $460.83 billion, reversing the $593.87 million in net inflow a year earlier “due to certain domestic and international developments, such as the US air strike against Syria, global terrorist attacks, interest rate increases by the US Federal Reserve, political turmoil in the US, and the closure order for several mining companies in the country,” the central bank said.
Data showed that 82 percent of hot money was invested in securities listed on the Philippine Stock Exchange, mainly in holding firms, property companies, telecommunications firms, banks, and utilities companies; 17.2 percent went to peso government securities (GS); and 0.8 percent was placed in other peso debt instruments (OPDIs).
“Transactions in PSE-listed securities and OPDIs yielded net inflows of $311 million and $16 million, respectively, while investments in peso GS resulted in net outflows of $247 million,” the BSP said.
The United States, United Kingdom, Singapore, Malaysia and Luxembourg were the top five investor-countries in the Philippines, with a combined share-to-total of 80.1 percent. The United States again accounted for the largest amount of repatriated funds, totaling 64.7 percent of the money transfers from the Philippines.
Foreign portfolio investment is also called hot money because of the ease with which the funds move in and out of a country. The investment does not necessarily create jobs, unlike foreign direct investments, which are used to build factories and buy capital equipment.

domain: economics


The death toll in the collapse of a three-storey church building in eastern Ghana rose to 21, rescue officials said on Friday, as more bodies were pulled from the rubble.
“The deceased include 12 women and nine men,” the National Disaster Management Organization said in a statement, upping the toll from 17 dead on Thursday.
It said eight people had been rescued alive at the scene since rescue workers began combing the disaster site.
The Church of Prosperity collapsed on Tuesday in the town of Akyem Batabi just after worshippers had concluded a prayer meeting.
One survivor told local media that some 60 people were inside the building when it came down.
The causes of the accident were still unknown but the church’s founder, Prophet Isaac Ofori, popularly known as Akoa Isaac, was assisting police with its investigation.
Coronavirus cons: Fake COVID-19 cures sold by doctors in Africa’s Ghana, finds BBCDodgy doctors and con men have been selling fake coronavirus cures in Ghana, many of which contain dangerous levels of bacteria and mold, revealed a ... Coronavirus
Coronavirus: French oil giant Total abandons plan to buy Occidental’s Ghana assetsTotal SA abandoned a plan to acquire assets from Occidental Petroleum Corp. in Ghana, walking away after a proposal to buy Algerian projects from the ... Coronavirus
Coronavirus: One person in Ghana infected 533 others at fish factoryA worker at a fish-processing factory in Ghana's Atlantic seafront city of Tema infected 533 other workers at the facility with the coronavirus, ... Coronavirus
Two Canadian women abducted in Ghana are rescuedSecurity forces rescued two Canadian women who were abducted in Ghana earlier this month and arrested eight people in connection with their kidnapping ... World News
At least 60 people killed in Ghana bus collisionAt least 60 people were killed on Friday in a head-on collision between two buses in Ghana, police said, in the latest deadly crash on the ... World News
Tomato truck crash leaves 61 dead in GhanaThe crash is considered one of the country’s deadliest ever road disasters North Africa
Nine dead in Eid party stampede in GhanaNine people were killed in a stampede at a party in central Ghana to mark Eid al-Fitr North Africa


The instant messaging application Telegram has introduced a number of new features in its latest update, including search filters, anonymous administrators, and channel comments.
The new search filters feature allows users to search for what they want based on type, be it media, links, files, music, voice memos or chats.
Users will also be able to search in a specific period of time, they will only have to write the date they want, such as “August 14” or “Yesterday”, in the search engine, as well as search according to the source, writing the name of anyone , group, channel or bot.
All these search filters can also be combined, allowing you to search for things in a more specific way, according to Telegram on its website.
Another function that Telegram has added is that of anonymous administrators in groups. To do this, users will have to select ‘Be anonymous’ in the administrator permissions to activate the ‘Batman’ mode and be hidden in the list of chat members.
This novelty is focused on protecting people in the protests, since, in addition to hiding their identity, the messages they publish will appear with the group’s signature.
Telegram has also added a button to comment on channel posts. These comments will appear in their own thread that will reach the group conversation to keep everyone up to date.
Users will be able to test the channel comments feature on a limited basis by selecting ‘Channel Settings’ and then ‘Conversations’. Also, the app has introduced more animated emojis.
For Telegram users with Android operating system, the application has new animations, as well as the function of displaying or hiding the keyboard, changing day and night themes and seeing animated pop-up windows when they delete messages or change notifications, among other things. .
© 2020 Europa Press.


Mike McGarry pointed at one of the 35,000 vines at Sugarloaf Mountain Vineyard in Dickerson, his scissors following the scaly bark.
“You don’t want a stagnant microclimate,” he said, snipping bunches of grapes off the vine as he explained how more sunlight and breezes make for healthier, hardier grapes.
McGarry and a handful of workers were in the middle of the season’s final harvest, part of the legacy of a tightknit family with Maryland roots that has worked the 92-acre farm for half a century.
McGarry is one the four co-owners of the vineyard and winery, the only one in Montgomery County open to the public. When it began production, Sugarloaf was one of about 12 wineries in Maryland, according to Maryland Wineries Executive Director Kevin Atticks. Maryland’s wine industry has exploded since then, he said, to 60 wineries today.
“There was still a perception in general in the wine community — that it was hard to make good wine in Maryland,” when Sugarloaf began breaking ground 10 years ago, Atticks said.
Sugarloaf leases its fields from Windmill Farm, jointly owned by McGarry’s wife, Carol, and her three siblings, the children of Dan and Polly O’Donoghue, who bought the land in 1962. Dan O’Donoghue was a trust and estates lawyer in his mid-50s when he and his wife began spending their weekends there. They raised Black Angus cattle and winter wheat.
Their four children — Dan, Phil, Carol and Lois — visited as teenagers and adults, while on college break or with their spouses and young children.
They began transforming the former cattle and wheat farm into an award-winning vineyard in 2002, just before their father died in 2003. Polly had died a few years earlier, in 1999, son Dan O’Donoghue said.
Everybody chips in to help run the vineyard, McGarry said. Carol, for instance, worked as an accountant for many years, so she does the vineyard’s tax filings. Other siblings or their spouses help in sales, or by doing the vineyard’s legal work.
Building a vineyard from scratch was challenging, too, Carol McGarry said, but “we’re still enjoying each other.”
“The weather is the biggest challenge for everybody,” she said. “It surprises us people still come out on rainy days.”
The vineyard and its owners have come a long way since they first conceived the idea 10 years ago.
When they started, “We knew nothing,” Mike McGarry said.
They hired a viticulturist — an expert in the cultivation of grapes — selected vines, tested the farm’s soil, planted their grapes and set to work making wine.
The vineyard grows the five Bordeaux grapes (cabernet franc, cabernet sauvignon, merlot, malbec and petit verdot), along with three whites (chardonnay, pinot grigio and Viognier). The French vines were grafted onto American stems, which resist vine-killing pests, Mike McGarry said.
They harvested 5 tons of grapes their first year; this year it was about 50 tons, he said. A volatile spring killed off some of the vineyard’s more delicate grapes, lowering yields from 4 tons per acre to about 2.5 tons per acre.
“The rest of the summer was great, so we had big yields for our reds,” he added. “This was a very good year, probably a vintage year.”
The vineyard can produce up to 6,600 cases of wine per year, or about 73,000 bottles, though they have been bottling about 5,000 cases (60,000 bottles). A majority of the wine is sold at the vineyard, which holds tastings year-round.
The four couples, who live in Bethesda, Chevy Chase and Washington, D.C., still gather every year at the farm to celebrate Thanksgiving with their relatives, about 40 people in all.
“We didn’t want to sell the land,” said Jim McKenna, who married Dan and Polly’s daughter Lois. “We really didn’t want to see green space go away.”
The whole adventure has required a can-do attitude, hard work and faith, sprinkled with a dose of luck.
“My father would have been nervous when we were starting out,” Dan O’Donoghue said. “But my mother would really have been pleased — she was the one who insisted the family be together, to live in the same town. She would have been ecstatic — she treasured family.”
The best part of the venture has been “learning the high quality wine we can make; we weren’t sure at first,” Carol McGarry said. “The first year we won the double gold — we went, ‘Wow! ... We can make good wine!’”
When they started, the family had to pay a “huge initial outlay,” Mike McGarry said, preparing the land and planting the vineyard’s thousands of vines, and didn’t expect to make any wine until their third year. But the grapes grew better than they expected, and they harvested their first batch of grapes after just two years. The first wine produced — a cabernet franc — won a double gold award in an international competition in New York state in 2007.
The youngest wines — some of the white wines and a red or two — sit for roughly nine months before the vineyard offers it to the public, while the more seasoned reds age for up to two years before becoming available to the public, McGarry said.
“It says a whole lot about Sugarloaf and their careful planning — they knew enough to know they had to do it right from the start,” said Atticks, the Maryland Wineries executive director.


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What is Gary Oldman Net Worth?
Gary Oldman is an American actor who has starred in many movies and television shows. Some of his best known works include The Nightmare Before Christmas and The Dark Knight Rises. He was also nominated for a Golden Globe for his role in the film 127 Hours. However, there is more to his life than
Gary Oldman is an American actor who has starred in many movies and television shows. Some of his best known works include The Nightmare Before Christmas and The Dark Knight Rises. He was also nominated for a Golden Globe for his role in the film 127 Hours. However, there is more to his life than just his acting.
Gary Oldman is a British actor. He has starred in films such as Nil by Mouth, Leon: The Professional, and Darkest Hour. Aside from his acting, he has also been a producer on several movies.
He has received numerous accolades for his role in the film Nil by Mouth. His performance ranked as 62 on Premiere’s list of 100 best performances. It was a heartbreaking look at working class life in south London.
Oldman has also starred in films such as JFK (1991), Air Force One (1997), and David Bowie’s music video, “The Next Day” (2013). He has been awarded sole physical custody of his children.
Gary Oldman grew up in a working-class family in London. He started his acting career at the age of 16. He attended Rose Bruford College of Theatre and Performance. However, he dropped out at sixteen.
Gary Oldman has starred in several films, including the dark and eerie drama Nil by Mouth, the ominous thriller Child 44, and the conspiracy-driven JFK. Some of his other credits include Bram Stoker’s Dracula (1992), and the controversial crime film Criminal Law.
Nominated for a Golden Globe
The Golden Globes are an annual awards ceremony that celebrates the best in the film and television industries. This year, the 80th edition will be broadcast live from the Beverly Hilton in California. These events are presented by the Hollywood Foreign Press Association.
The organization, which has 105 members, is known for recognizing excellence in the international film industry. There are two separate ceremonies, one for dramas and another for comedies. The awards will air on January 10 and will be hosted by stand-up comedian Jerrod Carmichael.
The list of nominees for the 80th Golden Globes has been announced. Nominations are separated into categories, such as best actor, actress, director, and best picture. Also, there is a special award named the Cecil B. DeMille Award.
Some of the nominees for this year’s Golden Globes include Baz Luhrmann’s biopic Elvis, and the dramas House of the Dragon and The Fabelmans. In addition, the comedy series Abbott Elementary is nominated for five Globes, including best supporting actor.
Gary Oldman is a successful British actor who has been in many roles over the years. He has appeared in a wide range of films, including The Dark Knight, Batman Begins, Drexl in True Romance, and Tinker Tailor Soldier Spy.
Gary Oldman has been married five times. His most recent marriage is to art curator Gisele Schmidt. It was a private ceremony held at a home in Beverly Hills, California. They are currently co-parenting two sons from their previous marriages.
One of Gary Oldman’s previous wives was a successful model named Donya. She worked with Revlon and Van Halen, and she had two kids. Sadly, she suffered from addictions to alcohol and painkillers. In 2001, when she was pregnant with her second child, Charlie, she had a bad accident. She subsequently filed for divorce from Gary Oldman.
There is no telling what may come next for Gary. His latest wife may be the one that changes him.
Gary Oldman is an English actor and filmmaker. He was born on March 21, 1958 in London, England. As an actor, he has appeared in a wide range of films and television shows. He also has a background as a musician and pianist.
Oldman graduated from Rose Bruford College of Speech and Drama in Kent. After graduating from college, he went on to appear in a number of plays. In the late 1980s, he appeared in a number of television programs, including “Murder in the First,” “Chattahoochee,” and “The Pope’s Wedding.”
Oldman began his career in theatre in 1979. His first appearance in a film was in the 1982 film Remembrance. Throughout his acting career, Oldman has starred in many films, including Harry Potter and the Prisoner of Azkaban (2004), Harry Potter and the Deathly Hallows – Part 2 (2011), and The Fifth Element (1997).
Gary Oldman has had an incredibly varied and successful career as an actor. Although he has had numerous major roles in Hollywood, he is known for his portrayal of villains and over-the-top antagonists.

domain: artistic