Methods:  This is a retrospective study on patients with prolifer

Methods:  This is a retrospective study on patients with proliferative lupus nephritis who had received PSI treatment. Results:  Seven patients were included. Two patients had concomitant membranous lupus nephropathy. The indications for PSI included mycophenolate mofetil intolerance (n = 4), history of malignancy (n = 2) and leucopoenia (n = 1). Five patients were given PSI when disease was active. Two had treatment discontinued because of acute cholecystitis and leucopoenia, respectively. In the other three patients combined steroid and PSI treatment as induction therapy led to improvements in serology, see more renal function and

proteinuria. In two patients treated with PSI and low-dose steroid as maintenance immunosuppression, both maintained stable lupus serology, renal function and proteinuria over 18 months. Side-effects included dyslipidemia and oral ulcers. Conclusion:  Proliferation signal inhibitors warrants buy Osimertinib further investigation as an alternative immunosuppressive treatment in lupus nephritis. “
“Aim:  The aims of the study were to translate the Kidney Disease Quality of Life – Short

Form version 1.3 (KDQOL-SF ver. 1.3) questionnaire into Iranian (Farsi), and to then assess it in terms of validity and reliability on Iranian patients. Methods:  The questionnaire was first translated into Farsi by two independent translators, and then subsequently translated back into English. After translation disparities had been reconciled, the final Iranian questionnaire was tested. An initial test–retest reliability evaluation was performed over a 10 day period on a sample of 20 patients recruited from a larger group (212 patients with end-stage renal disease on haemodialysis). Afterwards, reliability was estimated by internal consistency, and validity was assessed

using Methocarbamol known group comparisons and constructs for the patient group as a whole. Finally, the factor structure of the questionnaire was extracted by performing exploratory factor analysis. Results:  All of the scales in the questionnaire showed good test–retest reliability (i.e. intraclass correlations between test and retest scores were >0.7). All of the scales met the minimal criteria (0.7) for internal consistency and Cronbach’s-α ranged 0.71–0.93. Furthermore, results from a discriminate validity evaluation showed that the questionnaire could be used to discriminate between subgroups of the patients. Finally, a principal component analysis of the disease-specific scales indicated that this part of the questionnaire could be summarized into an 11 factor structure that jointly accounted for 79.81% of the variance. Conclusion:  The Iranian version of the KDQOL-SF questionnaire is both highly reliable and valid for use with Iranian patients on haemodialysis. “
“Aim:  Alport syndrome (AS) is a progressive renal disease characterized by hematuria and progressive renal failure.

Bacterial genomic DNA was extracted from the strains using Wizard

Bacterial genomic DNA was extracted from the strains using Wizard Genomic DNA Purification kit (Promega, Madison, WI). We confirmed the appropriate extraction of the bacterial DNA by PCR using universal primers (Tamura et al., 2005) and P. gingivalis-16S rRNA-specific Everolimus primers (Amano et al., 1999). The PCR amplification was performed in a total volume of 20 μL consisting of PCR Pre-Mix (STD02-M50h; SolGent, Korea), 0.5 μM each primer, and 5 μL of the template DNA solution in sterile distilled water. The amplification reaction was performed in a thermal cycler (Model 9700;

Applied Biosystems, Branchburg, NJ) with the following cycling parameters: an initial denaturation at 95 °C for 5 min, 30 cycles consisting of 94 °C for 30 s, 55 °C for

30 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min. Positive and negative controls were included in each PCR set and in the processing of all samples. The PCR products were electrophoresed on 1.8% agarose gels. The previous type II primers hybridized to the DNA of P. gingivalis strains harboring type Ib as well as type II fimA, while the new primers specifically amplified only the DNA fragment of type II fimA-P. gingivalis (Table 1). The genotype specificity of the type II (new) primers was further confirmed using the pure culture of strain HG1691 (type Ib) and the mixed culture of strains A7A1-28 (type II) and HG1691. The type Ib primers were used as a positive control. The previous type II primers generated a 257-bp PCR product from the DNA of HG1691, while the new primers did not (Fig. 1a). The sensitivity of the type II and type II (new) primers LGK-974 molecular weight was compared using a decreasing amount (5000–0.5 pg) of A7A1-28 genomic DNA. These two sets of the primers generated PCR products of the expected sizes, 257 and 292-bp, respectively, from as low as 5 pg of the DNA (Fig. 1b). But, the band intensity of the PCR product amplified from 5 pg of the DNA using the new primers, which analyzed using ImageJ (NIH), was 3.14 ± 0.85 (mean ± SD)-fold higher than that amplified using the previous type II primers. Rebamipide Using both the sets of

the primers, we determined the prevalence of type II fimA in the patients with peri-implantitis (PI), which is the destructive inflammatory process affecting the soft and hard tissues surrounding dental implants, as the associated microbiota resembles that found in periodontitis (Becker et al., 1990; Rams et al., 1991). According to an ethically acceptable protocol approved by the IRB Committee of Kyung Hee University School of Dentistry (approval number: KHUSD 1009-02), subgingival plaque samples were taken from 171 Korean adults with PI; two paper points were inserted into the PI pocket for 30 s and then removed and placed in a sterile tube with 1 mL of sterile phosphate buffered saline. Bacterial genomic DNA was extracted from the samples as described earlier.

, Amesbury, Wiltshire, UK) has been demonstrated to


, Amesbury, Wiltshire, UK) has been demonstrated to

allow both characterization of exosome size, as well as direct quantification of exosomes.[41, 42] There are particular considerations required in the purification and storage of urinary exosomes. Tamm-Horsfall protein (uromodulin) can form fibrillary aggregates in urine especially at low temperature which can entrap exosomes and prevent their efficient isolation and purification by centrifugation. The entrapment can be eliminated by using the reducing agent dithiothreitol (DTT).[43] Currently, there is no standard protocol for collection, processing and storage of urine samples that will allow correct, find more comparable and reproducible urinary exosome analyses. Protease inhibitors and storage at −70°C gave a better recovery of urinary exosomes than at −20°C.[44] Nephrotic urine contains a large amount of proteins that

tend to be retained after ultracentrifugation, which learn more can affect the detection of exosomal proteins. Recent studies have demonstrated that ultracentrifugation followed by size exclusion chromatography can enrich and purify exosomes in nephrotic urine sample.[45] Despite being first described in the early 1980s,[46, 47] exosomes garnered minimal scientific attention as their role was considered little more than to discard unwanted cellular components, until the 2000s. As a result, their biological and physiological roles are still being discovered. Currently, exosomes are known to play significant roles in intercellular communication, non-classical protein secretion, immunomodulation, pathogen biology and cancer progression. Intercellular communication was previously thought to be limited to cell-to-cell adhesion contact (gap junctions) or secreted

signals such as hormones, neurotransmitters, and cytokines released from cells and acting in an autocrine or paracrine manner. acetylcholine Exosomes can mediate a novel intercellular communication mechanism. They can be transported between different cells and adhere to target cells with high specificity via receptor or adhesion molecules but without membrane fusion leading to receptor activation and downstream signalling. Alternatively, exosomes can fuse with target cells or be incorporated by target cells via endocytosis.[10, 48] Transferred RNAs can affect protein production and gene expression in target cells.[49] The exosomal lipid bilayer protects proteins, mRNAs and miRNAs from degradation, which may make this intercellular communication pathway more reliable in comparison with free floating proteins and RNAs and enable targeted delivery of a higher concentration of messenger. A physiological role for exosomes was first described in the maturation process of erythrocytes from reticulocytes.[14, 50] It is known that transferrin receptors are lost during this maturation process.

The strength of the mAb 20 1-induced stimulation obtained with BT

The strength of the mAb 20.1-induced stimulation obtained with BTN3A1-transduced CHO cells is remarkable and considerably higher than that observed with CHO Chr6 BTN3A1 cells (Fig. 1) or RAJI cells [8]. To some extent this might be due to the three- to fourfold higher BTN3A1 expression by CHO BTN3A1 compared with CHO Chr6 BTN3A1 cells,

but it cannot be excluded that Chr6 negatively affects this type of activation. Other cell type-specific features of the presenting cells may also act on reporter cell activation, e.g. BTN3A1-transduced murine L929 cells which, like CHO BTN3A1 cells, do not present PAg, induce a much weaker, although statistically significant, mAb 20.1-triggered response (data not shown). The strong mAb 20.1-induced stimulation observed with CHO BTN3A1 cells as presenters and Vγ9Vδ2 TCR53/4-CD28+ H 89 T cells as reporter cells contrasts with Vavassori et al. [12], who found an excellent PAg- but not mAb 20.1-induced activation of Vγ9Vδ2 selleck compound TCR-transgenic mouse reporter cells. Again differences between PAg- and mAb 20.1-induced activation could be due to differences between the presenting cells (human

A375 cells versus rodent cells), but could also be due to different reporter cell origin (cell line versus artificially in vivo-matured Vγ9Vδ2 T cells [12, 14]) and, finally, different TCR specificities might also play a role. Surprisingly, CHO Chr6 BTN3A1 cells, as well as several of the Chr6-containing hybridomas (Fig. 1), lacked the capacity to activate the reporter cells in the presence of the sec-butylamine. Alkylamines and aminobisphosphonates inhibit FPPS and therefore have been proposed to act by causing accumulation of IPP in the presenting cells [5]. Thus, either inhibition of FPPS activity is not the common feature that makes both substances Vγ9Vδ2 T-cell activators or alkylamines require additional cellular compound(s), which are missing in CHO Chr6 BTN3A1 cells, to exert FPPS inhibition. Independent evidence that, in addition to BTN3A1, human Chr6 is needed to convert rodent

cells into mediators of PAg-dependent Vγ9Vδ2 T cell activation was obtained using total human PBMCs as reporter cells. In this experiment we did not include mAb 20.1, since binding to BTN3A1 expressed by the γδ T cells would have complicated interpretation. medroxyprogesterone Figure 2 summarizes data comparing zoledronate-pulsed CHO Chr6 and CHO Chr6 BTN3A1 cells for induction of CD69 expression by the Vγ9Vδ2 T-cell population contained in total PBMCs. Most importantly, only the Vδ2+ T cells (essentially identical to Vγ9Vδ2 T-cell population) were activated. Furthermore, activation was better with CHO Chr6 BTN3A1 than with CHO Chr6 cells, while CHO (not shown) and CHO BTN3A1 cells failed to induce a PAg response. This experiment provides independent experimental proof that, in addition to BTN3A1, other gene(s) on Chr6 are indeed mandatory for PAg action.

The results demonstrate that the highest percentage of inhibition

The results demonstrate that the highest percentage of inhibition by GPC81–95 treatment is observed 24 hr after LPS stimulation (Fig. 5d). The inhibitory effect of GPC81–95 treatment on the secretion of TNF-α was analysed in 10 independent experiments (performed on different days) and demonstrates that GPC81–95 treatment significantly suppresses TNF-α production (P = 0·0002). The average inhibition observed in each experiment is shown (Fig. 5f). To compare the inhibitory effects of recombinant TGF-β1 and GPC81–95, PBMCs were treated with different concentrations of rTGF-β1, GPC81–95, or PBS diluents (as negative control) for 5 hr and the cells were stimulated with

LPS. The percentage of TNF-α inhibition by GPC81–95 treatment DAPT purchase was equivalent to the percentage of inhibition seen with a high dose of recombinant TGF-β1 (Fig. 5e). The inhibitory effects of GPC81–95 and VIP, which has been shown to possess anti-inflammatory properties in

vitro and in vivo,22–25 were confirmed in our system (Fig. 5g). To study the role of TGF-β1 in GPC81–95-mediated inhibition, anti-TGF-β1 monoclonal antibody (mouse IgG1) was added to the culture and the results demonstrate that this blocking antibody abrogated the inhibition seen with GPC81–95 treatment. The inhibitory effects of GPC81–95 treatment were not diminished when a mouse see more IgG1 isotype control (Fig. 5h), or when anti-LAP (TGF-β1) monoclonal antibody (mouse IgG1) was added to the culture (data not shown). The results demonstrate that GPC81–95 suppress TLR4-ligand-induced TNF-α production in a TGF-β1-dependent manner. The depletion of CD4+ T cells from the PBMCs also abolished the inhibitory effects of GPC81–95 (Fig. 5i), suggesting that the anti-inflammatory effect of GPC81–95 is mainly mediated by CD4+ T

cells. In this study, we demonstrate Flavopiridol (Alvocidib) that a 15-mer GPC-derived peptide (GPC81–95) has the intrinsic ability to stimulate the expression of LAP (TGF-β1) on CD4+ T cells. The bioactivity of GPC81–95 could not be attributed to potential contaminants such as non-GPC81–95 peptide derivatives produced during peptide synthesis or TLR ligands. Finally, we show that GPC81–95 suppresses TLR4 ligand-induced TNF-α secretion, which is dependent on the presence of both TGF-β1 and CD4+ T cells. Our data show that GPC81–95 does not induce cell death, which has previously been shown to stimulate TGF-β1 release and thereby suppresses the production of pro-inflammatory cytokines by monocytes.21 GPC81–95 suppresses TNF-α production but does not inhibit the production of other pro-inflammatory cytokines including IL-1β by PBMCs stimulated with LPS. This is in accordance with the results demonstrating that recombinant TGF-β1 inhibits LPS-induced TNF-α production but does not alter the levels of IL-1α and IL-1β production.

Although the standard deviations were high in all groups, our res

Although the standard deviations were high in all groups, our results are statistically significant in all relevant comparisons: when comparing changes in BPI-ANCA values in patients with or without EIGSS, patients with or without LTX and BPI-ANCA values before and after EIGSS and LTX. The non-operated group included more chronically infected patients than the EIGSS

group, and consequently, this group had higher BPI-ANCA levels, but those were unchanged over time. Based on experience from patients with granulomatosis with polyangiitis (Wegener’s) (GPA), where IgG ANCA is also associated with disease activity [21], it must be expected Metformin mw that the levels of BPI-ANCA may depend on the assay methodology [22]. At present, the assays available DNA Damage inhibitor for the detection of BPI-ANCA have not been standardized. As patients with CF have more positive IgA than IgG BPI-ANCA, it may be necessary to further investigate whether or not this difference is real and also whether or not different assays might be more sensitive. ANCA is a family

of autoantibodies directed at different components in the granules of the cytoplasm of human neutrophils. It is of interest that the presumed mechanism for BPI-ANCA production is a costimulation of dendritic cells with BPI complexed to P. aeruginosa surface antigens [9] and other Gram-negative bacteria, Venetoclax research buy whereas the presumed mechanisms for production of PR3-ANCA include molecular mimicry [23], a disrupted balance between the naturally occurring PR3-ANCA and its anti-idiotypic antibody [24], and epigenetic modifications leading to inappropriate expression of PR3 [25]. Another aspect is the possible pathogenic role of ANCA. In microscopic polyangiitis (MPA), ANCA is mainly directed against myeloperoxidase (MPO). MPO-ANCA and to a lesser extent Pr3-ANCA has been shown to activate TNF-primed neutrophils

[26]. Later, it has been possible to mimic MPA manifestations in experimental animals by infusing MPO-ANCA [27], and recently, a similar observation has been made by the same author, inducing GPA-like manifestations in mice with a humanized immune system using PR3-ANCA [28]. So far, a pathogenic role for BPI-ANCA has not been reported, but BPI-ANCA may also play a pathogenic role by neutralizing BPI, which is a potent inhibitor of Gram-negative bacteria [5, 8]. No standardized guidelines exist regarding the criteria for sinus surgery in patients with CF [10, 29]. Our results indicate that EIGSS with the intensive postoperative treatment regimen should be performed in selected CF patients with sinus infection. The possible long-term benefit of EIGSS in patients with CF has to await postoperative follow-up studies on the quality of life, frequencies of lung colonizations and the need for LTX.

Such documents are peer-reviewed, but not copy-edited or typeset

Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Airway remodelling contributes to increased morbidity and mortality in asthma. We have reported that triptolide, the major component responsible for the immunosuppressive and anti-inflammatory effects of Tripterygium wilfordii Hook F, inhibited pulmonary inflammation in patients with steroid-resistant asthma. In the present study, we investigated whether triptolide inhibits airway remodelling

in a mouse asthma model and observed the effects of triptolide on selleck products the transforming growth factor-β1 (TGF-β1)/Smad pathway in ovalbumin (OVA) -sensitized mice. BALB/c mice were sensitized to intraperitoneal OVA followed by repetitive OVA challenge for 8 weeks. Treatments included triptolide (40 μg/kg) and dexamethasone (2 mg/kg). The area of bronchial airway (WAt/basement membrane perimeter) and smooth muscle (WAm/basement membrane perimeter), mucus index and collagen area were assessed 24 hr after the final OVA challenge. Levels of TGF-β1 were assessed by immunohistology and ELISA, levels of TGF-β1 mRNA

were measured by RT-PCR, and levels of pSmad2/3 and Smad7 were assessed by Western blot. Triptolide and dexamethasone significantly reduced allergen-induced increases in the thickness of bronchial airway and smooth muscle, mucous gland hypertrophy, goblet cell hyperplasia and collagen deposition. Levels of lung TGF-β1, TGF-β1 mRNA and pSmad2/3 were significantly reduced in mice treated with triptolide and dexamethasone, and this was associated MLN0128 ic50 with Farnesyltransferase a significant increase in levels of Smad7. Triptolide may function as an inhibitor of asthma airway remodelling. It may be a potential drug for the treatment of patients with a severe asthma airway. Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The morbidity and mortality of asthma have increased sharply worldwide and it has become a severe global public health problem.1 The frequent occurrence of injury and repair initiated

by chronic inflammation could lead to structura1 changes in the airway, collectively termed airway remodelling. Airway remodelling is characterized by airway wall thickening, subepithelial fibrosis, increased smooth muscle mass, angiogenesis and increased mucous glands.2,3 Generally, airway remodelling is thought to contribute to airway hyper-responsiveness and irreversible airflow limitation. Severe asthma has a distinct pathophysiology including airway remodelling that contributes to the decreased effectiveness of standard therapy. The treatment strategy for asthma airway remodelling consists mainly of the use of bronchodilators (such as β-agonists, theophylline, anti-cholinergics and anti-leukotrienes).

Initially, Xiao et al demonstrated that wild type

Initially, Xiao et al. demonstrated that wild type AZD2014 in vivo or C4-deficient mice exhibited symptoms of ANCA-associated glomerulonephritis while C5 or fB-deficient mice did not develop disease.65 Further investigation also demonstrated that this anti-MPO antibody-induced disease could also be prevented by administering a C5 inhibitory antibody.69 The involvement of complement is also supported by several clinical studies that showed the presence of complement components in renal biopsies from ANCA-associated glomerulonephritis patients.70,71 The mechanistic link between

ANCA-induced neutrophil activation and initiation of the AP complement system remains to be elucidated, and whether anti-complement therapy might be effective clinically is yet to be established. Unlike systemic causes of glomerulonephritis, MPGN is defined by mesangial cell proliferation and double contours in the GBM from rapid expansion.72 Subendothelial

or intramembranous deposits in glomeruli cause these morphological changes, and the location and contents of these deposits distinguish the subclasses of MPGN.57,72 MPGN type I has subendothelial immune complexes with C1q and is associated with classical pathway complement activation.72,73 Some consider MPGN type III a subset of type I, as it has the same features of type I with additional subepithelial deposits.72 MPGN type II, sometimes called dense deposit disease, does not have immune complexes, but instead is identified by electron-dense intramembranous deposits.74,75 MPGN is a rare disease, observed in USA and western Europe in 2–7% of renal biopsies, but in certain populations Y-27632 nmr of eastern European, African and Asian descent it has been found in up to BCKDHA 30% of renal biopsies.73 Regardless of its incidence, the prognosis for MPGN is poor as treatments are limited and often unsuccessful. While type I MPGN

has been linked to the classical pathway, type II MPGN is associated with overactive AP complement activity,76 often due to the presence of an immunoglobulin termed C3 nephritic factor that binds to the AP C3 convertase and delays its inactivation.72 Interestingly, many cases of MPGNII have also been documented where patients have defective or deficient fH.77,78 Many MPGNII patients also have ocular drusen deposits, which are linked to uncontrolled AP activity and age-related macular degeneration (AMD) pathogenesis.75,78,79 Animal studies have confirmed the role of overactive AP activity in the development of MPGNII. Both pigs with a natural mutation of fH80 and mice engineered by gene targeting to be deficient in fH developed MPGN that resembled the human disease.64 fH knockout mice had low circulating levels of C3 but strong C3 and C9 deposition within the kidney, especially along the capillary walls and mesangium in glomeruli.64 By 8 months the fH knockout mice had spontaneously developed electron-dense deposits similar to those seen in MPGNII patients.

[49] In rats and mice, the HPA axis expresses important differenc

[49] In rats and mice, the HPA axis expresses important differences from that found in humans. For example, the major product of HPA axis activation in humans is cortisol, while that in most rodents is corticosterone.[50] Moreover, the development of the fetal adrenal gland in rats and mice is markedly different with major relative deficiencies in important enzymes and preference for different substrates. In these species, EMD 1214063 the response to stress may lead to fundamentally different means of pregnancy failure, including a decreased level of circulating progesterone.[51] While

rodent models may not be ideal for the examination of the role of HPA axis in normal pregnancy, evolving rodent models may be of interest in understanding the interaction of the HPA axis and stress in parental behavior.[52] Sheep have been used as a model of maternal[53] and fetal HPA axis function during pregnancy. In this animal model, it is the development and activation of the fetal HPA that is the primary driver of parturition,[54] and stresses such as hypoxia activate the HPA axis in sheep and lead to preterm labor.[55] The maternal–fetal interface in humans includes close contact

between maternal and fetal cells not only within the placenta and uterus[8] but also within the maternal and fetal circulations, as cellular traffic has been shown in either direction.[56, 57] The expression of proteins unique to the mother on fetal cells has raised a decades-long Epacadostat debate over the critical pathways and mechanisms needed to assure both immune tolerance

C-X-C chemokine receptor type 7 (CXCR-7) and protection of the fetus from infection.[58] Humans can mount an immune response against fetal antigens during pregnancy,[59] and it is clear that there is an intricate interaction between maternal immune cells and trophoblast.[60, 61] This interaction may be of benefit to the evolving conceptus[62] or may be involved in early pregnancy loss or other adverse pregnancy outcomes.[63] Activation of local innate immunity within the myometrium is thought to play a role in parturition[64] and in premature uterine contractions.[65] In humans, certain pathogens are more deleterious during pregnancy as compared to the non-pregnant state,[66] while others are not,[67] and the role of the placenta as a safe harbor for evolving pathogens has been described.[68] Some infection syndromes that occur in humans occur only under contrived conditions in animals.[69] Moreover, some organisms, such as CMV, are different in different hosts.[70] Both the peculiarities of the immune response and the infectious agent must be taken into consideration when using an animal model to understand the function of the immune response during pregnancy.

Size and luminance of all stimuli were also matched to ensure tha

Size and luminance of all stimuli were also matched to ensure that high- and low-level stimulus differences could not influence this study (see Figure 1). Participants were seated on a chair in front of a 17” TFT Tobii T60 monitor. Images were presented on the monitor using Tobii Studio software (Tobii Technology AB; During stimulus presentation, the Tobii monitor recorded gaze location for both eyes based on the reflection

of near-infrared light from the cornea and pupil. Gaze information was sampled at a frequency of 60 Hz. Monitor specifications included an accuracy of 0.5 degrees of the visual angle and a tolerance of head movements within a range of 44 × 22 × 30 cm. Electroencephalogram (EEG) was recorded continuously throughout the ERP task using a 128-channel HydroCel Geodesic Sensor Net (HCGSN), which was referenced on-line to vertex (Cz). For a subset of participants, a

NetAmps INCB024360 solubility dmso 200 amplifier was used and the Pexidartinib electrical signal was amplified with a .1- to 100-Hz band pass filter. For the remaining infants, a NetAmps 300 amplifier was used with no band pass filter (an analysis using amplifier type as a between-subjects factor is described in the ERP results section). All data were digitized at a 500 Hz sampling rate and stored on a computer disk for further processing and analysis. E-Prime software was used for stimulus presentation, and NetStation software was used for EEG data acquisition and postprocessing. The eye-tracking and ERP tasks took place over a 2-day

period for each infant. On Day 1, infants completed the initial portions of the eye-tracking task. Participants were seated in a chair in front of a Tobii T60 monitor in an electrically and sound-shielded testing room with dim lighting. The chair was positioned such that each participant’s eyes were approximately 60 cm from the monitor. Before beginning the eye-tracking experiment, participants completed a calibration procedure to ensure the eye-tracker was adequately tracking gaze. In this calibration Inositol monophosphatase 1 procedure, a red dot appeared at 5 locations: Each of the four corners of the monitor and the center of the screen. Following calibration, the Tobii Studio program reported whether the eye-tracker successfully picked up gaze at the five locations. If calibration was successful, the experimental procedure was begun. If calibration was unsuccessful, the monitor and chair were adjusted and the calibration procedure was rerun until it successfully picked up on all five locations of gaze. Following calibration, infants began the Day 1 portion of the visual paired comparison task (VPC). During all phases of the VPC, faces were presented side by side, each measuring 14 × 14.5 cm on the screen and separated by a distance of 3.5 cm. With infants positioned at 60 cm from the screen, this resulted in each face subtending a visual angle of 13 degrees.