Introduction: Pediatric supracondylar humerus fractures are the most common elbow fractures in pediatric patients. Surgical fixation using pins is the primary treatment for displaced fractures. Pin site infections may follow supracondylar humerus fracture fixation; the previously reported incidence rate in the literature is 2.34%, but there is significant variability in reported incidence rates of pin site infection. This study aims to define the incidence rate and determine pre-, peri-, and postoperative factors that may contribute to pin site infection following operative reduction, pinning, and casting.Methods: A retrospective chart analysis was performed over a one-year period on patients that developed pin site infection. A cast care form was added to Nemours’ electronic medical records (EMR) system  to identify pin site infections for retrospective review. The cast care form noted any inflamed or infected pins. Patients with inflamed or infected pin sites underwent a detailed chart review. Preoperative antibiotic use, number and size of pins used, method of postoperative immobilization, pin dressings, whether postoperative immobilization was changed prior to pin removal, and length of time pins were in place was recorded.Results: A total of 369 patients underwent operative reduction, pinning, and casting. Three patients developed a pin site infection. The pin site infection incidence rate was 3/369=0.81%. Descriptive statistics were reported for the three patients that developed pin site infections and three patients that developed pin site complications.Conclusion: Pin site infection development is low. Factors that may contribute to the development of pin site infection include preoperative antibiotic use, length of time pins are left in, and changing the cast prior to pin removal. Pediatric supracondylar humerus fractures (PSCHF) constitute approximately 17% of all fractures in children and are the most common elbow fracture . Type I Pin site infection after PSCHF may occur when pins are buried underneath the skin or when the ends of the pins are left exposed . Some stPostoperatively, casts or splints are typically used, but studies have not shown an association between a specific type of immobilization associated with a higher incidence of pin site infections . In addiThe type of skin preparation and draping used for closed reduction and pinning, and the dressing used to cover the pin site after surgery, vary in use after PSCHF. Percutaneous pinning using a semi-sterile surgical technique reported no incidence of pin site infection, with historical controls being sterile surgical preparation and draping. The semi-sterile technique saves time and money compared to a sterile surgical technique, which is currently commonplace . The stuA significant risk factor for pin infection is the length of time the pins are left in place, as longer periods of time increase the opportunity time for an infection to develop . TypicalA gap in the literature exists due to the variability in reported incidence rates of pin site infection following percutaneous pinning to treat PSCHFs. This study seeks to analyze data collected at the time of pin and cast removal following surgical treatment of PSCHF to define the incidence rate of pin site infection and skin complications following percutaneous pin fixation. A retrospective chart review of identified cases was conducted to evaluate the pre-, peri-, and postoperative factors that contribute to the development of pin site infections and complications. The null hypothesis states that there is no difference in the reported incidence rate of pin site infection in the literature and the study incidence rate. The alternative hypothesis states that the study incidence rate of pin site infection is lower than the pin site infection rate reported in the literature.Approval for this study was obtained from Nemours Institutional Review Board . Patient informed consent was obtained.A retrospective chart analysis was performed on pediatric patients (age range: one to 18 years) who were identified with pin site infection and complication following supracondylar humerus fracture fixation. Patients who had supracondylar humerus fracture fixation that was performed at Nemours’ clinical sites in Orlando, FL, Jacksonville, FL, Pensacola, FL, or Wilmington, DE, and developed pin site infection, were included in this study. There were no specific exclusion criteria other than age. Data collection for this study ranged from December 1, 2014, through December 1, 2015.Patient charts were accessed using Nemours' EMR system. A standardized cast form within the medical record was used to identify patients who developed pin site infection upon cast removal. This form was completed by an orthopedic technician at the time of cast removal on every patient in the system, and it specifically collected data on the presence or absence of pins beneath a cast. For each patient with K-wires beneath the cast, the form requires evaluation of the pin site either as normal, inflamed, or infected. Inflamed pins are those that are erythematous or have granulation tissue present, but do not require antibiotic treatment and are treated only with pin removal. Any pin site that is treated with oral or intravenous antibiotics or with surgical debridement, is defined as infected. To establish a denominator and calculate the incidence of pin site infection, all patients with PSCHFs who underwent surgery at the four sites were identified by a search of our coding system for the current procedural terminology (CPT) codes for closed and open treatment of supracondylar humerus fractures . Wound healing complications were recorded if occurring in the 30 days following pin removal. Data were abstracted into an Excel spreadsheet. Data collected for analysis were diagnosis, type of fracture , age, sex, race, type of surgical preparation used, draping types, antibiotic used, and timing. Details about pin use were recorded: pin size, number of pins, pin entry site , pin site dressing, and pin site care instructions given to the patient . PeriopeDescriptive statistics were calculated. The incidence rate of pin site infection was expressed as a frequency and percentage. Our pin site infection rate was compared to historic data from Iobst et al. , who perThree hundred sixty-nine pediatric patients underwent surgical reduction and pin fixation for PSCHF during the data collection period. Three patients developed a pin site infection for a 0.81% (3/369) incidence rate. Our rate of 0.81% was not statistically significantly different from the rate of 2.34% reported by Iobst et al. . A post hoc power analysis suggested the power to detect the difference in infection rates (1.53%) was about 55%.A detailed chart review of the three patients who developed a pin site infection was performed. Additionally, three patients whose pin site was noted to be inflamed developed an alteration in skin integrity (pressure sore or ulcer). Their pin sites were not infected and therefore did not contribute to the reported incidence of pin site infection. A detailed chart review of these inflamed ulcer and pressure sore cases was also performed to determine factors that contribute to altered pin site skin integrity and complication development. Demographic variables of these cases are shown in Tables We found the incidence of pin site infections after surgical treatment of PSCHF to be low (3/369=0.81%), and not significantly different from rates reported by others . The powThere is varied opinion in the literature on the use of preoperative antibiotics in PSCHF surgical procedures. Bashyal et al. found no advantage to the use of preoperative antibiotic use in preventing pin site infections . The thrOne patient developed a pressure sore around the pins after presenting late for cast removal (37 days postoperatively). The patient complained that his distal humerus was still tender to palpation when the cast was taken off at this visit. Another cast was applied, and the patient was instructed to come back in one week for final cast and pin removal. The patient was then lost to follow-up for one month, and when the patient presented at clinic, the pins had been in place for 70 days. The suggested time for pin removal following PSCHF is between three and four weeks . This caTwo patients developed superficial ulcers when they presented postoperatively for cast removal. After removing the casts, dirt and water was noted underneath of the casts. Wet casts and foreign objects have both been shown to contribute to the development of infection, skin breakdown, and skin irritation . FurtherLimitations of this study include the relatively low sample size and resultant low post hoc power. Causation cannot be proven between the previously mentioned variables  and the development of pin site infection and complication. This study only included data from the four Nemours clinical sites in Orlando, FL, Jacksonville, FL, Pensacola, FL, and Wilmington, DE. This data may be representative of the pediatric patients in these locations, but may not be representative of the national pediatric population.This study reports a very low incidence of pin site infections. The three pin site infection patients did not receive pre-operative antibiotics, and although the use of pre-operative antibiotics may have a role in the prevention of pin site infections following PSCHF, a much larger clinical trial would be needed to verify that assumption. Limiting the length of time that pins are left in is important in the development of pin site complications, and this study provides further evidence to support this practice. Finally, the importance of proper cast-care and patient education is important in the prevention of pin site complications.Pin site infection development following PSCHF at Nemours’ clinical sites is low. Factors that may contribute to the development of pin site infection include preoperative antibiotic use, length of time pins are left in, inappropriate cast care, and changing the cast prior to pin removal.


Immunoreceptors can transduce either inhibitory or activatory signals depending on ligand avidity and phosphorylation status, which is modulated by the protein kinases Lyn and Fyn. Here we show that Lyn and Fyn control immune receptor signaling status. SHP-1 tyrosine 536 phosphorylation by Lyn activates the phosphatase promoting inhibitory signaling through the immunoreceptor. By contrast, Fyn-dependent phosphorylation of SHP-1 serine 591 inactivates the phosphatase, enabling activatory immunoreceptor signaling. These SHP-1 signatures are relevant in vivo, as Lyn deficiency exacerbates nephritis and arthritis in mice, whereas Fyn deficiency is protective. Similarly, Fyn-activating signature is detected in patients with lupus nephritis, underlining the importance of this Lyn–Fyn balance. These data show how receptors discriminate negative from positive signals that respectively result in homeostatic or inflammatory conditions. Src-family kinases Fyn and Lyn are signaling components downstream of ITAM-bearing antigen receptors. Here the authors show that by phosphorylating SHP-1 at different residues, Lyn and Fyn can have opposing regulatory effects on ITAM receptors. An important axis of regulation comprises immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptors, such as the T-cell receptors (TCR) and B-cell receptors (BCR), Fc receptors (FcR)2. ITAMs are defined by two consecutive Yxx[L/I] sequences separated by 6 to 12 amino acids, and are present in the cytoplasmic domains of several transmembrane adapter molecules, such as the common γ subunit of FcR (FcRγ), the Igα and Igβ subunits of the BCR, the γ, δ, ε, and ζ subunits of the TCR-associated CD3 complex3, and in FcγRIIA4. Cellular responses after FcR triggering depend on ligand avidity. Receptor clustering mediated by high avidity ligand interaction induces phosphorylation on ITAM tyrosine residues by membrane-anchored and receptor-associated Src-family kinases (SFK). Phosphorylated ITAMs are docking sites for recruitment of the tyrosine kinases Syk or Zap70, which launch inflammatory responses and restore homeostasis. However, in case of dysregulation or chronic stimulation, ITAM signal can also result in autoimmune and inflammatory diseases5. Both in innate and adaptive immunity, the activation of ITAMs-bearing immune receptors is actively counteracted by the action of ITIM-bearing inhibitory receptors such as FcγRIIB with the ITIM being defined by a single [I/V/L/S]xYxx[L/V] sequence. This regulation generally involves co-aggregation of inhibitory and targeted activated receptors and is promoted through recruitment of relevant phosphatases such as Src homology region 2 domain-containing inositol 5′ phosphatases (SHIP-1 and SHIP-2)6. In addition to this inhibitory feedback targeting co-aggregated activated receptors, a continuously active inhibitory mechanism generated by ITAM-bearing receptors following low avidity interactions has been described that acts towards a whole array of activating receptors without the requirement for co-aggregation12. This mechanism has been named inhibitory ITAM (ITAMi) and is thought to be involved in the maintenance of homeostasis13. Various FcRs, such as FcαRI, FcγRIIA, and FcγRIIIA can function as such bi-functional receptors to trigger inhibitory signals, a property that can be exploited to reduce the susceptibility to autoimmune and inflammatory diseases11.The immune system is controlled by a finely tuned network of regulatory mechanisms that maintain homeostasis or can initiate inflammatory responses14. BCRs and TCRs have been also reported to recruit SHP-1 upon interaction with low avidity ligands16. Moreover, deletion of SHP-1 in haematopoietic lineages, including T cells, neutrophils, and dendritic cells, is associated with a variety of pathologies20. Together, these evidences support an important role of SHP-1 in the maintenance of immune homeostasis.Induction of FcR ITAMi signals by weakly binding ligands with low avidity depends on the recruitment of the Src homology region 2 domain-containing tyrosine phosphatase SHP-121. In B cells, Lyn was reported to have both positive and negative roles in BCR-mediated signaling22. Aged Lyn-deficient mice have high levels of serum immunoglobulins (including autoantibodies) and their B cells are hyper-responsive to IL-4 and CD40 engagement25 demonstrating a defective homeostasis due to a deficient negative regulation. Therefore, the individual function and coordination of Lyn and Fyn in the control of ITAM signaling pathways mediated by the engagement of FcRs or BCRs by either high or low avidity ligands is unclear.SFKs, such as Lyn and Fyn, are implicated in the initiation of ITAM-receptor-mediated signaling. These kinases are responsible for ITAM phosphorylation upon receptor aggregation leading to Syk recruitment initiating further signal propagation via downstream effectors such as PI3-kinase and phospholipase C-γHere we report that Lyn and Fyn have important and non-redundant roles in the ligand-sensing threshold between ITAM and ITAMi signals mediated by immunoreceptors, able to differentially translate outside ligand interactions into opposite signals. Our results show that, although Lyn is crucial in coupling to ITAMi signals, Fyn is the essential effector molecule coupling to ITAM signals following FcR, but also BCR, engagement. This ITAM switch involves a capacity to differentially control SHP-1 by shifting its phosphorylation status. These signaling signatures are confirmed in inflammatory and auto-immune diseases involving an imbalance between ITAM and ITAMi signals.11. The ITAMi molecular signature was characterized by transient Syk recruitment followed by stable and prolonged SHP-1 recruitment. This required the presence of Lyn but not Fyn. In contrast, multivalent crosslinking of these receptors inducing an ITAM activation signal molecular signature with a stable recruitment of Syk (but not SHP1), required the recruitment of Fyn nal Fig. . FurtherLyn Fig. . By contLyn Fig. . These r28. Hence we analyzed whether Lyn and Fyn could control SHP-1 function through differential phosphorylation of this phosphatase. Stimulation of bone marrow-derived macrophages (BMDM) from FcγRIIATg (R131 isoform) mice29 under ITAMi conditions showed that Lyn induced SHP-1 Y536 phosphorylation. By contrast, receptor multivalent aggregation induced a Fyn-dependent SHP-1 S591 phosphorylation  model via administration of a rabbit anti-mouse glomerular basement membrane (anti-GBM) serum. A mild NTN model was chosen because FcγRIIAses Fig. . Renal dein Fig.  and blooein Fig. . They alein Fig. . Glomeruein Fig. . These eein Fig. . This waTg and Fyn-deficient FcαRITg mice despite similar serum levels of injected mouse anti-collagen antibodies  using two transgenic animals expressing either hFcγRIIA or hFcαRI. The aggravating role of Fyn was highlighted by the absence of lesions in both Fyn-deficient FcγRIIAies Fig. . By contion Fig. . To deteFyn Fig. . We nextion Fig. . Moreove591 Fig. . AltogetS591 phosphorylation were associated with immune complex-mediated disease via the FcγRIIA in patients with a given inflammatory disease, we analyzed blood leukocytes from untreated patients with lupus nephritis at different stages of renal involvement morphologically classified as class IV-A (severe nephritis with immune deposits and leukocyte infiltration) and pure class V (membranous immune deposits only). As shown in Fig. S591 and pPKCα were exclusively observed in patient cell lysates, and were not associated with FcγRIIA. Consistent with the role of Lyn/SHP-1 axis in homeostasis, Lyn and pSHP-1Y536 were strongly associated with FcγRIIA in healthy individuals only, underlining the inhibitory ITAM homeostatic phenotype, whereas Fyn and Syk were exclusively associated with FcγRIIA in patients highlighting the deleterious role of ITAM signaling in this inflammatory disease. Moreover, tissue analysis of phosphorylated phosphatases in situ of renal biopsies from untreated patients with lupus nephritis at different stages of disease show that the phosphorylation status of SHP-1S591 residue was linked to disease activity in proximity of phalloidin+ renal cells  or inhibitory (ITAMi) signaling depending on ligand avidity.Here we characterize the mechanism by which SFKs and SHP-1 control the balance between the activating and inhibitory ITAM signaling. Initially described for their activating function, evidences have accumulated that FcRs bearing an ITAM motif could generate opposite signals, known as ITAMi. Yet, a black box remains in regard to the inhibitory signaling mechanism involved. In this study, we show that low valency aggregations of BCR by anti-CD79a F(ab′)11, and of SHP-1 recruitment. In contrast, upon multivalent crosslinking of FcγRIIA, Fyn but not Lyn, plays a crucial role in FcγRIIA-ITAM-mediated cytokine production. This Lyn/Fyn switch is not affected by other SFK such as Hck and Fgr. Interestingly, it has been shown that FcγRs can mediate a well-established ITAM-dependent function, the phagocytosis, in the absence of Lyn, Hck, and Fgr35. Our data highlight that Lyn and Fyn are essential for ITAM responses as silencing of both SFK render the cell unresponsive for FcγRIIA activating or inhibitory stimuli.Our study further demonstrates that single receptors switch between ITAM and ITAMi signals using distinct SFKs fulfilling opposite signaling functions, such as described here for FcR and BCR. Upon divalent targeting of immunoreceptors Lyn, but not Fyn, is recruited to the receptor, leading to ITAM partial phosphorylation on tyrosine, as we previously reported for FcγRIIA36. Of note, the two SH2 domains of SHP-1 can cooperate to bind phosphotyrosines on adjacent molecules37, a configuration expected to be that of ITAMi. This recruitment would enable ITAMi-mediated SHP-1 phosphatase activity to inactivate signal effectors mobilized by heterologous receptors38. In contrast, multivalent crosslinking of immunoreceptors results in the recruitment of the SFKs Lyn and Fyn to the receptor leading to full phosphorylation of the ITAMs. These would serve as “docking” sites for Syk preventing SHP-1 recruitment, which is associated with its phosphorylation on S591 in the cytosol. Thus, Fyn but not Lyn redirects SHP-1 towards its inactivated form allowing simultaneously full ITAM phosphorylation and receptor activation signaling by Syk. As Fyn is a tyrosine, but not a serine, kinase we explored the mechanism involved. SHP-1 is constitutively associated with PKCα27. Upon cell activation, PKCα phosphorylates SHP-1 on S591 in its C terminus thereby negatively regulating the activity of this phosphatase27. Using a specific inhibitory and siRNA strategy, we found that a Fyn–PI3K–PKCα axis induced the phosphorylation of SHP-1 on S591 upon crosslinking of FcγRIIA in the presence of Fyn, despite the recruitment of Lyn. Since S591 phosphorylation on SHP-1 keeps the phosphatase in a closed conformation36, our results suggest that while S591 residue is phosphorylated by Fyn, the Y536 residue is inaccessible to Lyn. Moreover, this study demonstrates also that the absence of Fyn favors the phosphorylation of SHP-1 on Y536 in the presence of Lyn, despite the extensive crosslinking of FcγRIIA. These results indicate that the selective absence or inhibition of Fyn may abolish inflammation during autoimmune and proinflammatory processes. Whether Fyn–PI3K–PKCα axis is involved in previously described Fyn-dependent humoral responses39 remains to be demonstrated.The mechanism by which Lyn and Fyn alternatively switch ITAM-bearing receptor function relies on their control of SHP-1 phosphorylation status. It is now clear that SHP-1 phosphorylation at Y536 induces a conformational change easing SHP-1 recruitment through its SH2 domains to phosphotyrosine residues, thereby lifting the inhibition of the phosphatase domain by its N-SH2 domain41. Interestingly, similar to Lyn-deficient mice, R131H-FcγRIIATg mice develop spontaneously autoimmunity at old age, with a complex spectrum of symptoms similar to those found in human rheumatoid arthritis (erosive pannus) and SLE 42. Several studies have reported that Lyn, but not Fyn, controls IgG-mediated or IgE-mediated systemic anaphylaxis44. Our data elucidate the role of these SFKs in the development of autoimmune and proinflammatory disease. Indeed, Fyn and Lyn deletion had opposing effects on the regulation of inflammation observed in R131H-FcγRIIATg mice. In the mild model of NTN, ablation of Fyn had no effect on young mice, but that of Lyn induced lethal nephritis associated with the activation of Syk and in situ phosphorylation of SHP-1 on S591. It is unlikely that SHIP-1 plays a negative role in this model through a putative engagement of FcγRIIB on infiltrating macrophages by mouse anti-rabbit Ig antibodies anti-GBM, as no phosphorylation of SHIP-1 was observed in situ. In addition, FcγRIIB function is Lyn dependent45 and our results show that Lyn-deficient mice did not develop any severe nephritis as compared to WT mice after NTN induction. The ablation of Fyn, however, protected the R131H-FcγRIIATg or FcαRITg mice against spontaneous renal autoimmune disease and arthritis development. In agreement with our in vitro data, this was associated with the phosphorylation of SHP-1 on Y536 but not on S591 and with the inactivation of Syk. Consistent with the role of Lyn in the ITAMi-mediated homeostasis, Lyn was strongly associated with FcγRIIA in leukocytes of healthy individuals. In contrast, in lupus nephritis patients, while Lyn weakly associated to FcγRIIA, Fyn, and Syk were highly associated to the receptor. According to our in vitro and in vivo data, the recruitment of Lyn to FcγRIIA was associated with the pSHP-1Y536 in healthy individuals. On the opposite, blood leukocytes from lupus nephritis patients display a strong recruitment of Fyn to FcγRIIA, which was associated with pSHP-1S591, pPKCα and weak recruitment of Lyn. The selective targeting of Fyn activity may thus become a challenge to open new therapeutic strategies for autoimmune diseases. Moreover, the inactive form of SHP-1 (pSHP-1S591) was linked to the severity of inflammatory glomerular lesions in biopsies from lupus nephritis patients and was observed in kidneys of FcγRIIATg mice with severe spontaneous nephritis. Interestingly, intra-glomerular CD68+ macrophages in class IV lupus nephritis patients with high disease activity expressed pSHP-1S591 suggesting that SHP-1 status may contribute to disease pathogenesis by failing to downregulate FcγRIIA activity. We cannot determine from our study whether the pathogenic role for pSHP-1S591 involved infiltrating or kidney resident macrophages, as described recently46.R131H FcγRIIA polymorphism has a strong correlation with the pathogenesis, development and increased susceptibility to several autoimmune diseases, in particular systemic lupus erythematosus (SLE) and rheumatoid arthritisThis study uncovers how a given receptor discriminates negative or positive signals leading to homeostatic or inflammatory conditions. SHP-1 emerges as a master molecule translating the sensing by distinct SFKs into opposite, activating, or inhibitory, signals Fig. . This me47 presenting with active disease with nephritis proven by kidney biopsy (5 at class IV and 5 at class V) and in whom peripheral blood by venepuncture was obtained immediately prior to immunosuppressive therapy administration. All patients were female with age varying between 25 and 42. Ethical approval for this study was obtained from the Bichat Hospital Local Research Ethics Committee and informed consent was obtained from all patients enrolled. Data collection and analyses were performed anonymously.Eighteen individuals  were studied. The SLE group was composed of 10 patients attending or referred to the Bichat’s Hospital specialist nephrology unit between July 2014 and January 2016 meeting at least four ACR SLE criteriaTg mice expressing the WT human FcγRIIA on CD11b-positive cells were from Jackson Laboratory . Fyn-deficient FcγRIIATg, Lyn-deficient FcγRIIATg were obtained by the intercross of FcγRIIATg mice with mice knockout for Fyn (JAX) or for Lyn (previously described in ref. 44). All mice were of C57BL/6 strain, female gender between 8–10 weeks old. All mice carrying the FcγRIIA transgene were used as heterozygous animals. Mice were bred and maintained at the mouse facilities of the Bichat Medical School campus. All experiments were performed in accordance with the French Council of Animal Care guidelines and national ethical guidelines of INSERM Animal Care Committee .C57BL/6 human FcγRIIANTN was induced by i.p. injection (200 μl/20 g body weight) of rabbit anti-mouse GBM in 8–10-week-old mice. Briefly, mice were preimmunized i.p. with normal rabbit IgG (0.5 mg/20 g body weight) in CFA 5 days prior to i.p. administration of NTN serum. Blood samples were collected and animals were killed at day 7 following NTN injection. Renal function parameters (urinary proteins and BUN), histological and immunohistological parameters were studied.32 using the Arthrogen-CIA Arthritogenic Monoclonal Antibody kit . Mice were injected i.v. with anti-CII Ab cocktail (Day 0) followed by LPS (i.p.) 3 days later. Animals were injected i.p. with 10 mg/20 g body weight of 500 μg serum human IgA /20 g body weight or 100 µg AT-10 F(ab′)2 or irrelevant mAb F(ab′)2 (clone 320) for 10 days at 2-day intervals. The first dose was administered 2 days prior to anti-CII Ab cocktail injection. Paw thickness was measured with a pocket thickness gauge. On day 10, animals were sacrificed and hind paws and knees were fixed in formalin or snap-frozen.Arthritis was induced as described+-CD14+ cell lines (kindly provided by Novimmune)48 were maintained in RPMI-1640, 10% FCS and 50 µM β-mercaptoethanol or supplemented with 200 µg/ml Zeocin, 10 µg/ml blasticidin and 2 µg/ml puromycin . Ramos human cell line  was maintained in RPMI-1640 supplemented with 10% FCS and antibiotics. FCS was removed from the culture medium immediately before stimulation. Human blood samples (12 ml) were first submitted to red cell lysis and pellets of 107 leukocytes were subjected RIPA buffer treatment (see below). BMDM from 6-week-old to 8-week-old mice were obtained after a 7-day culture with M-CSF (R&D systems). Cell lines (THP-1 and Ramos as well as transfectants) were studied. For reagents: Mouse mAbs anti-human FcγRII (clones IV.3 and AT-10), anti-human CD79a (clone ZL7-4) were purchased from Santa Cruz and used in their F(ab′)2 fragment forms. Mouse mAb anti-human FcαRI (clone A77) and control mAb (320) were purified in-house and were used as F(ab′)2, as previously described11. For biochemical studies, rabbit polyclonal anti-Syk, anti–SHP-1, anti-Lyn, anti-Fyn, anti-ERK  at dilution 1/500 in 5% w/v nonfat dry milk, anti-SHP1 (phospho–Y536)  at dilution 1/1000 in 5% w/v nonfat dry milk, anti-SHP1 (phospho–S591)  at 1/1000 in 5% w/v BSA and rabbit polyclonal anti-phosphotyrosine  antibodies were used. Anti-pERK, anti-pAKT, anti-pPKCα, anti-AKT, and anti-PKCα were from Cell Signalling and catalog # 4377, 4060, 9375, 9272, and 2056, respectively.THP-1  and THP1-FcγRIIA-R1316 of monocytic cell lines (THP-1-CD14+-FcγRIIA+ or THP-1-CD14+), and Ramos B cell lines (transfected with different siRNA) were incubated for 30 min with 10 µg/ml of anti-FcγRIIA (clone IV.3) or anti-CD79a (clone ZL7-4) F(ab′)2 fragments at 37 °C, respectively. Cells were then incubated with or without LPS (10 ng/ml) as described8 or Pam3csk4 (1 µg/ml) for 1 h for monocytic cell lines and 18 h for Ramos. For ITAM signaling, cells were incubated with 10 µg/ml of anti-FcγRIIA (clone IV.3) or anti-CD79a (clone ZL7-4) F(ab′)2 at 4 °C followed by an anti-κ light chain F(ab′)2 at 37 °C for 18 h for cytokine measurement. For kinase inhibitor experiments, cells were incubated overnight with inhibitors for kinases such as PI3K (LY294002), ERK (PD98059) , and PKC (GF109203X) (from Sigma Aldrich) or with vehicle alone (1:1000 diluted DMSO in PBS), followed by induction of FcγRIIA-mediated ITAM signals.For ITAMi signaling, 5 × 106 to 107) were solubilized in RIPA lysis buffer containing 1% Nonidet P-40/0.1% sodium dodecyl sulfate (SDS) as described8. For immunoprecipitation, cell lysates were incubated with 2 μg/ml of IV.3 anti-FcγRIIA, A77 anti-FcαRI or ZL7-4 anti-CD79a mAbs and immunoprecipitated overnight at 4 °C with Protein G-Sepharose . Samples were resolved by SDS polyacrylamide gel electrophoresis (10%), transferred to nitrocellulose membranes and immunoblotted with rabbit antibodies followed by goat anti-rabbit IgG  coupled to horseradish peroxidase. Membranes were developed by enhanced chemical luminescence treatment (Amersham Biosciences). All uncut Western blots are available in Supplementary Fig. Cells 5 × 106 to 107 IL-8 was measured in the supernatants of stimulated cells using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer’s instructions. Anti-type I and II collagen mouse IgG antibodies were measured by an ELISA kit with TMB according to the manufacturer’s instructions .RNA purification from homogenized kidneys was performed by using RNAble (Eurobio). cDNA was obtained by reverse transcription using Moloney murine leukaemia virus (Invitrogen). Samples were analyzed by real-time PCR with Taq Man Gene Expression Master Mix (Applied Biosystem). Primers were purchased from Eurofins. Gene quantification was performed using a Chrom o4 Real-Time PCR Detection System (Bio-Rad Laboratories). Data were normalized to β-actin values. For primers and probe sequences see Supplementary Table Experiments were performed using predesigned HP GenomeWide  siRNAs. For targets DNA sequences and siRNAs sense and anti-sense see Supplementary Table Y525–526 AF488  and anti-phalloidin AF568 (Life Technologies) followed by incubation with streptavidin-Alexa Fluor 488 (Life Technologies) at room temperature. Tissue sections were mounted with Vectashield (Vector Laboratories). Slides were read with a laser-scanning confocal microscope  using a 63× objective and the LSM Image Browser (Carl Zeiss). For formalin-fixed hind paws and mouse knees, the samples were decalcified and embedded in paraffin. Sagittal sections were stained with haematoxylin and eosin. Histological score of peri-articular exudates, synovitis and tissue loss were graded as 0 , 3 (mild), 6 (moderate), or 9 (severe). The cumulative score of all features was used as histological score to represent overall disease severity. Analyses were made in a blind manner. Sections were incubated with rabbit primary antibodies against mouse phospho (p)-Syk (Y525-526)49, mouse p-SHP1 (S591) (both from Abcam), or p-SHP1 (Y536) (Assay Biotech). The presence of antibodies was detected with biotinylated anti-rabbit antibody (Southern Biotech) followed by peroxidase streptavidin and diaminobenzidine successive stains. Positive cells were quantified in hind paw sections with analysis software CaloPix  after scanning with Aperio ScanScope CS . For immunofluorescence studies, frozen kidney slides or cytospined cells were incubated in blocking buffer  for 30 min, and then with anti-phospho SHP-1Y536 AF488, SHP-1S591 AF647 , anti-CD68 AF405 (Life Technologies 1/50 dilution), and anti-Phaloidin AF568  for 2 h. Fluorescence was detected by confocal laser scanning microscopy .For the kidney, paraffin-embedded sections 4 µm in thickness were stained with PAS for morphological analysis. For immunohistochemistry, frozen kidney sections were incubated with biotinylated antibodies against rabbit IgG or monoclonal antibodies anti-mouse CD11b, anti-mouse F4/80, anti-mouse CD3, and anti-mouse Ly6G (Becton Dickinson) for 1 h at room temperature. When necessary, the primary antibody incubation was followed by incubation with anti-rabbit IgG or anti-goat IgG (Southern Biotech Associates). Slides were mounted with the Eukitt mounting medium (Electron Microscopy Sciences) and read with an upright microscope  using the IM50 software (Leica). For colocalization experiments, frozen kidney sections were incubated overnight successively with each antibody  with Bonferroni’s, Holm–Sidak’s, or Newman–Keuls multiple comparisons test were used to evaluate multiple groups. Sample sizes were higher than five per group. P-values of 0.05 were considered significant; values <0.05 are indicated in the figure legends.All data were expressed as mean ± SEM. Statistical significance between two groups was examined by the Student’s The data that support the findings of this study are available from the corresponding authors on request.Supplementary Information


Screw-rod constructs have been widely used to correct spinal deformities, but the effects of different screw-rod systems on anti-rotational torque have not been determined. This study aimed to analyze the biomechanical effect of different rod-screw constructs on anti-rotational torque.Three conventional spinal screw-rod systems  were used to test the anti-rotational torque in the material test machine. ANOVA was performed to evaluate the anti-rotational capacity of different pedicle screws-rod constructs.P value lower than 0.05. This results indicated that the Legacy screws-rod construct could provide a highest anti-rotation capacity, which is 68% and 210% greater than RF-F-10 screw-rod construct and USSII screw-rod respectively.The anti-rotational torque of Legacy group, RF-F-10 group and USSII group were 12.3 ± 1.9 Nm, 6.8 ± 0.4 Nm, and 3.9 ± 0.8 Nm, with a The anti-rotational torque may be mainly affected by screw cap and groove design. Our result showed the anti-rotational torque are: Legacy system > RF-F-10 system > USSII system, suggesting that appropriate rod-screw constructs selection in surgery may be vital for anti-rotational torque improvement and preventing derotation correction loss. Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional 3D) anomaly of the spine in the coronal, sagittal, and axial planes. Rotational deformity is an important part of AIS and can affect mental health and cause cosmetic defects. Cotrel-Dubousset (CD) instrumentation and rod derotation are excellent techniques for the coronal and sagittal realignment of deformities, but it provides poor rotational improvement for a weak posteromedialization effect , 2. ModeD anomalyThe purpose of this study was to evaluate anti-derotational torque, that is, how much tolerance a screw-rod construct has when twisting the rod in the pedicle screw groove. Here, we assessed the anti-rotational capacities of three different screw-rod constructs . Each group comprised seven monoaxial pedicle screws and one round rod . The anti-rotational torque of three groups are presented as mean ± S.D. Analyses of variance (ANOVAs) were performed to evaluate the posthoc anti-rotational capacities of different pedicle screw-rod constructs. P < 0.01). Posthoc testing comparing pairs of groups revealed that all were statistically different . The anti-rotational torque of the Legacy screw-rod construct was larger than that of RF-F-10, and the USSII screw-rod construct was the lowest  is a complex 3D anomaly of the spine. Deformity in a single plane does not develop in isolation; rather, it is dependent on the co-development of curvature, translation, and rotation in other planes –18. CurvA good 3D correction for scoliosis requires both spinal instrumentation systems and corrective techniques. In the early 1980s, Cotrel-Dubousset (CD) instrumentation with rod derotation was introduced to enable 3D scoliosis correction surgery. However, recent reports suggest that rotational correction is variable (<25%) . CompareIn this study, we biomechanically tested the anti-rotational torque of three different pedicle screws-rod constructs. The Legacy screw-rod construct provided the best anti-rotation capacity compared with the RF-F-10 and USSII screw-rod constructs by 68% and 210%, respectively. This suggests that the Legacy screw-rod construct can increase derotational power and better sustain potential derotational correction than the other two spinal instrumentation systems. As the same type of round rods are used in all the three, it is suspected that the anti-rotational torque differences may be caused by differences in pedicle screw design, especially in the screw head and cap part. Indeed, the cap of the Legacy screw is considered to be the main reason for its excellent anti-rotational torque.The biomechanical testing results demonstrated that the Legacy screw-rod construct provides greater capacity for anti-rotational torque compared to the other two systems. This desirable property is likely affected by different screw cap and groove designs; thus, selecting an appropriate screw-rod system is very important for increasing the anti-rotational torque of a screw-rod construct, and derotational correction loss after AIS surgery is thereby reduced. However, the actual anti-rotational capacity of these three constructs need to be confirmed in clinical comparisons.It is important to discuss the limitations of the current study. Only one pedicle screw with straight rod was tested in each simulation, which is both biologically and mechanically different from the situation in which multiple pedicle screws connected with curved rod during surgery. The second limitation was that polyaxial screws test was not considered in this study, although we thought it will more useful for easying the rod placement in clinical practice but not derotation. Furthermore, our findings do not clarify whether the resulting stress concentration by the cap tip on the rod in the Legacy screw-rod construct would cause fatigue failure in the rods Fig. . Thus, fIn summary, we successfully assessed three conventional spinal screw-rod fixations for anti-rotational torque with the MTS 858 Testing System. The results showed that Legacy screws-rod construct can provide better anti-rotational capacity, possibly due to the screw cap and groove designs. The preliminary experimental results demonstrated that appropriate rod-screw construct selection in the clinic is very important for improving anti-rotational torque and preventing derotation correction loss.


There is still an ongoing discussion on the prognostic value of cystatin C in assessment of kidney function. Accordingly, the present study aimed to conduct a meta-analysis to provide evidence for the prognostic value of this biomarker for acute kidney injury (AKI) in children.An extensive search was performed in electronic databases of Medline, Embase, ISI Web of Science, Cochrane library and Scopus until the end of 2015. Standardized mean difference (SMD) with a 95% of confidence interval (95% CI) and the prognostic performance characteristics of cystatin C in prediction of AKI were assessed. Analyses were stratified based on the sample in which the level of cystatin C was measured (serum vs. urine).p < 0.0001) and urine  levels of cystatin C were significantly higher in children with AKI. Overall area under the curve of serum cystatin C and urine cystatin C in prediction of AKI were 0.83 (95% CI: 0.80-0.86) and 0.85 (95% CI: 0.81-0.88), respectively. The best sensitivity  and specificity , were observed for the serum concentration of this protein and in the cut-off points between 0.4-1.0 mg/L.A total of 24 articles were included in the meta-analysis [1948 children (1302 non-AKI children and 645 AKI cases)]. Serum (SMD = 0.96; 95% CI: 0.68-1.24; The findings of the present study showed that cystatin C has an acceptable prognostic value for prediction of AKI in children. Since the serum level of cystatin C rises within the first 24 h of admission in patients with AKI, this biomarker can be a suitable alternative for traditional diagnostic measures. Acute kidney injury (AKI) is considered as one of the main public health issues all over the world. The precise prevalence of AKI in children and adolescents is unknown. However, evidences showed that the prevalence of pediatric AKI is increasing –4. This Multiple diagnostic methods have been proposed for detection of AKI in children but none can accurately predict the outcome , 12. In Cystatin C is a 13 kilo Dalton proteinase inhibitor from the cystatin super family of cysteine protease inhibitors which plays and important role in intra-cellular catabolism of proteins and peptides. Some studies have shown that this biomarker is a better indicator of kidney function compared to creatinine , 17, butThis study was designed based on the Meta-analysis of Observational Studies in Epidemiology statement . The seaIn order to find further articles or non-indexed data, hand-search in the reference lists of relevant studies was done. Google search engine, Google scholar and ProQuest were also manually searched.Both retrospective and prospective, cohort and cross-sectional studies evaluating the prognostic value of cystatin C in detection of AKI in children (age < 18 years old) were included in the present study. Measuring the level of cystatin C after the final diagnosis of AKI, evaluating animals and using a reference test incompatible with standard criteria were considered as the exclusion criteria.The results of the search in databases were pooled and repetitive articles were removed using EndNote  software. Studies were evaluated and controlled regarding their research methodology and the summary of extracted resources were assessed independently by two separate reviewers. The reason for dismissal of any articles was recorded. In case of disagreement between the two reviewers, a third reviewer studied the article and solved the disagreement through discussion (inter-rate reliability = 0.91). Results of the search were recorded in a checklist according to the guidelines designed by PRISMA statement . ExtractQuality status of the articles was assessed using the 14-item Quality Assessment of Diagnostic Accuracy Studies (QUADAS2) tool . Only stArticles in which an appropriate reference test was not used for defining acute kidney injury were considered as having a poor quality and were excluded from the study. Good articles were the ones in which all the seven QUADAS-2 items were found to be acceptable (have low risk of bias). Even if an article had used an appropriate reference test but at least one of the other QUADAS-2 items has high risk of bias or was unclear, it was considered as having a fair quality.2 tests and a p value of less than 0.1 was considered as statistically significant (indicative of heterogeneity) [All the results of the studies were summarized into means and standard deviations, TP, FP, TN and FN. Heterogeneity between the studies was evaluated using Chi-squared and Igeneity) . If the Analyses were carried out in two steps. In the first step the mean level of cystatin C was compared between the two groups AKI and non-AKI by calculating standardized mean difference (SMD) with a confidence interval of 95% (95% CI) based on Hedge g. in this step, publication bias was also assessed through drawing funnel plots using Egger’s and Begg’s tests .In the second step, TP, FP, TN and FN cases in AKI and non-AKI patients were recorded separately for each study. These values were used for calculation of area under the curve (AUC), sensitivity, specificity, positive likelihood ratio and negative likelihood ratio with a 95% confidence interval (95% CI). Mixed-effects binary regression model, a type of random effect model was used in this section. This strategy was selected due to the significant heterogeneity between the articles. Subgroup analysis was performed to identify the sources of heterogeneity using a bivariate mixed-effects binary regression model. Deeks’ funnel plot asymmetry test was used for evaluation of publication bias.Analyses were stratified based on the samples in which cystatin C level was measured (serum and urine). STATA version 11.0  was used for data analysis.After the initial screening of the search results, full texts of 115 articles were studied and a total of 24 articles were included in the meta-analysis  while minor heterogeneity was found in the second part of our analyses evaluating the urine concentration of cystatin C . In overall analysis no publication bias was found in assessment of the relation between serum level  and urine level  of cystatin C with AKI.Analyses are presented in two separate sections evaluating the correlation between the serum level of cystatin C with occurrence of AKI and that of the urine concentration of this protein. In assessing the relation between serum level of cystatin C with AKI, a significant heterogeneity was observed between the studies . Serum cystatin C level measured on arrival , after 6 h , after 12 h  and after 24 h  were all significantly higher in AKI patients  and the ones who developed AKI following cardiac surgery  was significantly higher than non-AKI children while in other settings  no significant difference was observed between AKI and non-AKI children . The temperature at which serum was stored before conducting protein assays was another factor affecting the prognostic value of serum cystatin C level. The results showed a significant difference in the levels of cystatin C between AKI and non-AKI children when their serum were stored at the temperatures of -70 °C  and -80 °C  but such association was not observed in patients whose sera were stored at -20 °C .It seems that the setting of the study affects the serum level of cystatin C in patients. As presented in Table p = 0.44), AKI definition criteria (p = 0.79), and sample size of study (p = 0.23) had no significant effects on the difference between AKI and non-AKI children regarding their reported serum levels of cystatin C in the serum.Timing of AKI definition (p < 0.0001). Cystatin C levels on arrival  and after 12 h  were significantly higher in AKI patients but the differences in its levels between AKI and non AKI subjects after 24 h  were not statistically significant , AKI definition criteria (p = 0.11), sample size of study (p = 0.34), storage degree (p = 0.26) and setting of study (p = 0.21) did not affect the difference in urine cystatin C levels between AKI and non-AKI children.In addition, timing of AKI definition Table The best sensitivity and specificity were observed for the serum concentration of cystatin C and in the cut-off points between 0.4-1.0 mg/L, calculated to be 0.85 (95% CI:0.78-0.90) and 0.61 (95% CI:0.48-0.73), respectively.The present study aimed to gather information on the predictive value of cystatin C for AKI in children. The results for both urine and serum concentrations of this biomarker showed that cystatin C has an acceptable prognostic value for prediction of AKI in children. We included studies that had evaluated the serum or urine level of cystatin C in the first 24 h after admission or surgery of the patients. In most of these studies AKI had been diagnosed by conventional biomarkers such as serum creatinine level in the second, third or fourth day (24-96 h) post-admission or surgery. So it seems that cystatin C rises earlier than serum creatinine in response to AKI and can be a potential substitute for creatinine concentration.It seems that the serum level of cystatin C has a higher predictive value for AKI compared to its urine concentration. However, the number of studies with cystatin C urine concentration assessment was limited , 55, 62,Two factors should be evaluated when the prognostic value of a biomarker in prediction of AKI is being assessed. The first one is the time of assessment and the second is the cut-off point. A suitable biomarker should be able to diagnose a condition in the shortest amount of time with a high accuracy , 67. In Moreover, it should be noted that in the majority of included studies in this meta-analysis, AKI was diagnosed based on the rise in serum creatinine level between 24 to 96 h post-admission or surgery while the findings of this survey showed that serum cystatin C levels measured in the first 24 h, can predict development of AKI in the next few days. So it seems that cystatin C levels respond to AKI earlier than the conventional biomarkers such as creatinine and urine output, one of the strengths of this biomarker compared to others. To confirm this hypothesis, direct comparison between cystatin C and creatinine levels could not be performed due to the limited number of studies which had included this direct comparison in their analyses. Nevertheless, AKI was defined based on serum creatinine levels in the included studies and so an indirect comparison has already been conducted between the two biomarkers.The cut off points also differed between included studies (ranging from 0.4 mg/L to 2.5 mg/L) so we categorized these articles into two groups of a cut-off point less than 1.0 mg/L and a cut-off point off higher than 1.0 mg/L (serum level). The cut-off level of 1 mg/L was selected based on the previous studies showing that the normal serum level of this biomarker in children aged 1 to 17 is always less than this level .The extensive search in electronic databases and using hand-search to yield maximum relevant articles add to the strength of the present study. Another strength of this survey was the subgroup analysis performed based on two important factors of measuring time and cut-off point. The significant heterogeneity observed between the included studies was one of the weaknesses in this survey, the source of which was identified to be the setting of these studies. Moreover, in assessing the diagnostic value of cystatin C urine concentration, only six studies were included which makes the yielded results susceptible to publication bias. It should also be mentioned that all the included studies were observational surveys which increases the possibility of selection bias in these studies.Finally, it should be mentioned that the definition of AKI differed between the studies which might have affected the results of the survey. On the other hand, AKI had been diagnosed at different times (between 24 to 720 h) which made interpretation of the findings quite difficult and their generalization doubtful. However, subgroup analysis and meta-regression showed that the differences in definition and timing of AKI do not affect the prognostic value of cystatin C in predicting development of AKI.The present meta-analysis is the first to assess the prognostic value of cystatin C in detection of AKI in pediatric population. The findings of this study showed that cystatin C has an acceptable prognostic value for prediction of AKI in children, with its serum concentration diagnostic value being higher than that of its urine level. So, measuring cystatin C serum level in the first 24 h and considering a cut-off point between 0.4-1.0 mg/L provides the highest value in predicting AKI.


The central premise for the commercialization of diets with hydrolyzed ingredients is that the small-sized digested peptides would be unable to crosslink allergen-specific IgE at the surface of tissue mast cells and induce their degranulation. Evidence for the validity of this concept to diagnose food allergies in dogs and cats is limited, however. Our objectives were to study the recognition of standard and variably hydrolyzed poultry extracts by sera from dogs and cats with elevated chicken-specific serum IgE.Forty sera from dogs and 40 from cats with undetectable, low, medium or high serum levels of chicken-specific IgE were tested by ELISA on plates coated with the positive controls chicken, duck and turkey meat extracts and the negative controls beef meat (dogs) or wheat (cats). Plates were also coated with a non-hydrolyzed chicken meal, and mildly- or extensively-hydrolyzed poultry feather extracts. The frequencies of dogs with positive IgE against the various extracts were: chicken meat: 100%, duck and turkey meats: 97%, beef meat: 3%, non-hydrolyzed chicken meal: 73%, mildly-hydrolyzed poultry feathers: 37% and extensively-hydrolyzed poultry feathers: 0%. For cats, these respective percentages were (with wheat replacing beef as a negative control): 100, 84, 97, 7, 7, 0 and 0%. To detect any allergenic cross-reactivity between poultry meat-based and feather hydrolysate-derived extracts, an IgE ELISA inhibition was also done. Ten canine sera with the highest level of anti-poultry IgE in the previous experiment were incubated overnight with a previously optimized 50 μg amount of each of the extracts used above. We performed ELISA on plates coated with chicken, duck or turkey meats with or without inhibitors. The median inhibition percentages after incubation with the non-hydrolyzed chicken meal were ~22%, with the mildly-hydrolyzed poultry feathers: 14–22%, and those with the extensively-hydrolyzed poultry feathers: 5 to 10%; the last inhibition level was similar to that of the beef meat negative control.Altogether, these results suggest that an extensive—but not partial—hydrolyzation of the poultry feather extract is necessary to prevent the recognition of allergenic epitopes by poultry-specific IgE.The online version of this article (doi:10.1186/s12917-017-1183-4) contains supplementary material, which is available to authorized users. In dogs and cats, as in humans, the diagnosis of adverse food reactions (AFRs) relies on the recurrence of clinical signs after provocation with causative food ingredients. Such food re-challenges are typically done after a lengthy period of dietary restriction (i.e. “elimination”) to permit the disappearance of existing clinical signs and the return to a state of normalcy that allows the detection of positive provocations . In compFinding a diet with “novel” ingredients, be it home-cooked or commercially available, is becoming increasingly challenging because of the diverse—often unusual and sometimes even “exotic”—regimes eaten by today’s pets and the rising complexity of currently available commercial foods. Furthermore, there is evidence that some commercial diets are contaminated by ingredients not listed in their composition label –7. FinalTo alleviate the need for a perpetual quest to identify new ingredients and to reduce problems of allergenic cross-reactivity, pet food manufacturers created diets containing proteins hydrolyzed into peptides of a size supposedly insufficient to induce IgE-mediated mast cell activation (reviewed in ). For maTo further study if protein hydrolyzation results in reduced immunological allergenicity in companion animals, as it does for food allergic humans, we set out to investigate if partially- or extensively-hydrolyzed poultry feather extracts were recognized by serum IgE from dogs and cats sensitized to poultry meats. We will demonstrate herein that only the extensive hydrolyzation of the poultry feather extract results in a lack of IgE recognition by poultry-specific IgE, thereby confirming the validity of extensive hydrolyzation for hypoallergenic pet food development.405 uncorrected for background) in the Sensitest commercially available ELISA (Avacta).Surplus canine and feline sera submitted to Avacta Animal Health  by veterinarians for food allergen-specific IgE serological testing were used for this study. For dogs, the panel consisted of beef, pork, lamb, duck, chicken, turkey, wheat, soybean, barley, rice, potato, corn, oat, cow’s milk, whole hen’s egg, white fish, venison, salmon and rabbit. For cats, the panel included beef, pork, lamb, duck, chicken, turkey, rabbit, salmon, tuna, white fish, wheat, soybean, rice, corn, cow’s milk and whole hen’s egg. All dog and cat sera had been stored at −80 °C for a maximum of two and six years, respectively. Canine sera were selected according to their original levels of serum chicken-specific IgE D1-MCR: ten dogs with moderate IgE chicken reactivity (OD=405 > 1.0)D1-HCR: ten dogs with high chicken IgE reactivity (ODThe D1 group (30 dogs) contained the following sera:In their previous ELISAs done at the time of the original submission, all of the dogs in this group had detectable IgE to at least one food extract; one dog was only positive to chicken and none had any detectable IgE reactivity to beef.405 < 0.3). By previous ELISAs, these dogs had no visible IgE reactivity to any food tested on the panel.D2-NCR: This group included sera from ten dogs with undetectable levels of IgE to chicken .Similarly, we selected feline sera based upon their previously determined levels of serum chicken-specific IgE (OD405: ~0.2–0.4)C1-LCR: eleven cats with low chicken IgE reactivity (OD405: ~0.5–1.0)C1-MCR: ten cats with moderate chicken IgE reactivity (OD=405: >1.0)C1-HCR: ten cats with high chicken IgE reactivity . By previous ELISAs, there was no IgE reactivity to any food in these cats.Finally, the C2-NCR group comprised sera from nine cats with undetectable levels of IgE to chicken in the Sensitest ELISA , turkey meat (TMT), duck meat (DMT), beef meat (BMT) and wheat (WHT) extracts were purchased from Greer Laboratories . Non-hydrolysed chicken meal , mildly-hydrolysed poultry feathers (MHPF) and extensively-hydrolysed poultry feathers (EHPF) were obtained from Royal Canin. The main difference between the MHPF and EHPF was the presence of residual proteins of molecular weight superior to 10 kDa in the former, but not in the latter; the EHPF were those used in the RCA. The commercial-grade material was defatted in acetone, filtered and then dried. Allergens were obtained after two series of successive extractions in phosphate-buffered saline (PBS), followed by centrifugation and, ultimately, by dialyzing the supernatant against distilled water. The protein concentration of each extract was determined at 280 nm by Trinean DropSense96 . The final protein concentrations of the NHCM, MHPF and EHPF extracts were 6.8, 3.1 and 2.4 mg/ml, respectively. Extracts were stored at 4 °C before use.Canine and feline food allergen-specific IgE serum levels were determined by ELISA. Microtiter plates  were coated overnight at 2–8 °C with 50 μL/well of each extract, diluted to a concentration of 5 μg/mL of protein in 0.05 M carbonate/bicarbonate buffer, pH 9.6.405: 1.8) was found on a 0.5 μg/mL dog IgE coat without any concurrent detection (OD405: 0.11) of dog IgG coated at up to 20 μg/mL. After the final three washes with TBST, plates were developed with 50 μL/well of an alkaline phosphatase substrate  for 30 min at room temperature. The reactions were stopped by the addition of 50 μL/well 1 M NaOH, and OD405 were determined using a microplate reader . For both ELISAs, standard curves were generated by fitting the mean standard uncorrected OD405 to a sigmoidal 4PL curve  with log10 concentrations of the standard dog or cat serum pools.Plates were washed three times with 150 μL/well Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) and blocked with 150 μL/well TBS containing 0.5% sucrose and 0.5% PVP10 (block) for 2 h at room temperature. After removal of the block, plates were dried at 37 °C for 2 h. Canine and feline sera were diluted 1:10 in TBST, and incubated with coated extracts overnight at 2–8 °C  or chicken (for cat tests) were included on each plate. Undiluted, these two standard pools were assigned a value of 500 arbitrary units (AU). Positive and negative serum controls, with moderate and negligible levels of beef- (dog tests) or wheat- (cat tests) specific IgE, respectively, were also diluted at 1:10 in TBST and included on each plate. Plates were washed with TBST, as before, and incubated with 0.5 μg/mL alkaline phosphatase-labelled anti-dog IgE  for 2 h at RT. This mouse monoclonal antibody has been described previously  and showInhibition ELISAs were performed to determine if NHCM, MHPF and EHPF extracts could block serum IgE binding to poultry extracts, thereby demonstrating the presence of shared IgE binding epitopes. Ten dog sera that showed the highest IgE reactivity to CMT, DMT and TMT in the ELISAs described above (i.e. the HCR sera) were selected for the inhibition assays. Due to insufficient volumes available, inhibitions were not performed with the feline sera. Inhibitor solutions of NHCM, MHPF and EHPF were prepared in TBST, each to a concentration of 500, 1000 and 2000 μg/mL . To serve as the positive inhibitor, the CMT, DMT and TMT were combined, as a three-meat extract in a 1:1:1 ratio of equal protein levels, to the same concentrations as that of the extracts above. As a negative inhibitor, we diluted the BMT extract to the same concentrations. Dog sera were pre-incubated overnight at 2–8 °C, at a dilution of 1:10, with 50 μL each of the three increasing concentrations of the test and control inhibitor solutions. Additionally, each dog serum was diluted at 1:10 in TBST in the absence of inhibitor. All serum-inhibitor mixes and no-inhibitor controls were then tested at 50 μL/well by ELISA on the CMT, DMT and TMT extracts, as described above. For any given concentration of inhibitor, the percentage inhibition was calculated as follows: 100 – ((OD405 of serum with inhibitor/OD405 of serum without inhibitor) ×100) where OD405 represents the background-corrected absorbance at 405 nm.405: 1.0, by previous ELISAs), diluted at 1/10 in TBST. The membrane was incubated with serum at RT overnight. After washing three times with TBST, the membrane was incubated with alkaline phosphatase-conjugated anti-dog IgE antibody  at room temperature for 2 h with shaking. The membrane was washed three times with TBST and rinsed twice prior to addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (Thermo Scientific). The reaction was stopped with deionised water and blots were then dried at room temperature. Images of gels and blots were captured using a G:BOX Chemi-XR5 gel imaging system .The extracts above, thawed after two years of freezing, were heated at 70 °C for 10 min in the presence of 5% Criterion XT reducing agent  before sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins (5 μg per lane) were separated on 4–12% Bis-Tris Criterion XT precast gels (Bio-Rad) at 180 V using MES (4-morpholineethane-sulfonic acid) running buffer on a Criterion Cell system (Bio-Rad). Molecular weight markers  were run in parallel to the extracts. Following electrophoresis, the gels were either stained using InstantBlue Coomassie stain  to verify protein loading and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad). This was performed at 100 V for 1 h in Tris/glycine transfer buffer (Bio-Rad) containing 20% methanol using Criterion Blotter apparatus (Bio-Rad). The membrane was blocked with TBS containing 0.5% PVP10 at room temperature for 2 h, and then incubated with a serum pool from three dogs with high chicken-specific IgE levels . The frequencies of canine or feline positive ELISA tests with the various extracts were compared with permutation tests-based on Fisher’s exact test with a resampling size of 1000 times. Then, the alpha inflation risk subsequent to multiple extract comparisons was controlled with false discovery rate adjustments. The level of statistical significance was set at405 plus three standard deviations of a large number (n = 95) of canine sera without detectable anti-poultry IgE.The canine ELISA standard curve had an accuracy of between 99 and 117% at concentrations varying between 3.7 and 100 AU. The inter-assay coefficients of variation (made from 14 measurements over seven plates) of the negative and positive controls were 12 and 7%, respectively. We established the positive threshold of 8.1 AU as the mean ODFor the feline ELISA, the standard curve was 99–106% accurate over the same concentration range as above. The inter-assay coefficient of variation of the positive control was 6%. The positive threshold—determined as for the canine ELISA, but with a similar number and type of feline sera—was calculated to be 6.2 AU.The frequencies of group D1 and D2 sera testing positive on the various extracts are shown in Fig. Twenty-two sera (73%) were positive with the NHCM, and these were also positive for the CMT. When tested on the two hydrolysates, 11 (37%) and no dogs (0%) reacted to the MHPF and EHPF extracts, respectively; sera positive on the MHPF were also positive when tested on the CMT and NHCM extracts. Details on the actual frequencies of reactivity of the various D1 serum subgroups are provided in the Additional file The proportions of positive canine sera tested on the various extracts were compared statistically and results are shown in Table In the group D2 (NCR), nine of ten sera (90%) had negative results for all tested extracts; one (10%) was a very weak positive  when tested on the MHPF extract.All frequencies of positive tests using C1 and C2 sera and the various extracts are depicted in Fig. All 31 feline sera from the C1 group tested positive on the CMT; 30 (97%) reacted positively to TMT and 26 (84%) to the DMT. Two sera (7%) had a positive test on the NHCM extract, and these were also positive for the CMT, DMT and TMT; two reacted to the WHT extract (7%). In contrast, none of the sera reacted to either the MHPF or the EHPF extracts. The proportions of positive sera within the three C1 serum subgroups are available in the Additional file The proportions of C1 sera testing positive to CMT, DMT, and TMT extracts were not significantly different from each other Table . SimilarFinally, none of the nine feline sera from the C2 (NCR) group reacted to any of the food extracts tested.The percentage inhibition of the three meat positive inhibitor on CMT, DMT and TMT plates correlated positively with the increasing concentrations of inhibitor used; the inhibition with the negative control (BMT) also increased proportionally, due to nonspecific binding. At this concentration, the inhibition of the negative inhibitor background was minimal (<13%), whilst a strong inhibition (>50%) was present with the three-meat positive inhibitor. Consequently, we present herein the results of ELISAs obtained after incubation of the canine sera with 50 μg of the various inhibitors (50 μL of the serum + inhibitor mixtures), that is using a 1000 μg/mL inhibitor solution.The percentage inhibition with the positive control (three meats) varied between 86 and 91% depending upon the extract on which it was tested Fig. ; that wiSodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate the extract proteins under reduced and denatured conditions. Six distinct bands of approximately 17, 42, 48, 51, 62 and 69 kDa molecular weight were visible in the BMT extract Fig.  lane 2. Western blot analysis was then performed to evaluate the presence of IgE binding proteins in the same extracts. Canine chicken-reacting sera contained IgE that recognized only one protein of approximately 48 kDa molecular weigth in the CMT extract Fig. , lane 3.With these experiments, we provide evidence that only the extensive, but not the partial, hydrolyzation of poultry feathers prevents their recognition by serum IgE from dogs and cats sensitized to poultry. Furthermore, we found the EHPF to be unable to inhibit any of the IgE reactivity to chicken, duck or turkey meats, thereby suggesting that such hydrolysate could be of value as a restrictive commercial diet, even in dogs or cats with IgE hypersensitivity to the meats of any of these three poultry species.Using sera from AFR-suspected dogs and cats with undetectable, low, moderate or high levels of chicken-specific IgE, we first performed ELISAs using chicken, duck or turkey meat extracts. As could be expected from the results of a recent serological study , betweenAs these three meat extracts come from taxonomically related species, the co-reactivity we observed likely represents true allergenic cross-reactivity rather than improbably frequent co-sensitizations. Further ELISA inhibition studies with poultry meats and eggs should be performed to expand on the newly-proven cross-reactivity that exists between commonly eaten mammalian meats , 10. OurTo confirm these ELISA results, SDS-PAGE and immunoblotting were performed. Electrophoresis confirmed the lack of detectable bands in the NHCM, MHPF and EHPF extracts, which suggests that residual proteins, if any, would likely be below the lowest resolution allowed by this gel, that is 10 kDa. Immunoblotting done with a pool of three dogs with high chicken-specific IgE confirmed the lack of 10 to 190 kDa proteins targeted by IgE in the NHCM, MHPF and EHPF extracts.Interestingly, immunoblotting revealed that IgE from the chicken-reactive canine sera identified a single 48 kDa in the chicken meat, and likely the same protein in the beef meat extract. While the identity of this protein remains unknown, a likely candidate is Gal d 9, the chicken muscle beta-enolase, a newly-characterized chicken allergen for food allergic humans . Whilst In this study, there was low-to-absent IgE recognition of the two tested poultry feather extracts, and the frequency of positive tests depended upon the degree of hydrolysis of the product. At first, we hypothesized that poultry feathers, which are different in protein composition from that of meat, would not be recognized by IgE from chicken-sensitized companion animals. While this suspicion was verified with the feline sera, we found that serum IgE from 40% of the dogs identified the partially-hydrolyzed extract. Furthermore, our IgE ELISA inhibition studies confirmed that the MHPF extract did block between 14 to 22% of the original reactivity to the chicken, duck or turkey meats. These results imply the existence of some residual allergen cross-reactivity between poultry meats and feathers. In fact, the “bird-egg syndrome” of humans is a well-known example of such cross-reactivity ). In theIn contrast to the observations above, the extensively-digested poultry feather extract was not identified by IgE from any of our canine and feline sera, even by those with the highest levels of chicken-specific IgE. Furthermore, the percentage of inhibition of the original reactivity to chicken, duck or turkey meats was lower than that of the negative beef meat inhibitor. These results suggest that the extensive hydrolyzation of poultry feathers leads to a complete or near-complete lack of IgE recognition, even in pets with high levels of serum poultry IgE. Our ELISA inhibition data also revealed the apparent absence of allergenic cross-reactivity of this EHPF with any of the three poultry types of meat tested. These serological testing results suggests the “anallergenicity” of this extract, and they are supported by the lack of clinical reactivity seen in ten chicken-allergic dogs that ate the EHPF-containing RCA/Ultamino commercial diet for two weeks .In summary, these ELISA studies suggest that it is only with an extensive hydrolyzation that a protein source is not recognized by serum IgE from sensitized animals, even in those with high levels of sensitization. As a result, commercial foods containing extensive hydrolysates, such as the RCA/Ultamino, would be valuable to diagnose AFRs in dogs and cats, as shown by our recent trial of ten chicken-allergic dogs challenged negatively with this diet . AlternaAdditional file 1:Details of selected animal sera. (DOCX 128 kb)Additional file 2:Positive test frequencies of canine and feline serum groups. (DOCX 74 kb)


PH) is a complex disease comprising a pathologic remodeling and thickening of the pulmonary vessels causing an after load on the right heart ventricle that can result in ventricular failure. Triggered by oxidative stress, episodes of hypoxia, and other undetermined causes, PH is associated with poor outcomes and a high rate of morbidity. In the neonate, this disease has a similar etiology but is further complicated by the transition to breathing after birth, which requires a reduction in vascular resistance. Persistent pulmonary hypertension of the newborn (PPHN) is one form of PH that is frequently unresponsive to current therapies including inhaled nitric oxide (due to lack of proper absorption and diffusion), and other therapeutics targeting signaling mediators in vascular endothelium and smooth muscle. The need for novel agents, which target distinct pathways in pulmonary hypertension, remains. Herein, we investigated the therapeutic effects of novel muscarinic receptor ligand C1213 in models of PH. We demonstrated that via M3 muscarinic receptors, C1213 induced activating‐ eNOS phosphorylation (serine‐1177), which is known to lead to nitric oxide (NO) production in endothelial cells. Using signaling pathway inhibitors, we discovered that AKT and calcium signaling contributed to eNOS phosphorylation induced by C1213. As expected for an eNOS‐stimulating agent, in ex vivo and in vivo models, C1213 triggered pulmonary vasodilation and induced both pulmonary artery and systemic blood pressure reductions demonstrating its potential value in PH and PPHN. In brief, this proof‐of‐concept study provides evidence that an M3 muscarinic receptor functionally selective ligand stimulates downstream pathways leading to antihypertensive effects using in vitro, ex vivo, and in vivo models of PH.Pulmonary hypertension ( Pulmonary hypertension (PH) is a chronic debilitating disease that elicits vascular remodeling, progressive hypoxemia, and right ventricular heart failure. Hypoxia leads to pulmonary vessel constriction, and persistent hypoxia causes uncontrolled proliferation of endothelial cells, smooth muscle cells (SMC), and adventitial fibroblasts. Each of these sequelae contribute to pulmonary vascular remodeling (vessel narrowing), which in turn chronically increases resistance to blood flow through the pulmonary circulation, leading to right ventricular failure and declining cardiac output., adrenergic receptors , dopamine receptors , opioid receptors , a serotonin transporter (Kd = 1.7 × 10−9), a histamine receptor (H2 Kd = 7 × 10−10), and muscarinic receptors (nonselective Kd = 1 × 10−10). More specific testing for functional agonism and antagonism was then carried out for the muscarinic acetylcholine receptors as they are known to play crucial roles in pulmonary artery vasoconstriction/vasodilation  is a tetravalent guanylhydrazone  or indicated vendors. Compound 1213 was synthesized by Dr. Al‐Abed and was ≥95% pure by HPLC and structure was confirmed by MS and NMR , and functional agonist, and antagonist  assays were performed by NovaScreen Biosciences  as a fee for service by standardized protocols using cell lines overexpressing respective receptors under investigation.Binding , and secondary antibodies  were used. Cells were washed with ice‐cold PBS twice and were immediately lysed in the plate using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail . Proteins were resolved on SDS‐PAGE and blotted on PVDF membrane. Beta‐actin  was used as a loading control for normalization. Different doses of C1213 were used to assess target dose in relation to maximum physiological function and 10 μM was selected for studies. Densitometry was performed using Image J software which is freely available at https://imagej.nih.gov/ij/index.html. cGMP as a product of NO production was also assayed in EC culture cells after treating them with vehicle or C1213 .Compound 1213 was added to EC culture immediately before hypoxia exposure. One hour later, phosphorylation of serine‐1177 endothelial nitric oxide synthetase (eNOS) was measured by Western blot in both ECs incubated in room air (RA) or exposed to hypoxia. Commercially available antibodies for serine‐1177 POμmol/L) and indicated blockers (antagonists) were added to EC tissue culture and the western experiment described above was repeated. The following receptor antagonists were used: Atropine at dose of 0.01 μmol/L was used as nonspecific muscarinic (M) receptor blockade, pirenzepine (M1 blocker) at doses 1,10 and 100 μmol/L; dareifenacin (M3 blocker) at doses 0.01, 0.1, and 1 μmol/L, and methyllycaconitine (MLA) nicotinic blocker at doses 0.1, 1, and 10 μmol/L. Pirenzepine dihydrochloride was purchased from Tocris Bioscience .We used a series of acetylcholine receptor blockers and measured (serine‐1177) phosphorylated eNOS protein  as a read out. Both C1213 , dorsomorphin dihydrochloride aka “compound C” (AMPK inhibitor), and BAPTA‐AM (Ca+2 chelator). Wortmannin, compound C, and BAPTA‐AM were purchased from Tocris Bioscience . Western blot for AKT, pAKT, and actin was performed as described above. Commercially available primary antibodies for AKT, pAKT, and secondary antibodies were used in westerns .μM).Physiological function assessments were performed on explanted main intralobar pulmonary arteries with intact endotheliumas previously described in mice  in time‐dated pregnant sheep  was calculated using the following equation: PVR (mmHg.mL−1.min.kg body weight) =  47 pulmonary blood flow corrected for body weight . Systemic and pulmonary pressure, pulmonary and coronary blood flow, PaO2, and PaCO2 were recorded and once the animal was stable (about 2 h post partum), an IV bolus dose of 1213 (4 mg/kg – approx. 80 μmol/L in blood max.) was given. Readings of systemic and pulmonary vascular pressure and PO2/PCO2 were monitored postinjection of C1213.This study was approved by the Laboratory Animal Care Committee at the State University of New York (SUNY) Buffalo. Fetal ductal ligation was performed 20 days preterm at 125 days of gestation . T‐test was used in comparison between two groups. A μmol/L) in cells overexpressing certain receptors, C1213 induced nonspecific calcium flux through an unelucidated mechanism of action. This finding is difficult to interpret as these cells are engineered (proprietary) to stimulate calcium release as a readout of GPCR activation and also express much higher levels of receptors than a native cell, alternatively, compound solubility issues in the specialized buffer may have been the cause.Neurohumoral/neurotransmitter receptor functional activation was explored using a Novascreen G‐protein coupled receptor (GPCR) calcium flux assay . For agonist studies, C1213  stimulation of calcium flux for muscarinic receptors M1, M2, and M3, with IC‐50's shown in Figure Our data demonstrated that hypoxia led to an increase in the serine‐1177 eNOS phosphorylation as expected. Addition of C1213 for 1 h elicited a significant induction of ser‐1177 eNOS phosphorylation in the endothelial cell‐line HPMEC‐ST1.6R in both normal  and hypoxic (FiO2 1% for 60 min) conditions Fig. .These studies investigate the specific neurohumoral receptor subtype responsible for C1213 effects on serine‐1177 eNOS phosphorylation, which is a known stimulator of eNOS activity . We next examined the effect of C1213 on AKT activation by assessing its phosphorylation state in the presence of the same kinase inhibitors. When normalized to total AKT, C1213 induced AKT phosphorylation and as anticipated, only wortmannin profoundly inhibited AKT phosphorylation . Taken together, these finding begin to decipher pathways by which C1213 stimulates NO production: it increases the phosphorylation and activation of eNOS mainly through AKT and Ca+2 signaling mechanisms, but not through AMPK.Protein kinases (AKT and AMPK) activate NO production through phosphorylation of eNOS at specific amino acid residues. We investigated the functional involvement of AKT‐ and AMPK‐dependent and ‐independent pathways in C1213‐induced eNOS phosphorylation (ser‐1177) by looking at cell lysates of pulmonary endothelial cell cultures treated with C1213 for 1 h, in the presence and absence of various kinase inhibitors. When coincubated with wortmannin (AKT inhibitor) or BAPTA (Ca+2 chelator), eNOS phosphorylation was dampened, however, compound C (AMPK inhibitor) had no effect  rapidly (within 2 min) overcame the contraction induced by PE and the tone essentially dropped to the pre‐PE baseline level within 1–2 min  a rapid reflexive increase in PaO2 and corresponding decrease in PaCO2 is elicited which then normalizes.For our studies we used the gold standard model of PPHN in lambs, where PH is induced by ductal ligation in utero. The success of our model of PPHN was demonstrated by a peak PA pressure of close to 40 mmHg . In the initial phase of monitoring, systemic and PA pressures and ventilation status were stabilized allowing for acclimation Fig. C—plateau2 and NO inhalation therapy remains some of the most effective treatments for PPHN, this research has revealed the risks of oxidative stress that can be induced by inhaled oxygen  therapies  lead to the discovery of C1213, which was found to act as a functionally selective agonist on M3 receptors of pulmonary vascular endothelial cells. A summary of our main findings in endothelial cells and ex vivo and in vivo models of PPHN is presented in Figure Although OCombined, our results provide evidence for the continued investigation of C1213 as a new experimental vasodilatory agent, capable of stimulating endogenous NO production from endothelial vascular cells, leading to relaxation of smooth muscle pulmonary vasculature, and reduction in pulmonary hypertension. Additional testing indicated that C1213 may act as either a competitive antagonist or functionally selective agonist on muscarinic receptors, which was dependent on the downstream assay monitored and/or cell type used for study. Future studies will look at larger sample size of animal model with PPHN and look at the longer‐term effects of C1213 administration and help clarify other potential mechanisms  by which C1213 generates its effects on pulmonary arterial and systemic vascular pressure.None declared.