P < 0·05 was regarded as the significant level of probability throughout. Two trials designated Experiments 5 and 6 were conducted (Table 1), so numbered as they were part of a larger series of trials sharing the same design. Both experiments contained a group of sheep which had received a trickle immunising Romidepsin solubility dmso infection of 2000 T. circumcincta infective larvae three times per week for 8 weeks,

and a group of control sheep which had not received the trickle infection. All were dosed with fenbendazole one week prior to challenge with a single dose of 50 000 infective larvae, with surgery to cannulate the gastric lymph duct being carried out on 10 sheep in each experiment during the intervening week. Sheep were killed on days 5, 10 or 21 post-challenge. It was known from prior work using this experimental model that in previously infected sheep the cellular and humoral immune responses in lymph all occurred by day 9 after challenge. Therefore, lymph collection from the previously infected lambs was stopped GS-1101 mouse after 10 days. Large cells or lymphoblasts were determined as those with a diameter of >9 μm when measured by Coulter Counter, with small lymphocytes represented as those with a diameter of between 3 and 9 μm. During FACS analysis, small cells were those appearing within region R1 on a control sample Fsc vs. Ssc plot (Figure 1),

blast cells were designated as the gated lymphocytes which fell within region R2 and total lymphocytes within R3 (=R1 + R2). Downstream

FACS analyses of stained cells were gated to contain only those cells present in R3. Surface staining of lymphocytes from gastric lymph, and flow Tyrosine-protein kinase BLK cytometry, were carried out as detailed previously (6). Monoclonal antibodies that recognise border disease virus as isotype controls (clones VPM21 (isotype IgG1, 1/500 dilution) and VPM22 (isotype IgG2, 1/500) (25)), ovine CD4 (clone 17D, IgG1, 1/1000 (26)), CD8 (clone 7C2, IgG2a, 1/1000 (27)), γδ T cell receptor (clone 86D, IgG1, 1/1000 (28)), CD25 (an activated T cell marker, clone ILA111, IgG2a, 1/2000 (29)), CD21 (a pan B cell marker, clone CC21, IgG1, 1/10 (30)) and IgA (MCA628, Serotec, Oxford, UK, IgG1, 1/1000) were used. The percentage of total cells positive for the isotype control antibodies was observed to be below 0·15% for 99·3% of all samples. Detection and quantification of antibody in the gastric lymph was carried out as detailed previously (10). Briefly, total IgA was measured using a sandwich ELISA, with purified sIgA as a standard. Antigen specific IgA was measured for both somatic L4 antigen, and L4 excretory/secretory (ES) products, with a positive reference sample included on each plate. Previously infected lambs had significantly (P < 0·05) fewer parasites than controls on day 10 after challenge in both experiments (Figure 2a). However, on day 5 a significant difference (P < 0·05) was only observed within Experiment 5.

[74] Intravenous administration of miR-124 at the effector phase of disease ameliorated EAE and reduced neuroinflammation probably through its effect on macrophages, whereby miR-124 is able to promote a phenotypic switch from classically to alternatively activated macrophage, through indirect down-regulation of transcription factor PU.1, and thereby decreased expression of activation markers CD45, MHC class II and CD86, via inhibition of C/EBP-α.[74] Such a function is probably also Dabrafenib concentration at play in the maintenance of a quiescent microglial phenotype in the normal CNS. Alternatively activated microglia can secrete a wide range of molecules that can have a neuroprotective effect

in MS/EAE, either directly, such as insulin-like growth factor 1, which promotes proliferation and differentiation of neural progenitor cells,[75, 76] or indirectly through their anti-inflammatory effect, such as the anti-inflammatory cytokines

IL-4, IL-10 and TGF-β. In vitro studies have shown that IL-4-stimulated microglia are able to instruct neural progenitor cells to differentiate into oligodendrocytes, at least in part through release of insulin-like growth factor 1.[75] A number of disease-modifying drugs that have been, or are in the process of being, approved for MS, can potentially affect microglial phenotype directly or indirectly. We shall address this issue for the two most used first-line treatments for relapsing–remitting MS, IFN-β and glatiramer acetate (GA), and for the recently approved fingolimod and dimethyl fumarate (DMF). The precise mechanisms this website through which IFN-β exerts its immunomodulatory effect in

MS are still uncertain, but generally include inhibition and apoptosis of autoreactive T cells, induction of regulatory T cells, inhibition of leucocyte extravasation through the BBB, and modulation of cytokine expression.[77] Its effect on microglia has, as yet, been poorly investigated, with only scant in vitro studies reported. Kim et al.[78] showed that IFN-β induced the expression of chemokines such as RANTES and MIP-1b in primary human microglia, through activation of at least three different partially interconnected signalling cascades Nintedanib (BIBF 1120) including nuclear factor-κB, activator protein-1 and Janus kinase/signal transducer and activator of transcription. Kawanokuchi et al.[79] addressed the effect of IFN-β on murine microglial functions such as antigen presentation and secretion of inflammatory mediators; they showed that IFN-β inhibits the antigen-presenting function of microglia through suppression of IFN-γ-induced MHC class II expression and down-regulation of the co-stimulatory molecule B7-1, and suppresses differentiation of pathogenic autoreactive T helper type 1 T cells through down-regulation of microglial IL-12 production. Surprisingly, and in accordance with the study of Dasgupta et al.

The results we present here for purified memory-phenotype CD4+ T cells and for effector-memory Th17 cells derived from obstructed kidney indicate suppression of IL-17A secretion comparable to that of naïve CD4+ T cells. In the case of memory-phenotype CD4+ T cells activated in vitro under Th17-skewing conditions, MSC contact was also associated with inhibition of proliferation and of CD25 up-regulation. These results this website are in-line with the in vitro and in vivo findings of Rafei et al. for MSC effects on MOG-specific Th17 cells in mouse EAE 14. In addition, MSC-mediated suppression

of Th17 responses has been reported for antigen-specific Th17 cells in rat EAE and autoimmune myasthenia gravis and in established autoimmune diabetes mellitus in NOD mice 32, 33. Interestingly, however, evidence for enhancement of Th17 differentiation and IL-17A production

by MSCs and fibroblasts has also been presented in a small number of studies 34, 35. The reported results suggested that MSC production of IL-6 as well as stimulation of IL-1 and/or IL-23 secretion by APCs were responsible for the observations 34, 35. In our own experiments, we have observed that administration of a non-selective COX inhibitor in MSC/Th17 co-cultures is associated with enhancement of IL-17A secretion compared with control Th17 cultures (Fig. 5A and our unpublished observation). We have also confirmed production of IL-6 and TGF-β1 by MSCs co-cultured with activated T cells (our unpublished observation). Thus, it is important to consider that MSC Selleck FK506 next inhibition of Th17 cell differentiation and activation, while potent, is conditional, being dependent upon opportune MSC/T-cell contact and upon inducible mechanisms which, when absent or subject to blockade, may unmask a paradoxical

capacity for enhancement of Th17 activity. Furthermore, in the case of naturally occurring Th17 cells from obstructed kidney (or other sites of inflammation and autoimmunity), additional experimental work will be required to distinguish between direct and indirect MSC effects on this T-cell effector phenotype. From a mechanistic perspective, we provide compelling evidence that the induced production of PGE2 by MSCs in direct contact with CD4+ T cells undergoing activation was primarily responsible for suppressive effects on naïve- and memory-phenotype Th17 cells in vitro as well as on in vivo-derived effector-memory Th17 cells. This is consistent with the report of Ghannam et al. in which indomethacin reversed MSC-mediated suppression of Th17 differentiation from human naïve, cord-blood CD4+ T cells as well as IL-17A production by Th17 clones 9. By utilizing FACS to re-purify MSCs, we convincingly demonstrate significant up-regulation of COX-2 and production of PGE2 by these cells within 12–24 h of placement in Th17-skewing cultures.

Mean area of gelatin degradation was quantified by counting a degraded area in 15–20 different fields containing approximately the same number of cell nuclei. For Matrigel migration assays, BMDMs were detached and starved in DMEM without serum for a total of 3 h. After 2 h of starving, the cells were labeled with the fluorescent dye Celltracker Blue CMAC (Invitrogen) according to the producer instruction. A total of 105 cells in DMEM without serum were then plated on BioCoat Matrigel Invasion Chambers (BD Biosciences) for 24 h. Nonmigrated cells were removed and migrated cells were counted by reading the fluorescence on the bottom side of the inserts with a Victor Multilabel

Plate Reader (PerkinElmer). For trans-endothelial migration assays, H5V cells, an epithelial cell line kindly provided by E. Dejana (FIRC Institute Proteasome inhibitor of Molecular Oncology, Milan, Italy) were plated on FluoroBlok Inserts (Falcon) for 3 days until they formed a confluent monolayer, and then activated with 5 ng/mL TNF for 2 h in DMEM. find more A total of 105 BMDMs labeled with Celltracker Blue CMAC (Invitrogen) as above described for Matrigel assays, and resuspended in DMEM without serum, were then plated on FluoroBlok inserts coated with TNF-activated H5V cells for 22 h. Percentage of migrated cells was calculated by reading the fluorescence

with a Victor Multilabel Plate Reader (PerkinElmer). Cell migration in 2D was assessed by scraping a confluent monolayer of BMDMs with a pipette tip. Then the number of cells migrating into the open space was assessed microscopically [[12]]. Quantification of migrated cells was performed counting cells migrated into the wound in ten different fields. Cells were lysed with sample buffer: 25 mM Tris, pH 6.8, 50 mM β-mercaptoethanol, 1% SDS and 5% glycerol and then analyzed with Odyssey Infrared Imaging Astemizole System (Li-cor Biosciences, Nebraska, USA) using specific antibodies. The Student’s t-test has been applied to examine the statistical significance of differences between the data. Values of *p < 0.05 or **p < 0.01, ***p

< 0.001 were taken as significant. This work was supported by a grant from Italian Association for Cancer Research (AIRC) to GB (grant 2010). The authors are indebted to Clifford A Lowell (UCSF) for having made available to them mice with the genetic deficiency of Hck and/or Fgr generated in his laboratory. The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. "
“Macrophages (Mϕ) are professional antigen-presenting cells, but when they accumulate at sites of inflammation, they can inhibit T-cell proliferation. In experimental autoimmune uveoretinitis, this limits the expansion of T cells within the target organ.

42 The concentration of PGE prostaglandins in human semen is many times higher than in other areas of the body, and semen contains 19-hydroxy PGE, which is not found elsewhere. The effects of the seminal prostaglandins are two-fold.28,30 First, a cAMP-mediated effect on T cells inhibiting clonal proliferation, as well as natural killer cell function, and biasing CD4 cells to T-helper-2 pattern of cytokine production away from one

favoring a cell-mediated response. Second, PGE is a potent agent inducing a type 2 phenotype in dendritic cells, through its capacity to inhibit IL-12. Hence, at the level of the antigen-presenting cell, PGE and 19-hydroxy PGE alter the balance of cytokines, stimulating IL-10 and inhibiting IL-12 released by these cells, reinforcing CP-868596 in vivo its direct effects and inducing tolerance of antigens that are presented together with the IL-10. While necessary for the survival of the spermatozoa, such tolerance may have adverse effects, in the face of Alectinib in vivo infection. Viruses which can be transmitted in semen (such as HIV & HPV) and other invading organisms would benefit from this switch in cytokines and

the inhibition of the cell-mediated defenses. Not only is the initial immune response affected, but repeated exposure to semen could diminish immune surveillance and the removal of virally infected cells. TGF-beta is now known to be a principal mediator of oral tolerance.26,27 The seminal vesicle is the principal source of TGFβ in rodents, where its synthesis is regulated by testosterone. In contrast, the prostate has been identified as a major site of TGF-beta in men.43 The seminal fluid content of TGF-beta is high, approximately five-fold that of serum and similar to that of colostrum. The normal range for TGF-beta in fertile men has been shown to be approximately 40–150 ng/mL, which remains relatively constant

over time. Upon deposition in the female reproductive tract at coitus, seminal TGF-beta interacts with uterine and cervical epithelial cells, to initiate a cascade of downstream effects.44,45 It has been shown to be a principal agent in Selleck Cobimetinib the post-coital inflammatory response, in mice, resulting in the recruitment and activation of leukocytes, including neutrophils, macrophages, and dendritic cells. Epithelial cells up-regulate expression of several pro-inflammatory cytokines and chemokines within several hours of coitus. In humans, exposure to semen induces neutrophil recruitment into the superficial epithelial layers of the cervix. In addition to preventing aberrant immunity to spermatozoa, seminal fluid components derived from the seminal vesicles have been implicated in inducing an immune response that promotes embryo implantation. Robertson et al.

IL-10 KO mice naturally develop inflammation in the colon from 10 to 12 weeks of age [43]; however, in the present study, the NKG2D ligand expression on small IECs was investigated in the IL-10 KO mice before any development of clinical sign of colitis. Nonetheless, we cannot exclude that NKG2D

ligand upregulation is induced by an inflammatory molecule produced in these mice, especially as we in the present study found no alterations in the intestinal IL-10 levels of the antibiotic-treated mice. In addition, decreased level of IFN-γ and IL-15 in the small intestine was observed in the vancomycin-treated mice similar to the NKG2D ligand expression and IL-15 was furthermore increased in the ampicillin-treated mice as was the NKG2D ligand expression. https://www.selleckchem.com/products/ABT-263.html This is interesting, as IL-15 is known to be directly involved in NKG2D ligand upregulation on IELs during celiac disease [5], and it is thus tempting to speculate that a less proinflammatory state, kept in check by the commensal microbes, actively keeps the NKG2D ligand expression low, although such a scenario needs experimental verification. IL-17 was however downregulated in both ampicillin-

and vancomycin-treated mice which suggests that this cytokine is not involved in the regulation of NKG2D ligands on IECs. Instead, both antibiotic treatments most likely eradicated important bacteria, for example segmented filamentous bacteria which can induce IL-17 [31, CYC202 44]. The commensal microbiota may also directly express or secrete molecules that affect NKG2D ligand surface expression. We have previously shown that propionate from propionic bacteria is involved in the opposite scenario, as it increases

NKG2D ligand expression [17]. Further studies are however needed to establish the mechanisms behind these interesting Tangeritin observations. It is noteworthy that the level of NKG2D ligand expression was substantially lower in the B6 mice housed in the Novo Nordisk animal facility compared with that in B6 mice housed at the University of Copenhagen. Differences in gut microbiota compositions in the groups of untreated control mice because of the different facility environments, sex, and animal vendors from which the mice were purchased, may explain the observed differences in NKG2D ligand expression. In general, we believe that it is important to take differences in microbiota composition into account, when comparing levels of NKG2D ligands measured by different laboratories. This could, at least partly, explain differences observed in the past. NKG2D ligand regulation by microbial interaction is supported by a growing body of data. Tieng et al. [7] have shown increased expression of NKG2D ligands on IECs after infection with certain pathogenic strains of E. coli and IECs have also been shown to express NKG2D ligands upon TLR3-dependent poly I:C treatment [45].

We hypothesized that fibroblasts and possibly other abundant tissue cell types are major sources of sST2 protein in vivo and that deletion of the proximal promoter would result in less circulating sST2 and thus disruption of normal IL-33 regulation. Instead, we found that although loss of the proximal promoter abolished fibroblast-specific ST2 expression, it had no obvious impact on the amount of circulating sST2. Figure 1A is a map of the mouse ST2 locus illustrating the location of the two promoters, the intron-exon organization and the targeting strategy to generate the proximal promoter

and enhancer knockout. Figure 1B illustrates the alternative splicing whereby exons 9–11 are SB203580 supplier either included in the final spliced LDE225 order ST2L mRNA, or not included thereby leading to incorporation of an alternative stop codon and the generation of sST2. We selectively deleted the ST2 proximal promoter (with noncoding exon 1b) and its associated enhancer element. The resulting locus contains in their place a single loxP site, yet still retains the distal promoter and all coding exons. Homozygous knockout mice bred normally and nearly all animals lacked overt developmental or pathological

manifestations. However, interestingly, two homozygous knockout mice spontaneously developed what appeared to be subcutaneous tumors on Bay 11-7085 their neck and trunk and a third animal was found moribund due to unknown causes (not shown). Possibly relevant to these observations are previous findings that sST2 is correlated with progression of breast cancer [15] and that sST2 may modulate tumor cell activity in vitro [16]. Based on previous findings, we predicted that proximal promoter deletion would not disrupt expression of ST2L in immune cells. We performed a PCR designed to specifically amplify sST2 or

ST2L cDNAs, as indicated in Fig. 1A, and found that as expected ST2L mRNA was expressed similarly in both wild type and knockout splenocytes (Fig. 1C). Little to no expression of sST2 was detected in splenocytes. Therefore, consistent with previous data, we found splenocytes express predominantly the ST2L isoform and deletion of the proximal promoter did not abolish ST2L expression. We also found that deletion of the proximal promoter had minimal effects on the expression of ST2 in bone marrow-derived mast cells (BMMCs) (Fig. 1C). BMMCs express both sST2 and ST2L transcripts and neither isoform was affected by promoter deletion. Also, BMMCs from knockout mice developed normally in vitro (based on c-kit expression) and expressed equivalent amounts of ST2L on the cell surface compared with wild-type BMMCs (Fig. 1D). Moreover, knockout BMMCs responded to IL-33 by secreting equivalent amounts of IL-6 as compared with wild-type BMMCs (Fig. 1E).

“
“Please cite this paper as: Di Filippo, Monopoli, Ongini, Perretti and D’Amico (2010). The Cardio-Protective Properties of Ncx-6550, a Nitric Oxide Donating Pravastatin, in the Mouse. Microcirculation17(6), 417–426. Objective:  Determine the cardio-protective properties of a nitric oxide-releasing pravastatin (Ncx-6550), in comparison to pravastatin. Methods:  A mouse model of myocardial

infarct was used assessing tissue damage both at 2 and 24 hour post-reperfusion, administering compounds both prophylactically and therapeutically. Results:  Ncx-6550 induced a significant dose-dependent (2.24–22.4 μmol/kg i.p.) cardioprotection in the two hour reperfusion protocol. In vehicle-treated mice, infarct size (expressed as fraction of area at risk; Inhibitor Library high throughput IS/AR) was 41.2 ± 1%, and it was reduced to 22.2 ± 0.9% and 32.6 ± 0.9% following 22.4 and 6.72 μmol/kg Ncx-6550 (p < 0.05). 22.4 μmol/kg Ncx-6550 also increased cardiac levels of the enzyme heme oxygenase-1. Treatment of mice with pravastatin induced significant reduction of myocardial injury only at 22.4 μmol/kg (IS/AR value: 33.7 ± 0.9%). In a 24 hour

reperfusion protocol, Ncx-6550 and pravastatin were tested only at 22.4 μmol/kg i.p. being given either one hour prior to ischemia (prophylactic protocol) Acalabrutinib in vivo or one hour into reperfusion (therapeutic protocol). With either treatment scheme, Ncx-6550 produced higher cardioprotection compared to pravastatin, as reflected also by a reduction in the incidence of lethality as well as in circulating troponin I and interleukin-1β levels. Conclusions:  These results indicate Ncx-6550 as a novel therapeutic agent with a potential for the treatment of

myocardial infarct. “
“Three‐dimensional images of microvascular trees, within their surrounding tissue, are obtainable by micro‐computed tomography (micro‐CT) imaging of intact small animals or tissue specimens. With a resolution down to a few micrometers, these images can be used to measure the interbranch segment diameters, branching angles, volume of tissue perfused, and study the vascular anatomic relationships Exoribonuclease to organ microstructures such as glomeruli in kidney, hepatic lobules in liver, and so on. Such data can be used to model intravascular flow, endothelial shear stress, and altered branching geometry such as that which may occur in localized angiogenesis and around tissue infarction and tumors. Endothelial permeability can also be evaluated using cryostatic micro‐CT methods, and special contrast agents can be used to convey permeability and vascular lumen volumes. In this chapter, we provide background information of micro‐CT image systems, sample preparation methods such as ex vivo casting methods, in situ contrast agent injection techniques, special considerations pertaining to in vivo studies, and the use of probes (such as microspheres in “simulated embolization” experiments).

“
“Autologous microvascular breast reconstruction is an increasingly common procedure. While arterial

anastomoses are traditionally being hand-sewn, venous anastomoses are often completed with a coupler device. The largest coupler size possible should be used, as determined by the smaller of either the donor or recipient vein. While its efficacy check details has been shown using 3.0-mm size and greater couplers, little is known about the consequences of using coupler sizes less than or equal to 2.5 mm. Methods: A retrospective chart review of patients undergoing autologous breast reconstruction was conducted at NYU Medical Center between November 2007 and November 2011. Flaps were divided into cohorts based on coupler size used: 2.0 mm, 2.5 mm, and 3.0 mm. Outcomes included LY294002 solubility dmso incidence of arterial or venous insufficiency, hematoma, fat necrosis, partial flap loss, full flap loss, and need for future fat grafting. Results: One-hundred ninety-seven patients underwent 392 flaps during the study period. Patients were similar in age, type of flap, smoking status, and radiation history. Coupler size less than or equal to 2.0 mm was found to be a significant risk factor for venous insufficiency (P = 0.038), as well as for development of fat necrosis (P = 0.041) and future need for fat grafting (P = 0.050). In multivariate analysis, body mass index was found

to be an independent risk factor for skin flap necrosis (P = 0.010) and full flap loss (P = 0.035). Conclusions: DNA ligase Complications were significantly increased in patients where couplers of 2.0 mm or less were used, therefore to be avoided whenever possible. When needed, more aggressive vessel exposure through rib harvest, the use

of thoracodorsal vessels or hand-sewing the anastomosis should be considered in cases of internal mammary vein caliber of 2.0 mm or less. Therapeutic Level III. © 2013 Wiley Periodicals, Inc. Microsurgery 33:514–518, 2013. “
“Intraoperative near-infrared indocyanine-green (ICG) angiography enables the visualization of microvascular perfusion and may help in the early detection of complications. The purpose of the present study was to examine whether the effect of microvascular stenoses can be quantitatively assessed by analysis of ICG-angiography in a microvascular model. Graded stenoses and total vessel occlusion of the carotid, aorta, and femoral arteries were created in 25 Wistar rats. Stenoses were graded to reduce arterial flow by 25%, 50%, 75%, and 100% of baseline flow as measured by transit-time flowmeter analyzing the emission signal of the ICG detected and investigated by the mathematical software tool (FLOW 800). ICG angiography was performed to assess vessel perfusion and flow curves were analyzed and correlated with the stenosis rate. A total of 576 investigations were performed. The area under the curve (P < 0.001), first and second maximum (P < 0.001), and the maximum slope to the first maximum (P < 0.

As the asymmetrical pattern seems to merge some features of the other two—with infants paying attention to the mother’s focus, as in symmetrical, while refraining from acting together, as in unilateral—it has been presumed to work as a transitional state between the unilateral and the symmetrical.

Akt inhibitor With respect to the subcodes, we also expected symmetrical coregulation to change with advancing age, with affect sharing and action sharing occurring first and language sharing occurring later. In fact, the former patterns employ skills, like expressive and motor acts, that are already part of the infant’s repertoire at the beginning of the observational period, to communicate with others in person-focused interaction or to explore physical reality in object-focused interaction, respectively. By contrast, the latter pattern requires skills that infants still lack at the outset and that may be recruited for coregulation only in a subsequent period. Finally, as shown in previous studies on social play (Camaioni et al., 2003), we expected to see individual differences in the rate of developmental change. Because of the focus

on developmental change and individual differences, a multiple case study method (Camaioni et al., 2003; Fogel, 1990; Hsu & Fogel, 2001; Lavelli & Fogel, 2002) was used. This method implies a multiple timepoint design, providing a dual Edoxaban opportunity to make meaningful statements about the group and also to capture the rate and the shape of developmental trajectories for each case. Ten dyads were video-taped weekly at home, interacting with Ibrutinib cell line a toy tea set (dishes, forks, knives, spoons, cups, etc.) brought by the observer. Four girls and six boys were observed, with the girls belonging to dyads 1, 4, 8, 9 and the boys to dyads 2, 3, 5–7, 10. All of the infants were full term at birth; five of them were first borns, four were second borns, and one was third born. All children belonged to biparental middle-class families,

living in a town of central Italy. The observations started when infants were 10-months-old (M = 10.7 months) and continued until they were 24-months-old (M = 24.9 months). Each session lasted about 5 min (M = 5 min 2 sec). Mothers were sitting with their infants at their favorite table with the toy tea set at their disposal. No other instruction was given to them than to play as usual and to ignore the observer as much as possible. All the mothers were informed about the general interest of our study and all of them agreed to participate. At the end of the study, they received an edited tape of the observational periods as a gift for their intensive participation in the project. The Relational Coding Scheme developed by Alan Fogel (1993) was employed to assess mother–infant coregulation.