40 Recombinant antibodies Kinase Inhibitor Library for clinical therapeutic use in humans are expressed in low yields in mammalian cells, which accounts for their high cost. To cut costs, cPIPP was expressed as a periplasmic protein in tobacco leaves at a high yield of 20 mg of purified protein per Kg fresh tobacco leaves.41 Being given that it was expressed in endoplasmic reticulum of the leaves, plant-specific fucose and xylose residues were not loaded on the antibody.42 cPiPP had an affinity of 1.9 × 1010 m−1 for hCG. It was totally devoid of cross-reaction with hFSH and hTSH and had <5% cross-reaction with hLH. The antibody was fully competent to block hCG-induced gain

of uterine weight of immature mice in vivo and hCG-induced testosterone production by Leydig cells in vitro.40,41 Its efficacy was also tested in a human cell system. Placental villi cytotrophoblasts, isolated from placental villi of MTP cases, on culture in a medium containing anti-hCG antibodies failed to fuse into syncytium. Furthermore, the production of progesterone by the placental cells was fully blocked by cPiPP.26 These observations vouch for the suitability of cPiPP for use as a vacation contraceptive and for non-surgical termination of pregnancy. Choriocarcinoma trophoblast cells are known to make and secrete hCG.43,44 The cells carry receptors for hCG, by virtue of selleck monoclonal humanized antibody inhibitor which hCG

acts as an autocrine growth factor for these cells. Radio-iodinated PiPP bound to these cells in vitro. JEG cells administered to Nude mice form a cancerous implant. Injection of 131I-PiPP to such mice led to selective localization of radioactivity

at 3-mercaptopyruvate sulfurtransferase the tumor site, whereas the radioactivity of a similarly radio-iodinated non-relevant antibody is distributed randomly all over the body45 (Fig. 1a,b). The binding of the radio-iodinated PiPP to tumor cells is further confirmed by histioradiography (Fig. 1c). These studies clearly demonstrate the utility of the recombinant antibody for imaging and selective delivery of radiations to the tumor cells. It could be of particular utility for tracing of metastasis of such cancers. The curious phenomenon of cancer cells expressing hCG or its subunits has been discussed elsewhere in this article. We carried out studies on T-lymphoblastic leukemia MOLT-4 and lymphocytic leukemia U-937 cells, both available from ATCC. Both MOLT-4 and U-937 cells were bound with cPiPP. The binding as studied by flow cytometry was on the membranes and was specifically competed by authentic purified hCG.46 hCG was not picked up from other cells but was indeed synthesized by the cancer cells, as permeabilized MOLT-4 cells enabled the detection of the presence of hCG within the cells, to which the antibody permeating in the cells could bind. Incubation of MOLT-4 cells with anti-hCG antibodies did not however impair the viability and multiplication of these cells. Nor were the cells lysed by cPiPP in the presence of complement.

An accurate genetic diagnosis of AS is very important

for genetic counselling and even prenatal diagnosis. Methods:  We detected mutation of COL4An by amplifying the entire coding sequence mRNA buy Navitoclax of peripheral blood lymphocytes using polymerase chain reaction (PCR) in five Chinese AS families who asked for genetic counselling and prenatal diagnosis, then performed prenatal genetic diagnosis for four families. Mutation analysis of the foetus was made using DNA extracted from amniocytes. Foetus sex was determined by PCR amplification of SRY as well as karyotype analysis. Maternal cell contamination was excluded by linkage analysis. Results:  Four different COL4A5 gene variants and two COL4A3 gene variants were detected in the five families. Because there was a de novo mutation in family 2, prenatal diagnosis was performed for the other four families. Results showed a normal male foetus for family 1 and family Selisistat molecular weight 4, respectively. Results showed

an affected male foetus for families 3 and 5, and the pregnancies were terminated. Conclusion:  An easier, faster and efficacious method for COL4An gene mutation screening based on mRNA analysis from peripheral blood lymphocytes was established. Prenatal genetic diagnosis was performed in four AS families in China. “
“Aim:  Cardiovascular disease (CVD) is the leading cause of death among chronic

kidney disease (CKD) patients. The role of vitamin D remains controversial in this process. We evaluated the relationship between Epothilone B (EPO906, Patupilone) 25-hydroxyvitamin D, abnormal T helper cells (CD4+CD28null cells), systemic inflammation and atherosclerosis in CKD patients. Methods:  A total of 101 stage 4–5 non-dialysis CKD patients and 40 healthy controls were studied. Common carotid artery intima media thickness (CCA-IMT) was measured with an ultrasound system. 25(OH) vitamin D and highly sensitive C-reactive protein (hsCRP) were measured in serum by enzyme linked immunosorbent assay. The frequency of circulating CD4+CD28null cells was evaluated by flowcytometry. Results:  CKD subjects exhibited higher CCA-IMT (0.71 ± 0.01 vs 0.56 ± 0.01 mm, P < 0.0001), hsCRP (90.7 ± 5.8 vs 50.1 ± 8.6 µg/mL, P < 0.0001), CD4+CD28null cell frequency (9.1 ± 0.9 vs 3.6 ± 0.5%, P < 0.0001) and lower 25(OH) vitamin D levels (17.9 ± 1.9 vs 26.9 ± 3.5 ng/mL, P < 0.0001). In CKD subjects, serum 25 (OH) vitamin D level showed a strong inverse correlation with CCA-IMT (r = −0.729, P < 0.0001) and correlated with CD4+CD28null cell frequency (r = −0.249, P = 0.01) and hsCRP (r = −0.2, P = 0.047). We also noted correlation of IMT with patient age (r = 0.291, P = 0.

Hypertension and proteinuria may relate to the anti-angiotensin-11 receptor-1 agonist antibodies (AT1-AA) found in women with preeclampsia.40 Their exact role has not yet been fully elucidated41 but it is difficult to impune a direct hypertensive effect given the known decrease (rather than increase) in endogenous human angiotensin II and aldosterone activity.42 These autoantibodies may be another marker of widespread endothelial dysfunction and result from placental

ischaemia.43 While in experimental animals sFLT-1 can be induced by Small molecule library high throughput AT1-AA,44 the induction of both is possible from reduced uterine perfusion pressure and low dose cytokines infusion (tumour necrosis factor-α). It remains to be seen how these compounds indicate a causal sequence in human preeclampsia. However, an agonistic AII effect may partly explain the increases in angiotensin-11 sensitivity and even the decrease in K(f) seen in preeclampsia. This is yet to be determined. Preeclamptic nephropathy is widely considered to be a predominantly glomerular endothelial cell disorder.11 The term Selleckchem Sirolimus endotheliosis was first termed in 1959

by Spargo et al. who took advantage of the then, new technology of ultra thin sections and electron microscopy to identify the specific nature of these changes.45 They, and others have gone on to demonstrate that at the light microscopic level the glomeruli may appear normal at one extreme, to swollen and ischaemic with apparently thickened capillary walls PTK6 and reduction in capillary lumina at the other.46 The electron microscopic examination of the glomeruli typically reveals ‘endotheliosis’. Endotheliosis refers to the endothelial cell swelling resultant from the cytoplasmic expansion due to cytoplasmic vacuolation, droplet formation, cytoplasmic strands and membrane condensation.45

There is loss of the endothelial fenestrae as well as widening of the subendothelial space with deposition of hyaline material. Concordantly, the swollen endothelial cell encroaches on the capillary lumen and obliteration may occur.47 Given these changes, as well as the reduction in plasma volume and vasoconstriction, the oliguria associated with preeclampsia is not surprising12 Paramesangial deposition of fibrinoid material and mesangial expansion has also been noted.48 Although these renal histological changes have been considered pathognomonic for preeclampsia, this may not be the case. Several groups have performed antenatal renal biopsies in normal pregnant women and women with gestational hypertension.49–51 Strevenset al. demonstrated that five of 12 normal pregnant women had, albeit very mild, evidence of glomerular endotheliosis. These lesions resolve at variable rates post partum.

1 biotin (PK136), T-cell receptor (TCR) γδ (UC7-13D5), TCR-β (H57-597), CD127 Alexa Fluor 647/phycoerythrin (PE) (A7R34), CD25 FITC/APC (PC61.5), Streptavidin efluor 450, CD16/32 PE-Cy7 (93), CD4 PE-Cy7 (GK1.5), CD44 PE-Cy7 (IM7), CD23 (B3B4), CD21 (8D9), CD80 (16-10A1), MHC II

(M5/114.15.2), IgM (11/41), IgD (11-26), CD93 (AA4.1) and CD43 (R2/60). Immature, DN thymocytes were stained with a pool of antibodies recognizing lineage (Lin) markers. The lineage mix contained antibodies to B220, CD3ε, CD8β, CD8α, CD11b, Gr-1, CD11c, NK1.1, TCR-β, and TCR-γ as previously described.[21] The DN thymocytes, after lineage gating, were further characterized into DN1 (CD44+ CD25−), DN2 (CD44+ CD25+). DN3 (CD44− CD25+),

and DN4 (CD44− CD25−) populations.[22] Early T-lineage progenitors (ETPs) after lineage gating, were defined as CD44+ CD25− c-Kithi IL-7R−/lo.[21] KU-57788 concentration Effector/effector memory splenic T cells were defined as CD44hi CD62Llo, and central memory T cells were defined as CD44hi CD62Lhi.[23] Bone marrow B cells were defined based upon previously reported markers.[24, 25] Pre-pro B cells were defined as Erlotinib research buy B220+ CD19− CD43+ IgM−, pro-B cells were defined as B220+ CD19+ CD43+ IgM−, pre-B cells were defined as B220+ CD19+ CD43− IgM−, immature B cells were defined as B220+ CD19+ CD43− IgM+, and mature B cells were defined as B220+ IgM+ IgD+. In the spleen, B-cell subsets were defined as find more described by Allman and Pillai.[26] CD19+ B cells were defined as transitional (T) B-cell subsets; T1: B220+ AA4+ IgMhi CD23−; T2: B220+ AA4+ IgMhi CD23+; T3: B220+ AA4+ IgMlo CD23+ or marginal zone (MZ) B-cell subsets; MZ: B220+ AA4− IgMhi CD21hi CD23−; or marginal zone precursor (MZP): B220+ AA4− IgMhi CD21hi CD23+, or follicular (Fol) B-cell subsets were defined as Fol I: B220+ AA4− IgMlo CD21lo IgD+; or Fol II: B220+ AA4− IgMhi CD21lo IgD+. Compensation settings and lineage gates were based upon

single colour controls. Analysis was performed with FlowJo (Tree Star, Inc., Ashland, OR) Intracellular reactive oxygen species were analysed in selected subsets by using the oxidation sensitive dye dichlorodihydrofluorescein diacetate (DCFDA) as previously described.[6] Cells were incubated ex vivo with 2 μm DCFDA at 37° for 15 min, washed and surface stained. As a loading control, parallel samples were incubated with the oxidized control dye fluorescein diacetate (FDA) (0·01 μm) at 37° for 15 min, washed, and surface stained as described above. FACS analysis was performed immediately. DCFDA mean channel fluorescence was normalized to FDA uptake, and the data are shown as the per cent increase in DCFDA fluorescence in cells from Ts65Dn mice over euploid controls ± SEM. Intracellular glutathione levels were measured in progenitor subsets by flow cytometry using monochlorobimane (MCB) essentially as previously described.

Cellular immunoblotting has been validated multiple times to be able to distinguish type 1 diabetes patients from controls in blinded trials with excellent sensitivity and specificity [35,40]. PBMC find more reactivity to the islet cell proteins has also been demonstrated to have clinical relevance in identifying autoimmune diabetes patients with more severe loss of beta-cell function [41]. PBMCs from patients with T1D respond to between four and 18 molecular weight regions containing islet proteins, whereas normal control subjects respond to between zero and three molecular weight regions [42]. Disadvantages. Human islets are

needed to prepare the islet antigens. Twenty ml of blood is needed per patient. The antigen specificity of the T cell responses is not defined. 1 Normal human islet cells are placed into sodium dodecyl sulphate (SDS) sample buffer, boiled and then subjected to preparative one-dimensional 10% SDS-PAGE [43]. Background.  CFSE is a non-toxic fluorescent dye that is distributed evenly between daughter cells when a cell divides [44]. This dye can be used to determine the number of cells that have proliferated, in the presence or absence Pexidartinib nmr of antigen, by flow cytometry (see Fig. 2). Advantages.  This assay is more sensitive than [3H]-thymidine incorporation and the proliferation of different lineages of cells

can be determined directly by flow cytometry, making it well suited to measuring islet antigen-specific T cell responses to autoantigens [27]. Multi-colour flow cytometry can be used to gain further information on the phenotype of the cells that have proliferated,

such as their capacity to produce cytokines after a brief stimulation with anti-CD3 mAb. Alternatively, the proliferation of different cell lineages [B cells and natural killer (NK) cells, for example] can be measured in the same sample. Finally, the CFSE-based proliferation assay can be used to isolate T cell clones [45], allowing their specificity to be determined in detail [30,31]. Disadvantages.  Each sample must be analysed individually by flow cytometry. Because of the low precursor frequency of peptide and recombinant islet protein-specific T cells their responses can be variable between replicates. Dichloromethane dehalogenase This assay measures only cells capable of proliferating in vitro. 1 Draw blood into a heparin-containing tube (note: heparin is the recommended anti-coagulant because it does not interfere with immune function). Background.  Individual HLA–T cell receptor (TCR) contacts are low-affinity interactions [46]. However, cross-linking of multiple HLA/peptide complexes increases the avidity of the interaction allowing HLA/peptide multimers, such as tetramers and pentamers, to be used to stain antigen-specific T cells [47]. HLA class I tetramers were the first to be developed [22].

8,9 Herein we report the first case of a PJI due to Pseudallescheria apiosperma in an immunocompetent patient. A 61-year-old male immunocompetent farmer had a car accident in November 2000, ending up with his car in a fresh water canal. Besides a whiplash the patient had no injuries after the accident and water was not aspirated. Two months after the car accident he was ICG-001 purchase first seen suffering from increasing knee pain. Previous to the car accident, the patient had an unremarkable medical history. Orthopaedic investigation in January 2001 disclosed a gonarthrosis of his left knee which was probably not the result of the car accident (Fig. 1). Since the patient

had a severe form of osteoarthrosis, a hemi-prosthesis was implanted in March 2001, 1 month after his first clinical presentation. Five weeks after implantation the patient was admitted with pain in his left knee. The radiograph showed a well-positioned and well-fixed hemi prosthesis (Fig. 2). The initial blood test found a white blood cell count of 12.5 × 109 l−1, an erythrocyte sedimentation rate (ESR) of 102 mm h−1, and a C-reactive protein (CRP) of 200 mg l−1, suggesting inflammation of the

patient’s left knee. A drainage system was installed, but all routinely taken microbiological selleck chemicals llc cultures remained negative. The patient was treated with empirical antibiotics (3 dd 1000 mg cefazoline) for 2 weeks. One month later, in May 2001, he was re-admitted with fever (38.6 °C) and a red, swollen

and aching knee. Surgical drainage of the knee was started immediately with evacuation of approximately 100 ml foul-smelling, brownish-greyish pus. No bacteria were found using Gram staining, but in the blankophor preparation fungal elements were clearly visible. Cultures became positive with a fungus and routine morphological identification revealed a member of the Scedosporium/Pseudallescheria complex as causative agent. Based on good outcomes of itraconazole (ITZ)-treated Scedosporium-infections10–13 our patient started initially with ITZ 200 mg day−1 oral solution Ibrutinib chemical structure which was increased to 400 mg day−1 when the prosthesis remained in situ, as patient refused removal. Molecular identification was performed with ITS-sequencing. By BLAST analysis of the obtained sequence vs. a validated in-house Centraalbureau voor Schimmelcultures (CBS) research database the isolate was identified as P. apiosperma.14 The isolate has been deposited in the CBS collection under CBS 129357. The sequence of the ITS/D1D2 region of the isolate has been submitted to Genbank with accession number JF906010. During the next two months several pus-filled pockets and fistulas around the knee were drained and mycological cultures grew Pseudallescheria despite ongoing ITZ administration. Notably no grains were observed in the pus collections.

Interventional studies show that after drug cure, allergy may increase at the population level (80,81). Chemotherapy to remove intestinal helminths results, in some studies, in aggravated allergic responsiveness. In a recent double-blinded placebo-controlled interventional trial in an area of Vietnam where hookworm is the most common infection, the anthelmintic-treated group had a significantly increased selleck products incidence of skin allergy sensitivity

to house dust mite or cockroach allergens. This protection correlated with significantly higher levels of baseline IL-10 production to hookworm antigen, with a trend for decreased production of IL-10 after treatment (82). The idea that worm-induced immunomodulation could be used to treat immune dysregulation in the developed world has been gathering support in recent years. A turning point was a clinical trial in the USA, where Trichuris suis, the pig whipworm, was used to treat inflammatory bowel disease. The results of the trial were very encouraging, and the majority

of treated Selleck Copanlisib patients went into remission (83,84). However, the same therapy was ineffective against allergic rhinitis in humans (85). Humans are not a fully permissive host for T. suis, so the infection had to be boosted with larvae every 3 weeks to ensure continual presence of larvae in the gut (86). As a treatment for immune dysregulatory diseases, hookworm may be an attractive prospect – it is virtually asymptomatic in low-level experimental infections (40), it poses no risk of transmission in modern 4��8C sanitary environments and it survives for years within the human host, thus making

continual reinfection unnecessary. British and Australian researchers have used hookworm in seasonal hayfever, Crohn’s disease and coeliac disease, with varying success. The British trials showed that hookworm infection, despite the migratory stage through the lungs, does not exacerbate airway reactivity in allergic individuals; however, no suppression of allergic responses was detected (8,39). No suppression of inflammatory immune responses, as measured by production of IFN-γ or TNF-α, or induction of immunoregulatory mechanisms, as measured by levels of circulating CD4+CD25hiFoxp3+ Tregs or polyclonal CD4+ T-cell production of IL-10, was seen either (8). In contrast, the Australian Crohn’s disease trial led by John Croese showed a strong trend for suppression of Crohn’s disease symptoms after infection (87). However, caveats of this trial include a lack of blinding or a placebo control group, and continued and variable use of immunosuppressants. This trial is currently being extended by Croese and our group, to use hookworm to treat coeliac disease, a gluten-induced enteropathy dependent on a TH1/TH17 response (ms submitted).

We then addressed whether WT Mϕ inhibition of T-cell proliferation was a dominant effect. Addition of increasing numbers of WT Mϕ to cultures where OT-II T cells were activated by TNFR1−/− Mϕ led to a dose-dependent inhibition of proliferation. Adding WT Mϕ at a ratio of 1 : 1 with the TNFR1−/− Mϕ, prevented the proliferation induced by TNFR1−/− Mϕ (Fig. 1f). This TNF-α-dependent suppression of T-cell proliferation by naive Mϕ is similar to that induced by Mϕ in autoimmunity and by populations of myeloid-derived suppressor cells (MDSC), which prevent T-cell responses in tumour sites.13,16 The Mϕ from sites of autoimmune inflammation

and MDSC share phenotypic markers, including the expression Mitomycin C of CD11b, Gr-1 and CD31, which have been useful in identifying myeloid cells that can inhibit T-cell proliferation. As a consequence, we examined the phenotype of in vitro-generated naive Mϕ and observed that, consistent with in vivo-generated Mϕ, they expressed CD11b, CD31 and F4/80, but not Gr-1 (Supplementary Fig. S1). The activation HM781-36B research buy of BM-Mϕ with LPS or IFN-γ, in the absence of T cells, did not lead to the expression

of Gr-1 (data not shown). However, when BM-Mϕ were activated by co-culture with T cells and cognate peptide, both WT and TNFR1−/− Mϕ up-regulated Gr-1 (Fig. 2a), indicating a requirement for signals supplied by T cells for Gr-1 expression. Naive Mϕ from either mouse strain expressed CD31, which was down-regulated to a greater extent on TNFR1−/− Mϕ compared with WT Mϕ following activation (Fig. 2a). Interestingly, the mechanism by which Mϕ acquire a suppressive Gr-1+ phenotype appears to require cell–cell contact with activated T cells, rather than resulting from stimulation by soluble factors (Supplementary Fig. S2). The inhibition of T-cell proliferation in the presence of tumour-derived Mϕ has been associated with down-regulation of the ζ-chain of the CD3/TCR signal transduction complex.10,24 To determine the effects on the intracellular

expression of CD3ζ by crotamiton OT-II CD4+ cells, we examined cells stimulated by WT or TNFR1−/− BM-Mϕ. Compared with unstimulated T cells, activation with WT Mϕ led to lower levels of CD3ζ (Fig. 2b) consistent with T-cell inhibition,25 whereas activation with TNFR1−/− Mϕ led to CD3ζ up-regulation, consistent with normal activation26 (Fig. 2b). Since Mϕ in the local environment stimulate lymphocyte cytokine production but block the proliferation of T cells, we wished to ascertain the fate of T cells that escape from their presence. To do this, we tested whether co-culture with inhibitory BM-Mϕ produced a long-term unresponsive state in the T cells. OT-II CD4+ T cells were combined with BM-Mϕ and OVA peptide for 24 hr and then the non-adherent lymphocytes were removed and the T cells were re-plated in fresh medium. Cell proliferation was then assessed by [3H]thymidine incorporation.

Also, at equivalent amounts, whole gram-negative bacteria may deliver more lipopolysaccharide to the macrophage as compared with free lipopolysaccharide and would also stimulate other pathways (Nau et al., 2003). Nonetheless, it is striking Selleckchem p38 MAPK inhibitor how strongly every one of our treatments induced RCAN1-4, suggesting a common downstream pathway and mechanism of induction. This appears to involve calcium and ROS because both mediate gram-negative lipopolysaccharide effects (Figs 2 and 3), and we also observed calcium and ROS involvement in limited studies with our gram-positive agonists (data not shown). It is also important to note that commercial LTA and peptidoglycan

have been reported to contain TLR2 contaminants. These reports include evidence that lipoprotein-like compounds are responsible for the activity of the LTA fraction of Enterococcus hirae and S. aureus (Hashimoto et al., 2007); that bacterial compounds reported as TLR2 agonists are more likely contaminated with highly active natural lipoproteins and/or lipopeptides that are the true TLR2 agonists (Zähringer et al., 2008); and that proteoglycan

effects are actually due to the presence of LTAs (Travassos et al., 2004). Thus, we do not know for sure how much contribution either LTA or peptidoglycan provides in RCAN1-4 induction in our studies. Nonetheless, these contaminants, if present at significant levels in our peptidoglycan and LTA, are Seliciclib order still acting as TLR2 ligands, further

supporting that RCAN1-4 is induced by TLR2 stimulation. The in vivo studies revealed a strong effect of knocking out RCAN1, namely, cytokine induction in the lung. All of these same cytokines except IL-6 were also increased in day 7 spleen (data not shown). Interestingly, the cytokines analyzed (MCP-1, TNF-α, IL-6, and IFN-γ) can be upregulated by the calcineurin–NFAT pathway (Kiani et al., 2001; Satonaka et al., 2004; Keller et al., 2006), although these elevations appear to be cell and condition dependent as other systems show different responses (Ryeom et al., 2003; Keller et al., 2006). Nonetheless, our observation that MCP-1, TNF-α, IL-6, and IFN-γ are upregulated in the KOs are consistent with those reports that demonstrate their induction by this pathway Tangeritin because in the KO, the loss of RCAN1 and its inhibitory action would lead to elevated calcineurin activity and stimulation of target cytokine expression. Before our studies, there has only been limited characterization of RCAN1 regulation of cytokine expression. Specifically, Ryeom et al. (2003) observed decreased IFN-γ production in RCAN1 KO T-lymphocytes, although this was associated with a dying (FAS overexpressing) phenotype. Interestingly, they also observed that this effect was specific to Th1 T-helper cells, and that these cells had lower activation thresholds for IL-2, IFN-γ, and IL2 receptor as compared with WT cells.

g., noun- versus verb-phrase). Take for example the sentence “The beautiful baby smiled at her mother” which consists of two intonational phrases, with a boundary between “baby” and “smiled”. It is possible to create strong statistical cues between syllables that fall within an intonational phrase, as would be present in any natural language, or between syllables that span an intonational phase boundary, which almost never occurs in natural languages. This design was implemented in Shukla, White, and Aslin (2011) using nonsense syllables as in Saffran et al. (1996), but organized into short sentences rather than continuous streams. A family of such sentences was presented to

6-month-olds as they watched a video

display depicting PD0325901 in vitro three salient objects. One of the objects consistently underwent motion Romidepsin supplier across trials while the other two objects never moved, thereby drawing infants’ attention to the single moving object. The key feature of the design, implemented across two groups of infants, was that there were syllables with strong statistical links (i.e., words) and syllables with weak statistical links (i.e., part-words), but in only one of the two conditions were the strongly linked syllables within an intonational phrase. Thus, if infants attended only to syllable statistics, regardless of their positioning with respect to intonational phrases, both groups would extract these word-candidates and map them onto the single object in the video display that was moving. However, if infants were constrained to extract syllable statistics when they fell within an intonational phrase, then only

infants in the group where the ends of words were aligned with the ends of intonational phrases would map these syllable statistics to the moving object. That is precisely the outcome reported by Shukla et al. The main reason for describing the Shukla et al. (2011) study is that it illustrates how the statistical-learning mechanism of young infants is constrained in a principled way to reduce the computational complexity faced by a naïve learner in the language domain. Intonational phrases Immune system are universal characteristics of natural languages that presumably do not themselves have to be learned because they are based on low-level durational and pitch cues. But the Shukla et al. study also illustrates a second important point about the implications of designing laboratory experiments to test infants. As noted earlier, it is natural for experimentalists to eliminate all but one source of information to determine whether it alone is sufficient for learning; that was the goal of the Saffran et al. (1996) study that focused on syllable statistics while eliminating prosodic and repetition cues that are present in natural language input.