In keeping with the effects on angiogenesis induced by contact hy

In keeping with the effects on angiogenesis induced by contact hypersensitivity reactions in mouse ears, VS-I-treated mice revealed significantly reduced oedema formation, resulting from lower plasma leakage and inhibition of inflammation-associated vascular remodelling [66]. Intravital microscopy studies of inflamed ears showed a decrease

in the fraction of rolling leucocytes in VS-I-treated mice [66]. In addition to anti-microbial activity [67] Cgs may play important role in the neuroimmune interaction in relation to inflammatory function. This review will remain focused upon the function of Cgs in inflammatory responses in the gut. Circulating CgA levels, a marker for neuroendocrine tumours including carcinoids, have selleck products recently been found elevated in some patients with IBD [68]. In this context the disease activity and TNF-α levels influence the CgA pattern, which could reflect the neuroendocrine system activation in

response to inflammation [69]. In a recent letter addressed to the aforementioned study, Sidhu and collaborators [70,71] confirmed the observation of Sciolia et al.[69] of an elevated level of CgA Proteasome inhibitor serum in both IBD and diarrhoea-predominant IBS patients. The unifying hypothesis proposed could be the EC cell hyperplasia producing an elevated serum CgA levels, as reported previously [72]. The differential replication of EC cells in IBS patients could also explain why elevated levels are found only in a proportion of patients, and levels decline with time. Further studies of serial serum CgA measurements in both these conditions would strengthen our understanding of the plausible mechanisms behind these observations. In the context of experimental colitis, intrarectal injection of CAT can decrease the inflammatory markers [73]. Disease activity index, macroscopic and histological scores, as well Amylase as myeloperoxidase

(MPO) activity, were decreased significantly in mice treated with CAT compared to mice that received DSS only. Treatment decreased the onset of clinical disease as assessed by loose stools, weight loss and rectal bleeding. In addition, colonic tissue levels of IL-1β, IL-6 and TNF-α were decreased significantly in mice treated with CAT. Conversely, the biochemically modified fragment had no effect on the severity of colitis. These results support the hypothesis that Cgs-derived peptides modulate intestinal inflammation in a murine model of colitis by acting directly or indirectly on the microbiota and the immune system. Identification of the molecular and cellular mechanisms underlying the protective role of this peptide may lead to a novel therapeutic option in IBD.

It is recognized that

It is recognized that Galunisertib datasheet the microcirculation of the skin undergoes considerable modifications in the first few days or weeks of extrauterine life as the length and variability of the diameter of the capillaries increase [6] and BCD decreases progressively [39]. We have hypothesized that in singleton infants the abundant availability of nutrients after delivery presumably triggers a much more rapid and perhaps poorly controlled process of “capillary hyper-pruning,” culminating in these infants having capillary rarefaction by some

stage in later childhood [1, 14]. We speculate that this process of capillary hyperpruning may be less prominent or even absent in twin infants and this may explain the apparent lack of increased cardiovascular disease risk in these individuals in later life [20]. Another interesting observation in our study is the significantly lower family history of ischemic heart disease in the twin infants group. The significance of this finding is rather difficult to interpret but we cannot rule out a possible effect on the capillary density in these infants as we have previously reported that normotensive individuals with

Selleck Protease Inhibitor Library family history of essential hypertension have significant capillary rarefaction [4]. We acknowledge a major limitation in our study posed by the small numbers of twin infants but this emphasizes the difficulties in recruiting such infants. We also acknowledge the significant difference in the age of infants on the study day, as it was not always possible to perform capillaroscopy immediately after birth in the twin infants, who often had to be transferred to the neonatal unit. We feel it is essential to explain here how difficult it proved to study these new born infants who often wake up when they handled, and it then becomes difficult to proceed with the study as they become uncooperative and buy Ibrutinib their mothers restless and anxious, and we often then had to abandon the study in as much as 25% of potential subjects. In conclusion, twin infants born with LBW or NBW to normotensive mothers have significantly higher functional and structural

skin capillary densities at birth compared to singleton infants. Further longitudinal studies of skin capillary density and of retinal vascular parameters commencing from birth to various stages in early childhood are essential to identify the dynamics and the exact timing, if any, of the remodeling of microcirculation in these individuals. LBW is a risk factor for adult hypertension and cardiovascular disease and is associated with functional and structural microvascular disease. Twin infants as a group tend to have LBW, but do not appear to have increased risk of cardiovascular disease in later life. When examined at birth, twin infants do not have a reduction in microvascular density but rather higher capillary count.

Antigenic stimulation of PBMC for proliferation and cytokine secr

Antigenic stimulation of PBMC for proliferation and cytokine secretion was performed according to standard procedures (Mustafa 2009b). In brief, 2 × 105 PBMC suspended in 50 μL complete medium was seeded into the wells of 96-well tissue culture plates (Nunc, Roskilde, Denmark). Antigens

in 50 μL complete medium were added at optimal concentrations to the wells in triplicates. Whole bacilli were used at 10 μg mL−1 (wet weight) and all other antigens and peptides were used at an optimal concentration of 5 μg mL−1. The cells in the control wells did not receive any mycobacterial antigen/peptide. The final volume of the culture in each well was adjusted to 200 μL. Con A 10 μg mL−1 (Sigma Chemical,

St. Louis, MO) was used as a positive control. The plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. On day 6, culture GPCR Compound Library supernatants (100 μL) were collected from each well and frozen at −20 °C until used to determine cytokine concentrations. The remaining cultures were pulsed with 1 μCi 3H-thymidine (Amersham Life Science, Amersham, UK) and harvested (Skatron Instruments AS, Oslo, Norway) according to standard procedures (Al-Attiyah et al., 2003). The incorporated radioactivity was obtained as counts per minute (c.p.m.). selleck The average c.p.m. was calculated from triplicate cultures stimulated with each antigen or peptide pool, as well as from triplicate wells of negative control cultures lacking antigen. The cell proliferation results were presented as stimulation index (SI), where SI is the c.p.m. in antigen- or peptide-stimulated Methane monooxygenase cultures per c.p.m. in cultures lacking antigen or peptide. A patient was considered to be a responder to a given antigen if the PBMC yielded SI≥3 (Al-Attiyah et al., 2003). Positive responses ≥60% were considered strong, 40% to <60% moderate, and

<40% weak (Mustafa, 2009a, b). The supernatants, collected from the cultures of PBMC of TB patients (n=20) and healthy subjects (n=12) before 3H-thymidine pulse, were randomly selected for assays to determine concentrations of secreted IFN-γ and IL-10 using FlowCytomix kits (Bender Medsystems GmbH, Vienna, Austria), according to the manufacturer’s instructions (Al-Attiyah & Mustafa, 2008, 2009). These kits allow simultaneous quantification of cytokines including IFN-γ and IL-10. In brief, FlowCytomix technology is based on spectrally discrete microspheres that are used as solid phase in an immunoassay. The beads are internally dyed with Starfire Red, a far red (685–690 nm) emitting fluorochrome, which is excited by UV, argon or HeNe lasers. The test samples were analyzed by flow cytometry using Coulter EPICS FC500 (Beckman Coulter Inc., USA). For each analysis, up to 10 000 events were acquired. The mean concentration of each cytokine was expressed as pg mL−1.

[79] Dendritic cells and macrophages also

express a combi

[79] Dendritic cells and macrophages also

express a combination of surface markers that defines a particular function, for example antigen uptake and presentation. Antigen uptake induces the expression of the costimulatory molecules CD68, CD80 and CD86 on macrophages and DCs in mice and human kidneys.[91, 93, 101-104] Resident renal DCs capture antigens via phagocytosis, pinocytosis and receptor-mediated endocytosis, all functions typically ascribed to macrophages.[104] CH5424802 purchase Following antigen uptake, DCs migrate to secondary lymphoid organs where they reduce the expression of costimulatory molecules and gain the ability to activate and prime T cells through increased expression of MHC II. This specialized function of DCs, although potent, is not exclusive. Macrophages also migrate and present antigens,[105, 106] and display an almost complete overlap in MHC II expression with F4/80 mononuclear phagocytes in mucosal sites and kidneys.[102, 107] Macrophages and DCs also occupy overlapping anatomical sites within the normal kidney. During steady state, macrophages identified using F4/80 form an intimate relationship with renal TECs as they are found adjacent to the basement membrane of proximal TECs in the outer medulla.[108] DCs defined by CD11c expression are absent

from the glomerulus, but are localized to the tubulointerstitium with overlapping expression with F4/80 in mice.[91] Renal biopsies from normal human kidneys show an abundance of DCs in the tubulointerstitium this website that are absent in the glomeruli.[101] The localization of DCs strictly within the renal tubulointerstitium is

suggested to be optimal for antigen capture.[109, 110] Soos et al.[93] characterized the anatomy and phenotype of resident DCs SPTBN5 within normal mouse kidneys using the heterozygous CX3CR1GFP/+ mice. Laser scanning microscopy identified CX3CR1+ DCs throughout the entire renal interstitium, including the glomeruli, with dendrite processes extending between the TECs and into the tubular lumen. This DC population was defined by CX3CR1 expression alone. Profiling these cells by flow cytometry revealed that the majority of CX3CR1+ cells exhibited high levels of CD11c and F4/80, and low CD11b expression, thus raising concerns as to whether these cells only represented DCs. The cells expressing CX3CR1 are not fully defined in mice, but are generally homogeneous and indistinguishable from tissue macrophages and infiltrating monocytes, and in some settings cannot migrate to draining lymph nodes or present antigen.[111] In a more recent study using transgenic mice that express GFP and the diphtheria toxin receptor (DTR) driven by the CD11c promoter (CD11c-DTR) revealed CD11c-GFP cells displayed a typical DC morphology that localized to the tubulointerstitium, and not within the glomeruli, as consistent with previous reports.

001) In the single-pedicled flap group, there were no statistica

001). In the single-pedicled flap group, there were no statistical differences between survival flap areas of the non-rotated subgroup and the 90- and 180-degree rotation subgroups (P > 0.05), but the non-rotated subgroup had a statistically larger survival area compared to the 270-degree rotation subgroup (P = 0.003). Tyrosine Kinase Inhibitor Library In double-pedicled perforator flap group, the control subgroup had a statistically larger flap survival area compared to 90-degree, 180-degree, and 270-degree rotation subgroups (P = 0.004, P = 0.002, P = 0.001). Degenerative histological changes gradually increased in correlation with the rotation angle in both single-

and double-pedicled groups. When double- and single-pedicled

groups were compared; degenerative histology score displayed no statistical difference between control subgroups and rotated subgroups (P > 0.05). In this rat abdominal propeller perforator flap model, we found that double perforators without pedicle rotation could support larger flap survival when compared to the single pedicle. However, double perforators did not cause an increase of survival area when pedicles were rotated. In the single-pedicled perforator flap, the flap survival area did not significantly decrease until 180-degree pedicle rotation. In the double-pedicled perforator flap, the flap survival area decreased when the degree of rotation increased. The degenerative changes increased in correlation Methisazone with the rotation degree in both single- and double-pedicled perforator flaps. © 2014 Wiley Periodicals, Inc. Microsurgery 34:464–469, 2014. “
“Large osseous defects of the upper extremity can be a challenging problem for the reconstructive surgeon. There are numerous treatment options reported in the literature with variable results. We review our experience with the vascularized-fibular osteocutaneous graft for these complex defects with a focus on surgical techniques and outcomes. © 2009

Wiley-Liss, Inc. Microsurgery, 2011. “
“The reconstruction of complex soft tissue defects in hands remains a difficult challenge in reconstructive surgery. In this report, we introduce a combined medialis pedis and medial plantar fasciocutaneous flaps supplied by the lateral and medial branches of the medial plantar artery, which allows a one-stage reconstruction of multiple soft tissue defects in hand. Three combined medialis pedis and medial plantar fasciocutaneous flaps were transferred for repair of the soft tissue defects including palmar and dorsal areas of hand, thumb pulp, and the dorsum of index finger in three patients. All three flaps survived uneventfully with coverage matching the texture and color of the recipients. The donor sites healed without complication.

e inflammatory interstitial pneumonitis,

de novo glomeru

e. inflammatory interstitial pneumonitis,

de novo glomerulonephritis and systemic inflammatory response syndrome).[34] Together with the described role of the phosphatidylinositol 3-kinase–mTOR pathway in limiting the production of pro-inflammatory cytokines after stimulation by TLR agonists or CD40 ligand,[34] the relevance of mTOR pathways in M2 survival and M1 polarization could explain the distinct inflammatory side-effects observed during RAPA treatment. In conclusion, we demonstrate that RAPA affects M2 survival and unbalances this website to an M1-like inflammatory response both in vivo and in vitro. Consequently, our work proposes the mTOR pathway as a key regulator of macrophage polarization and offers a novel mechanistic insight in macrophage polarization. Due to the availability of mTOR inhibitors for clinical therapy, the effect on macrophage polarization may open the way for mTOR targeting and tailoring in M2-related human diseases. This work was supported by EU (HEALTH-F5-2009-241883-BetaCellTherapy), Juvenile Diabetes Research Foundation (JDRF Grant: 6-2006-1098, 31-2008-416, 4-2001-434, JT01Y01, 17-2011-601). The authors declare that they have no financial disclosures or competing interests. “
“Salivary host-defence peptides include defensins, histatins and cathelicidin. We have investigated the effects of these peptides on the microbial

composition of dental plaques. Salivary consortia, established within GDC-0068 mw hydroxyapatite disc models, were exposed during development to physiological levels of human neutrophil proteins (HNP) 1 and 2; human β defensins (hβD) 1, 2 and 3; histatins (His) 5 and 8; and cathelicidin (LL37). Effects on aggregation and microbial composition were determined using fluorescence microscopy; and differential culture with PCR-DGGE, respectively. LIVE/DEAD microscopic analysis indicated

that HDPs decreased total bacterial viability, whilst β defensins, paired HNPs, His 5, His 8 and the HDPs combined inhibited bacterial aggregation. According to differential culture, all test HDPs (except His 5) significantly decreased the abundance of Gram-negative Phosphatidylethanolamine N-methyltransferase anaerobes and lactobacilli (except HNP 2, hβD 1, paired HNPs and His 5). Combined HNPs and paired hβD 1 and 3 inhibited streptococci, whereas HNP 1, hβD 1, hβD 3, His 5 and LL37 increased streptococcal numbers. According to cluster analyses of DGGE profiles, HDP-exposed plaques were compositionally distinct from undosed controls. Thus, whilst HDPs reportedly exhibit variable potency against oral bacteria in endpoint susceptibly tests, exposure of nascent plaques can markedly influence bacterial viability, composition and microbial aggregation. Saliva contains a range of antimicrobial molecules of which over 45 have been characterized (reviewed by Gorr & Abdolhosseini (2011)).

If scenario 2 be the case, then each tissue must be able to produ

If scenario 2 be the case, then each tissue must be able to produce all three signals. Of course, a choice between the signals would have to depend on the characteristic of the pathogen–tissue interaction. Given coherence and independence of responsiveness, a decision between signals would be required. These are two extremes. However, they suggest a general case under which

each tissue has the potential to deliver all three signals but a given pathogen–tissue interaction would trigger only one of the three signals. Admittedly, there are many ambiguities here as tissues are composed of different cell types and themselves form organs. The relationship of pathogens to tissues will eventually have to deal with the relationship of pathogens to cells and organs. Further, implied is that the pathogenic universe itself is viewed by the adaptive immune system as Kinase Inhibitor Library chemical structure divided into four categories, each optimally responded to by one or the other of the four effector ecosystems. Lastly, if a given tissue traumatized

by different pathogens can deliver different signals (three are postulated), what might be the basis for the different interactions. One trauma signal might be determined by whether the pathogen is intracellular or extracellular (Signal 3a). Extracellular pathogens might be divided into those dependent on secreted toxins (Signal 3b) versus those that trigger and profit from immune subversion (Signal 3c) like a fulminating inflammatory response (i.e. immunopathology). The point being made, admittedly primitively, is see more that the postulate of a small number of effector ecosystems and corresponding class controlling trauma signals implies that evolution has classified the pathogenic universe

into a few categories that exert a similar selection pressure to which the evolution of the Farnesyltransferase host can respond. The Trauma Model is a theory of the regulation of expression of the effector ecosystems. Here, we will try to formulate one of several possible sets of postulates that would define such a model. Then, we will propose tests of these postulates: 1  The uptake by APCs of Eliminons that the germline-selected (‘innate’) repertoire cannot recognize requires an Eliminon-antibody aggregate. The source of this primer uptake antibody is the B cell, which must secrete, antigen-independently, primer antibody after undergoing a sorting of its repertoire ([6], see discussion of Hypothesis VII in ref. [46]). This limits the presentation of exogeneous self by APCs making the requirement for ARA at the level of the S-NS discrimination (Module 2) less stringent but not obviated (see earlier). The overwhelming belief that T-suppressors play their major role by regulating autoimmunity makes it necessary to point out that the Trauma Model redefines their normal role. Feedback regulation of the magnitude of the effector response is essential [47].

In the histological

analysis, distal colon showed edema,

In the histological

analysis, distal colon showed edema, hemorrhage, exudation and inflammatory infiltrations in the lamina propria. Orally immunized Depsipeptide supplier animals with heat-killed S. dysenteriae type 1 and S. flexneri type 2a strains showed high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies and conferred significant homologous protective immunity against subsequent challenges with the live strains. The direct administration of shigellae into the cecocolic junction induces acute inflammation, making this animal model useful for assessing shigellosis and evaluating the protective immunity of Shigella vaccine candidates. Bacillary dysentery or shigellosis is an acute colitis caused by enteroinvasive bacteria belonging to the genus Shigella. Shigellosis is an endemic disease throughout the world, particularly in the pediatric population between 1 and 5 years of age in developing countries (Phalipon et al., 2008). Shigellosis can be caused by any of the serotype belonging to four Cell Cycle inhibitor groups: Group A (Shigella dysenteriae), Group B (Shigella flexneri), Group

C (Shigella boydii) and Group D (Shigella sonnei). Worldwide, 164.7 million episodes of Shigella-mediated infections were reported each year, with ∼1.1 million deaths, mainly due to unhygienic conditions (Kotloff et al., 1999). Mucosally invasive shigellae, which often cause dysentery, are less amenable to the beneficial effects of oral rehydration than noninvasive pathogens,

such as Vibrio cholerae and enterotoxigenic Escherichia coli that cause acute watery diarrhea (Levine et al., 2007). In addition, increasing multi-antimicrobial resistance complicated the clinical management of shigellosis (Kotloff et al., 1999). Various in vitro cell culture models as well as studies in animal models including gastrointestinal infection in nonhuman primates have enriched our current understanding of Shigella pathogenesis (Cossart & Sansonetti, 2004; Sansonetti, 2006). Shigella targets the distal region of the colon and rectum (Anand et al., 1986), where the bacteria are captured by specialized M-cells located within the follicle-associated Selleck CHIR 99021 epithelium. The M-cells deliver bacterial antigens such as lipopolysaccharides and invasive plasmid antigen (Ipa) proteins to the underlying antigen-presenting macrophages and dendritic cells (Phalipon & Sansonetti, 2003). Shigella is phagocytosed by macrophages, but subsequently killed by the pathogen by apoptosis (Phalipon & Sansonetti, 2007). Before death, the infected macrophages release proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 (Chen et al., 1996). This helps to trigger a strong inflammatory response that leads to the migration of polymorphonuclear cells such as neutrophils (Anand et al., 1986), which infiltrate the infected site and destabilize the epithelium (Perdomo et al., 1994).

Detailed facts of importance to specialist readers are published

Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Macrophages play a crucial role in innate immune reactions, and Peritoneal Macrophages (PMs) guard the sterility

of this compartment mainly against microbial threat from the gut. Type-1 Diabetes (T1D) is an autoimmune disease in which gut microbiota and gut immune system appear to contribute to disease pathogenesis. We have recently reported elevated free radical production and increased permeability of gut epithelium in non-obese diabetic (NOD) mice. Impaired barrier function could lead learn more to bacterial leakage to the peritoneal cavity. To explore the consequences of impaired gut barrier function on extra-intestinal immune regulation, we characterized peritoneal

lavage cells from young newly weaned NOD mice. We detected a rapid increase in the number of macrophages 1-2 weeks after weaning in NOD mice compared to C57BL/6 and BALB/c mice. Interestingly, this increase in macrophages was abrogated in NOD mice that were fed an anti-diabetogenic diet (ProSobee), which improves gut barrier function. Macrophages in young (5 week old) NOD mice displayed a poor TNF-α cytokine response to LPS stimulation, and high expression of Toll-like Receptor (TLR) signalling pathway negative regulator, Interleukin-1 Associated Kinase–M (IRAK-M), indicating prior in vivo exposure to TLR-4 ligand(s). Furthermore, injection of however LPS intraperitoneally increased T-cell CD69

expression in pancreatic lymph node (PaLN), suggestive of T-cell activation. Leakage of bacterial components such as endotoxins into the peritoneal cavity may contribute to auto-reactive T-cell activation in the PaLN. This article is protected by copyright. All rights reserved. “
“The immune system evolved to require input from at least three sources that we collectively term the ‘old friends’: (i) the commensal microbiotas transmitted by mothers and other family members; (ii) organisms from the natural environment that modulate and diversify the commensal microbiotas; and (iii) the ‘old’ infections that could persist in small isolated hunter-gatherer groups as relatively harmless subclinical infections or carrier states. These categories of organism had to be tolerated and co-evolved roles in the development and regulation of the immune system. By contrast, the ‘crowd infections’ (such as childhood virus infections) evolved later, when urbanization led to large communities. They did not evolve immunoregulatory roles because they either killed the host or induced solid immunity, and could not persist in hunter-gatherer groups.

Fresh splenocytes

were prepared as described before 8 Cu

Fresh splenocytes

were prepared as described before 8. Cultured adherent cells were detached with disassociation buffer KU-60019 ic50 (GIBCO). Cells were stained with Abs (Supporting Information Table 2) at 4°C for 30 min. For two-step staining, biotinylated Ab were subsequently detected using streptavidin conjugates (Supporting Information Table 3). 1 μL of DAPI (0.25 μg/mL) and 20 000 of non-fluorescent particles (counting beads) (Spherotech) were added into each sample and the sample acquired using a CyAn flow cytometer. Data were analyzed by FlowJo software (TreeStar). For cell sorting, cells were prepared and stained as above. Cells were purified by MoFlo cell sorter (Dako Cytomation). Frozen IL-7−/− spleens kindly donated by Daniella Finke (University of Basel, Switzerland) were sectioned and prepared as described before 22. Cells were stained with the Abs (Supporting Information Tables 2 and 3). Anti-CD4 mAb was directly conjugated using the Alexa Fluor 647 mAb labeling kit (Invitrogen) according to the manufacturer’s instructions. Biotinylated Ab SCH 900776 were detected by streptavidin Alexa fluor 555 (Invitrogen). FITC-conjugated Ab were detected using rabbit anti-FITC Ab (Sigma), then goat anti-rabbit FITC Ab (Southern Biotech). Confocal images

were acquired using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss) and analyzed using LSM510 software. Cell suspension were made from spleens of Rag−/−γc−/− and CD3εtg mice as described previously 6. Briefly, CD4+ cells were enriched from CD11c+-depleted populations using MACS anti-mouse CD4 microbeads (Miltenyi Biotech) according to the manufacturer’s protocol. Enriched CD4+ splenocytes contained between 8 and 30% LTi-like cells 4. Briefly, 2–5×104 of CD4+-enriched cells were cultured and incubated for 4 days prior to further FACS analysis. SSCL and white pulp stromal cells were obtained as described previously

23. CD4+ enriched populations were cultured Fossariinae with DMEM with 10% FCS supplemented with 1% penicillin/streptomycin and 2 mM L-glutamine on irradiated (2000 Rad) CD45−podoplanin+ SSCL with or without 2 μg/mL blocking anti-IL-7 Ab (R & D). Controls were cultured in the absence of any stromal cells with or without 0.01 g/mL recombinant IL-7 (Pepro Tech). All cultures were incubated for 4 days. The recovered cell number calculated as: viable LTi-like cell number is equal to the DAPI−CD3−CD11c−B2220−CD4+ cell number shown in FACS plot divided by the bead number shown in FACS plot then times 20 000. CD45−podoplanin+ SSCL were frozen in liquid nitrogen, and high-purity cDNA was obtained from purified mRNA, using μMacs One-step cDNA synthesis kit, according to the manufacturer’s instructions (Miltenyi Biotech). β-Actin was used as the housekeeping gene for sample normalization, prior to amplifying the target genes of interest. RT-PCR was performed using SYBR Green with primers (Supporting Information Table 4).