Total Hip Arthroplasty in Protrusio Acetabuli: A Systematic Review.

Protrusio acetabuli, or abnormal protrusion of the femoral head into the acetabulum, requires performance of a total hip arthroplasty (THA) for which various reconstruction techniques and outcomes have been described. The aim of this systematic review is to provide a comprehensive analysis of the current evidence, evaluate treatment efficacy, compare surgical techniques, and identify topics for future research along with improving evidence-based decision-making, improving patient outcomes in the management of this condition. A thorough systematic review of the PubMed, Embase, Cochrane Library databases, and Scopus library was conducted, and articles describing techniques of THA for treatment of protrusion acetabuli were extracted. The initial search generated 751 results. After exclusion, 18 articles were included. Of these, eight were prospective studies and 10 were retrospective. Surgery was performed on 783 hips with a mean age of 60 years; 80% of females who mostly had inflammatory arthritis were followed up for 8.86 years (range, 2-15.4 years). Good outcomes have been achieved with THA using uncemented cups with bone graft; however, no conclusion could be drawn with regard to the femoral side. It can be concluded that the concept of restoration of the anatomical hip center of rotation is paramount for good outcome and better survival of the implant is important when using uncemented cups with a bone graft. In addition, screw augmentation for fixation is not recommended unless absolutely necessary. The most common complications were aseptic loosening and heterotopic ossification. While the former required revision, conservative management was administered for the latter.

Increased medial talar tilt may incite ankle pain and predispose ankle osteoarthritis after correction of severity of knee varus deformity among patients undergoing bilateral total knee arthroplasty: a prospective observation.

PURPOSE: Patients with varus knee osteoarthritis usually compensate at the ankle and typically walk with hindfoot valgus alignment. As the neutral weight-bearing axis of the lower limbs is restored with Total Knee Arthroplasty (TKA), ankle and hindfoot biomechanics also acutely change. This study aims to investigate whether any ankle clinical-radiographical changes occur as a result of bilateral mechanical TKA in patients with bilateral Osteoarthritis knee at a minimum follow-up of 6 months. METHODS: The prospective observational study included 61 patients (122 knees) undergoing simultaneous bilateral TKA (mechanical alignment). Tibio-talar angle(TTA), tibial Anterior Surface angle (TAS), lateral distal tibial angle (LDTA), talar-tilt angle (TT), anatomical talocrural angle (aTC), ground surface and distal tibial plafond angle (GP), ground surface and an upper surface of talus angle (GT)and tibial plateau and tibial plafond angle (PP) were measured on long-film radiographs to look for changes in the ankle, whereas functional assessment was done using American Foot and Ankle Society (AOFAS), Foot and Ankle Disability Index (FADI), and Forgotten Joint (FJS-12) scores. Patients were sub-grouped based on the Hip-Knee-Ankle (HKA) axis, and the effect of the severity of knee varus on the ankles after TKA was also analyzed. The minimum follow-up was 6 months. RESULTS: A significant decrease in the tibial plateau-tibial plafond (PP), ground-tibial plafond (GP), and ground-talar dome (GT) angles was noted after TKA (p-value < 0.05). Postoperative functional parameters were comparable to the preoperative status except for FADI, which significantly improved (p-value-0.03). Sub-group analysis based on the severity of knee varus (HKA) revealed GT to be most significantly reduced (p-value-0.036), while the talar tilt (TT) increased (p-value-0.044). Functional outcomes of the ankles clinically improved with the correction of severe knee varus after TKA. At a mean follow-up of 13.2 months post-TKA, 7 out of 61 (11.4%) patients complained of post-TKA ipsilateral ankle pain. CONCLUSION: Mechanically aligned bilateral TKA in severe varus deformity of the knee significantly decreases the GT angle but increases the varus tilt of the talus with lateral talar incongruency and under-coverage. Although the acute correction of severe knee varus deformity aligns the tibia more neutrally, resulting in an overall clinically evident improvement in ankle functional outcome, the increased varus talar tilt remains a deep concern. LEVEL OF EVIDENCE: Prospective, observational, comparative study Level II.

Anatomical Variations of the Optic Nerve in the Sphenoid Sinus: Do Ethnic Variations Matter?

There are several variations in the anatomical relations of the optic nerve with the sphenoid sinus. Proper understanding of these variations is clinically important to minimize injuries associated with surgical procedures that involve the sphenoid sinus. CT scans of paranasal sinuses obtained prior to any endoscopic surgery in the sphenoid sinus area is useful for designing operative strategies. Ethnic variations in sinonasal anatomy have been documented. The objective was to study the pattern of relationship between optic nerve and the sphenoid sinus using computerized tomographic imaging in a north Indian population, to compare our findings with previous studies in different ethnic groups and find out if ethnic variations in such a relationship matter. A prospective study was conducted on 300 patients who underwent computed tomography of the paranasal sinuses from Sept 2020 to June 2021. Relationship of optic nerves to the sphenoid sinuses was categorized according to DeLano classification. Pneumatization of the anterior clinoid process and bony dehiscence of optic nerve was also observed. Type 1 position of optic nerve was seen in 69.3%, Type 2 in 20.9%, Type 3 in 3% and Type 4 in 6.8% of sinuses. The pneumatization of anterior clinoid process (ACP) was observed in 10.5% and the bony dehiscence of optic nerve was noted in 6.5% sinuses. Bony dehiscence of optic canal had associated ACP pneumatization in 64.1% sinuses.The variability of the relation of optic nerve to the sphenoid sinus even in the persons of same ethnicity precludes a definite role of ethnicity in these variations. Other factors possibly contributing to such a relationship have been discussed.

Modern implants in total knee arthroplasty are more patellofemoral joint friendly compared to their traditional design: a systematic review and meta-analysis.

BACKGROUND: Implant designs for total knee arthroplasties (TKA) are continuously evolving to improve outcomes and satisfaction rates after TKA. The present systematic review and meta-analysis aimed to explore the evidence in the literature regarding the outcomes of the Attune knee system over the PFC Sigma knee design and investigate the advantage of one over the other. METHODS: A systematic review and meta-analysis of published studies till August 2021 was performed using the defined eligibility criteria. This was a systematic review of the literature published in the Cochrane Central Register of Controlled Trials (CENTRAL) Library, PUBMED, and EMBASE. The analysis included prospective and retrospective comparative trials comparing TKA by PFC sigma or Attune posterior stabilized (PS) or cruciate-retaining (CR), fixed bearing, or rotating platform systems. Patient-reported outcome measures (PROM) and postoperative patellar complications were analysed in the studies utilizing attune knee system (modern implants) to its counterpart PFC sigma (traditional implants) for TKA. Quality assessment was conducted using NIH Quality Assessment Tool for controlled intervention studies (RCTs and case-control studies). RESULTS: This review included 3 RCTs and the rest, 10 of which were non-RCT, including 5852 patients. ATTUNE designs suggested a statistically significant improvement in KSS over PFC Sigma designs. Other PROMs such as OKS and WOMAC scores yielded comparable results between the two groups. ATTUNE knee prosthesis did not result in better knee range of motion, patient satisfaction, or radiological outcomes than the PFC design. Regarding the complications, attune knee prosthesis demonstrated favourable results over PFC Sigma for anterior knee pain and patellofemoral (PF) crepitus. CONCLUSIONS: The present systematic review highlights better KSS and lesser chances of PF complications favouring a modern implant design over its traditional counterpart. Other patient-reported outcome analyses at a short-term follow-up period were comparable among patients undergoing total knee replacements with two different implant designs. Radiological outcomes for component positioning also suggested similar results among the two groups.

Chlorambucil-Loaded Graphene-Oxide-Based Nano-Vesicles for Cancer Therapy.

In this study, the authors have designed biocompatible nano-vesicles using graphene oxide (GO) for the release of chlorambucil (CHL) drugs targeting cancerous cells. The GO sheets were first sulfonated and conjugated with folic acid (FA) molecules for controlled release and high loading efficiency of CHL. The chlorambucil (CHL) drug loading onto the functionalized GO surface was performed through π-π stacking and hydrophobic interactions with the aromatic planes of GO. The drug loading and "in vitro" release from the nano-vesicles at different pH were studied. The average particle size, absorption, and loading efficiency (%) of FA-conjugated GO sheets (CHL-GO) were observed to be 300 nm, 58%, and 77%, respectively. The drug release study at different pH (i.e., 7.4 and 5.5) showed a slight deceleration at pH 7.4 over pH 5.5. The amount of drug released was very small at pH 7.4 in the first hour which progressively increased to 24% after 8 h. The rate of drug release was faster at pH 5.5; initially, 16% to 27% in the first 3 h, and finally it reached 73% after 9 h. These observations indicate that the drug is released more rapidly at acidic pH with a larger amount of drug-loading ability. The rate of drug release from the CHL-loaded GO was 25% and 75% after 24 h. The biotoxicity study in terms of % cell viability of CHL-free and CHL-loaded GO against human cervical adenocarcinoma cell line was found to have lower cytotoxicity of CHL-loaded nano-vesicles (IC50 = 18 µM) as compared to CHL-free (IC50 = 8 µM). It is concluded that a high drug-loading efficiency and controlled release with excellent biotoxicity of CHL-GO offers an excellent application in the biomedical field.

Use of Growth Rod Systems for Management of Early Onset Scoliosis in Cerebral Palsy: A Systematic Review.

AIM: To review the literature for the role and outcome of growing rod surgeries in patients with cerebral palsy associated neuromuscular scoliosis. MATERIAL AND METHODS: A systematic search was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Electronic literature search was conducted of PubMed and Embase databases. Patient demographics, type of growing rod used, lengthening and complications were analyzed from the included studies. RESULTS: A total of 11 studies with poor overall study quality (Level of evidence IV, V) were included in the study. A total of 181 patients with mean age 6.8 ± 1.3 (5-13 range) years at index surgery and mean follow-up of 3.02 ± 1.3 (2-5.8 range) years were included in the study, with a female preponderance. The most common curve and instrumentation was thoraco-lumbar and conventional dual growing rods respectively. All studies showed improvement in Cobb?s angle and pelvic obliquity. There was better improvement in pelvic obliquity if pelvis was included in instrumentation. Wound related complications (34.6%) were most commonly noted. CONCLUSION: Overall growing rod construct has shown questionable outcomes in cerebral palsy patients with scoliosis in terms of the complication rate observed although allowing growth of the spinal column with regular lengthenings. Magnetic controlled growth rods hold a bright promise for the future considering its ability to maintain correction as well as the lower rate of complications The benefits and risk of immediate fusion with respect to growth sparing surgeries should be considered before the decision.

Quantification of Volume Mismatch of Acetabulum and Femoral Head in Developmental Dysplasia of Hip.

Objectives: The sustained subluxation or dislocation of the femoral head over time does not permit normal development of acetabulum and results in predictable pattern of acetabular growth disturbance that is termed hip dysplasia. The primary aim of this study is to analyze and quantify the volume mismatch between acetabulum and femoral head of affected side as compared to normal hip. Methods: A prospective observational study was conducted by including isolated untreated unilateral idiopathic developmental dysplasia of hip (DDH). After routine clinical and radiographic examination, computed tomography (CT) of both hips was done with pre-determined radiation dosage within safe limits for the pediatric age group in 18 patients of median age 2 years (range 1-5 years). Results: A significant difference was noted between acetabular index (p<0.001), acetabular volume (p<0.001), femoral head volume (p<0.001), and acetabular anterior sectoral angle (p=0.002) of the affected and the normal hips. As compared to the normal side, the acetabulum is 2.6 times smaller than the normal side and femoral epiphysis volume by 3.8 times. A significant negative correlation (r=-0.66, p=0.04) was noted between posterior acetabular sectoral angle and acetabular volume of affected hip. Conclusion: CT is an important investigation in evaluation of late-presenting DDH. The absence of femoral head in its orthotopic location affects the volume of acetabulum as well as that of femoral head. The abnormality of the volume of acetabulum which is seen as related to the dysplasia should be studied and assessed in detail in a child of late-presenting DDH. This would guide us toward the coverage defect and type of osteotomy to be performed.

Characterization of Cationic Bolaamphiphile Vesicles for siRNA Delivery into Tumors and Brain.

Small interfering RNAs (siRNAs) are potential therapeutic substances due to their gene silencing capability as exemplified by the recent approval by the US Food and Drug Administration (FDA) of the first siRNA therapeutic agent (patisiran). However, the delivery of naked siRNAs is challenging because of their short plasma half-lives and poor cell penetrability. In this study, we used vesicles made from bolaamphiphiles (bolas), GLH-19 and GLH-20, to investigate their ability to protect siRNA from degradation by nucleases while delivering it to target cells, including cells in the brain. Based on computational and experimental studies, we found that GLH-19 vesicles have better delivery characteristics than do GLH-20 vesicles in terms of stability, binding affinity, protection against nucleases, and transfection efficiency, while GLH-20 vesicles contribute to efficient release of the delivered siRNAs, which become available for silencing. Our studies with vesicles made from a mixture of the two bolas (GLH-19 and GLH-20) show that they were able to deliver siRNAs into cultured cancer cells, into a flank tumor and into the brain. The vesicles penetrate cell membranes and the blood-brain barrier (BBB) by endocytosis and transcytosis, respectively, mainly through the caveolae-dependent pathway. These results suggest that GLH-19 strengthens vesicle stability, provides protection against nucleases, and enhances transfection efficiency, while GLH-20 makes the siRNA available for gene silencing.

Naturally Occurring Missense MRGPRX2 Variants Display Loss of Function Phenotype for Mast Cell Degranulation in Response to Substance P, Hemokinin-1, Human ß-Defensin-3, and Icatibant.

Human mast cells (MCs) express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR X2 (MRGPRX2). Activation of this receptor by a diverse group of cationic ligands such as neuropeptides, host defense peptides, and Food and Drug Administration-approved drugs contributes to chronic inflammatory diseases and pseudoallergic drug reactions. For most GPCRs, the extracellular (ECL) domains and their associated transmembrane (TM) domains display the greatest structural diversity and are responsible for binding different ligands. The goal of the current study was to determine if naturally occurring missense variants within MRGPRX2's ECL/TM domains contribute to gain or loss of function phenotype for MC degranulation in response to neuropeptides (substance P and hemokinin-1), a host defense peptide (human ß-defensin-3) and a Food and Drug Administration-approved cationic drug (bradykinin B2 receptor antagonist, icatibant). We have identified eight missense variants within MRGPRX2's ECL/TM domains from publicly available exome-sequencing databases. We investigated the ability of MRGPRX2 ligands to induce degranulation in rat basophilic leukemia-2H3 cells individually expressing these naturally occurring MRGPRX2 missense variants. Using stable and transient transfections, we found that all variants express in rat basophilic leukemia cells. However, four natural MRGPRX2 variants, G165E (rs141744602), D184H (rs372988289), W243R (rs150365137), and H259Y (rs140862085) failed to respond to any of the ligands tested. Thus, diverse MRGPRX2 ligands use common sites on the receptor to induce MC degranulation. These findings have important clinical implications for MRGPRX2 and MC-mediated pseudoallergy and chronic inflammatory diseases.

Cellular Delivery of siRNAs Using Bolaamphiphiles.

Discovery of RNA interference (RNAi) has opened up a new arena of therapeutic intervention for the treatment of cancerous as well as noncancerous diseases. The RNAi pathway utilizes RNAi inducers such as small interfering RNAs (siRNAs) to target and silence disease causing genes. However, efficient delivery of siRNAs for eliciting efficacious RNAi has remained a daunting challenge. Nonviral vectors such as lipids have shown great promise in delivering siRNAs. Recently, a novel class of cationic lipid molecules "bolaamphiphile lipids" or "bola lipids" has been shown to deliver siRNAs to cause effective gene silencing in cells. The present chapter showcases the ability of bola lipids to form micelles, bind with nucleic acids and protect nucleic acids against nucleases. Also, high in vitro transfection efficiency for silencing green fluorescent protein (GFP) using Dicer substrate siRNAs (dsiRNAs) designed against GFP at nontoxic dose in a human breast cancer model is demonstrated. Our results showed that these cationic bola lipids are promising siRNA delivery agents.

Functionalized non-viral cationic vectors for effective siRNA induced cancer therapy.

RNA interference (RNAi) has been regarded as a vital asset in the field of therapeutics as it has the capability to silence various disease causing genes including those that cause cancer. Small non-coding RNA molecules such as short interfering RNAs (siRNAs) are one of the extensively studied RNAi inducers for gene modulations. However, the delivery of RNAi inducers including siRNAs is compromised due to the barriers imposed by the biological system such as degradation by nucleases, rapid clearance, high anionic charge, immunogenicity and off-target effects. Viral vectors, in general exhibit high transfection efficiencies but are expensive and likely to confer immunological and safety issues. Therefore, non-viral cationic vectors (NVCVs) have received considerable attention to not only address these issues but also for developing efficacious siRNA delivery vectors. In this review, we will first discuss the historical development of various NVCVs and then will discuss functionalized NVCVs with linkers that provide stability, as well as respond to the cancer cell environment and with cancer cell receptor specific ligands to explicitly target them for improved siRNA efficacy. Multifunctional NVCVs (MNVCVs) that employ multiple synergistically working components to aid siRNA delivery efficacy are also discussed.

Roles of Mas-related G protein-coupled receptor X2 on mast cell-mediated host defense, pseudoallergic drug reactions, and chronic inflammatory diseases.

Mast cells (MCs), which are granulated tissue-resident cells of hematopoietic lineage, contribute to vascular homeostasis, innate/adaptive immunity, and wound healing. However, MCs are best known for their roles in allergic and inflammatory diseases, such as anaphylaxis, food allergy, rhinitis, itch, urticaria, atopic dermatitis, and asthma. In addition to the high-affinity IgE receptor (FcεRI), MCs express numerous G protein-coupled receptors (GPCRs), which are the largest group of membrane receptor proteins and the most common targets of drug therapy. Antimicrobial host defense peptides, neuropeptides, major basic protein, eosinophil peroxidase, and many US Food and Drug Administration-approved peptidergic drugs activate human MCs through a novel GPCR known as Mas-related G protein-coupled receptor X2 (MRGPRX2; formerly known as MrgX2). Unique features of MRGPRX2 that distinguish it from other GPCRs include their presence both on the plasma membrane and intracellular sites and their selective expression in MCs. In this article we review the possible roles of MRGPRX2 on host defense, drug-induced anaphylactoid reactions, neurogenic inflammation, pain, itch, and chronic inflammatory diseases, such as urticaria and asthma. We propose that host defense peptides that kill microbes directly and activate MCs through MRGPRX2 could serve as novel GPCR targets to modulate host defense against microbial infection. Furthermore, mAbs or small-molecule inhibitors of MRGPRX2 could be developed for the treatment of MC-dependent allergic and inflammatory disorders.

Modulation of host defense peptide-mediated human mast cell activation by LPS.

Human ß-defensin3 (hBD3) and the cathelicidin LL-37 are host defense peptides (HDPs) that directly kill microbes and display immunomodulatory/wound-healing properties via the activation of chemokine, formylpeptide and epidermal growth factor receptors on leukocytes and epithelial cells. A C-terminal 14 amino acid hBD3 peptide with all Cys residues replaced with Ser (CHRG01) and an LL-37 peptide consisting of residues 17-29 (FK-13) display antimicrobial activity but lack immunomodulatory property. Surprisingly, we found that CHRG01 and FK-13 caused Ca(2+) mobilization and degranulation in human mast cells via a novel G protein-coupled receptor known as Mas-related gene-X2 (MrgX2). At local sites of bacterial infection, the negatively charged LPS likely interacts with cationic HDPs to inhibit their activity and thus providing a mechanism for pathogens to escape host defense mechanisms. We found that LPS caused almost complete inhibition of hBD3 and LL-37-induced Ca(2+) mobilization and mast cell degranulation. In contrast, it had no effect on CHRG01 and FK-13-induced mast cell responses. These findings suggest that HDP derivatives that kill microbes, harness mast cell's host defense and wound-healing properties via the activation of MrgX2 but are resistant to inhibition by LPS could be utilized for the treatment of antibiotic-resistant microbial infections.

Activation of human mast cells by retrocyclin and protegrin highlight their immunomodulatory and antimicrobial properties.

Preclinical evaluation of Retrocyclins (RC-100, RC-101) and Protegrin-1 (PG-1) antimicrobial peptides (AMPs) is important because of their therapeutic potential against bacterial, fungal and viral infections. Human mast cells (HMCs) play important roles in host defense and wound healing but the abilities of retrocyclins and protegrin-1 to harness these functions have not been investigated. Here, we report that chemically synthesized RC-100 and PG-1 caused calcium mobilization and degranulation in HMCs but these responses were not blocked by an inhibitor of formyl peptide receptor-like 1 (FPRL1), a known receptor for AMPs. However, RC-100 and PG-1 induced degranulation in rat basophilic leukemia (RBL-2H3) cells stably expressing Mas related G protein coupled receptor X2 (MrgX2). Chemical synthesis of these AMPs is prohibitively expensive and post-synthesis modifications (cyclization, disulfide bonds, folding) are inadequate for optimal antimicrobial activity. Indeed, we found that synthetic RC-100, which caused mast cell degranulation via MrgX2, did not display any antimicrobial activity. Green-fluorescent protein (GFP)-tagged RC-101 (analog of RC-100) and GFP-tagged PG-1 purified from transgenic plant chloroplasts killed bacteria and induced mast cell degranulation. Furthermore, GFP-PG1 bound specifically to RBL-2H3 cells expressing MrgX2. These findings suggest that retrocyclins and protegrins activate HMCs independently of FPRL1 but via MrgX2. Harnessing this novel feature of AMPs to activate mast cell's host defense/wound healing properties in addition to their antimicrobial activities expands their clinical potential. Low cost production of AMPs in plants should facilitate their advancement to the clinic overcoming major hurdles in current production systems.

Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.

In this study we have investigated a new class of cationic lipids--"bolaamphiphiles" or "bolas"--for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5µg/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production.

Oxime ether lipids containing hydroxylated head groups are more superior siRNA delivery agents than their nonhydroxylated counterparts.

AIM: To evaluate the structure-activity relationship of oxime ether lipids (OELs) containing modifications in the hydrophobic domains (chain length, degree of unsaturation) and hydrophilic head groups (polar domain hydroxyl groups) toward complex formation with siRNA molecules and siRNA delivery efficiency of resulting complexes to a human breast cancer cell line (MDA-MB-231). MATERIALS & METHODS: Ability of lipoplex formation between oxime ether lipids with nucleic acids were examined using biophysical techniques. The potential of OELs to deliver nucleic acids and silence green fluorescent protein (GFP) gene was analyzed using MDA-MB-231 and MDA-MB-231/GFP cells, respectively. RESULTS & CONCLUSION: Introduction of hydroxyl groups to the polar domain of the OELs and unsaturation into the hydrophobic domain favor higher transfection and gene silencing in a cell culture system.

Regulation of Fc∈RI signaling in mast cells by G protein-coupled receptor kinase 2 and its RH domain.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCRkinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on Fc∈RI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2(flox/flox)/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ~50% decrease in antigen-induced Ca(2+) mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the Fc∈RI subunits (α, ß, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of Fc∈RIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates Fc∈RI signaling in mast cells via at least two mechanisms.One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt.

Mechanism of membrane permeation induced by synthetic ß-hairpin peptides.

We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form ß-hairpin structures under certain conditions, and a control non-ß-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ~50% of the liposomes that contain small (R(h) <1.5 nm) markers, only ~15% of those liposomes release a fluorescent dextran of 40 kDa. A multimeric model of the pore is presented based on these results. Atomistic molecular dynamics simulations show that barrels consisting of 10 ß-hairpin MAX1 and MAX35 peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action.

ß-Defensins activate human mast cells via Mas-related gene X2.

Human ß-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells in vitro and cause increased vascular permeability in rats in vivo. In this study, we sought to determine whether hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells (BMMC) in vitro and had no effect on vascular permeability in vivo. By contrast, these peptides induced sustained Ca(2+) mobilization and substantial degranulation in human mast cells, with hBD3 being more potent. Pertussis toxin (PTx) had no effect on hBD-induced Ca(2+) mobilization, but La(3+) and 2-aminoethoxydiphenyl borate (a dual inhibitor of inositol 1,4,5-triphosphate receptor and transient receptor potential channels) caused substantial inhibition of this response. Interestingly, degranulation induced by hBDs was substantially inhibited by PTx, La(3+), or 2-aminoethoxydiphenyl borate. Whereas human mast cells endogenously express G protein-coupled receptor, Mas-related gene X2 (MrgX2), rat basophilic leukemia, RBL-2H3 cells, and murine BMMCs do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD-induced degranulation, but had no effect on anaphylatoxin C3a-induced response. Furthermore, ectopic expression of MrgX2 in RBL-2H3 and murine BMMCs rendered these cells responsive to hBDs for degranulation. This study demonstrates that hBDs activate human mast cells via MrgX2, which couples to both PTx-sensitive and insensitive signaling pathways most likely involving Gαq and Gαi to induce degranulation. Furthermore, murine mast cells are resistant to hBDs for degranulation, and this reflects the absence of MrgX2 in these cells.