Enhanced arthrocentesis of the effusive knee with pneumatic compression.

AIM: Complete arthrocentesis of the effusive knee ameliorates patient pain, reduces intra-articular and intraosseous pressure, removes inflammatory cytokines, and has been shown to substantially improve the therapeutic outcomes of intra-articular injections. However, conventional arthrocentesis incompletely decompresses the knee, leaving considerable residual synovial fluid in the intra-articular space. The present study determined whether external pneumatic circumferential compression of the effusive knee permitted more successful arthrocentesis and complete joint decompression. METHODS: Using a paired sample design, 50 consecutive effusive knees underwent conventional arthrocentesis and then arthrocentesis with pneumatic compression. Pneumatic compression was applied to the superior knee using a conventional thigh blood pressure cuff inflated to 100 mm Hg which compressed the suprapatellar bursa and patellofemoral joint, forcing fluid from the superior knee to the anterolateral portal where the fluid could be accessed. Arthrocentesis success and fluid yield in mL before and after pneumatic compression were determined. RESULTS: Successful diagnostic arthrocentesis (≥3 mL) of the effusive knee was 82% (41/50) with conventional arthrocentesis and increased to 100% (50/50) with pneumatic compression (P = .001). Synovial fluid yields increased by 144% (19.8 ± 17.1 mL) with pneumatic compression (conventional arthrocentesis; 13.7 ± 16.4 mL, pneumatic compression: 33.4 ± 26.5 mL; 95% CI: 10.9 < 19.7 < 28.9 mL, P < .0001). CONCLUSIONS: Conventional arthrocentesis routinely does not fully decompress the effusive knee. External circumferential pneumatic compression markedly improves arthrocentesis success and fluid yield, and permits complete decompression of the effusive knee. Pneumatic compression of the effusive knee with a thigh blood pressure cuff is an inexpensive and widely available technique to improve arthrocentesis outcomes.

Neuronal delivery of antibodies has therapeutic effects in animal models of botulism.

Botulism is caused by a potent neurotoxin that blocks neuromuscular transmission, resulting in death by asphyxiation. Currently, the therapeutic options are limited and there is no antidote. Here, we harness the structural and trafficking properties of an atoxic derivative of botulinum neurotoxin (BoNT) to transport a function-blocking single-domain antibody into the neuronal cytosol where it can inhibit BoNT serotype A (BoNT/A1) molecular toxicity. Post-symptomatic treatment relieved toxic signs of botulism and rescued mice, guinea pigs, and nonhuman primates after lethal BoNT/A1 challenge. These data demonstrate that atoxic BoNT derivatives can be harnessed to deliver therapeutic protein moieties to the neuronal cytoplasm where they bind and neutralize intracellular targets in experimental models. The generalizability of this platform might enable delivery of antibodies and other protein-based therapeutics to previously inaccessible intraneuronal targets.

Extractable synovial fluid in inflammatory and non-inflammatory arthritis of the knee.

INTRODUCTION/OBJECTIVES: We hypothesized that mechanical compression of the knee in rheumatoid arthritis (RA) would mobilize occult extractable fluid and improve arthrocentesis success. METHODS: Sixty-seven consecutive knees with RA and 186 knees with OA and were included. Conventional arthrocentesis was performed and success and volume (milliliters) determined; the needle was left intraarticularly, and mechanical compression was applied with an elastomeric knee brace. Arthrocentesis was then resumed until fluid return ceased. Fluid was characterized as to volume and cell counts. RESULTS: In the RA, knee mechanical compression decreased failed diagnostic arthrocentesis from 56.7% (38/67) to 26.9% (18/67) (- 47.4%, p = 0.003) and increased absolute arthrocentesis yield from 4.7 ± 10.3 ml to 9.8 ± 9.8 ml (108% increase, 95% CI - 8.5 < - 5.1 < - 1.7 p = 0.0038). Total extractable fluid yield was 96% greater in RA (9.8 ± 9.8 ml) than OA (5.0 ± 9.4 ml, p = 0.0008), and occult extractable fluid was 77% greater in RA than OA (RA 5.3 ± 8.7 ml, OA 3.0 ± 5.5 ml, p = 0.046). Large effusions versus small effusions in RA demonstrated increased neutrophils in synovial fluid (p = 0.04) but no difference in radiologic arthritis grade (p = 0.87). In contrast, large effusions versus small effusions in OA demonstrated no difference in neutrophils in synovial fluid (p = 0.87) but significant different radiologic arthritis grade (p = 0.04). CONCLUSION: Mechanical compression improves the success of diagnostic and therapeutic knee arthrocentesis in both RA and OA. Large effusions in RA are associated with increased neutrophil counts but not arthritis grade; in contrast, large effusions in OA are associated with more severe arthritis grades but not increased neutrophil counts. Key points• Mechanical compression of the painful knee improves arthrocentesis success and fluid yield in both rheumatoid arthritis and osteoarthritis.• The painful rheumatoid knee contains approximately 100% more fluid than the osteoarthritic knee.• Large effusions in the osteoarthritic knee are characterized by higher grades of mechanical destruction but not increased neutrophil counts.• In contrast, large effusions in the rheumatoid knee are characterized by higher synovial fluid neutrophil counts but not the grade of mechanical destruction, indicating different mechanisms of effusion formation in rheumatoid arthritis versus osteoarthritis.

Can Diagnostic and Therapeutic Arthrocentesis Be Successfully Performed in the Flexed Knee?

BACKGROUND/OBJECTIVE: The objective of this study was to determine whether the extended or flexed knee positioning was superior for arthrocentesis and whether the flexed knee positioning could be improved by mechanical compression. METHODS: Fifty-five clinically effusive knees underwent arthrocentesis in a quality improvement intervention: 20 consecutive knees in the extended knee position using the superolateral approach, followed by 35 consecutive knees in the flexed knee position with and without an external compression brace placed on the suprapatellar bursa. Arthrocentesis success and fluid yield in milliliters were measured. RESULTS: Fluid yield for the extended knee was greater (191% greater) than the flexed knee (extended knee, 16.9 ± 15.7 mL; flexed knee, 5.8 ± 6.3 mL; P < 0.007). Successful diagnostic arthrocentesis (≥2 mL) was 95% (19/20) in the extended knee and 77% (27/35) in the flexed knee (P = 0.08). After mechanical compression was applied to the suprapatellar bursa and patellofemoral joint of the flexed knee, fluid yields were essentially identical (extended knee, 16.9 ± 15.7 mL; flexed knee, 16.7 ± 11.3 mL; P = 0.73), as were successful diagnostic arthrocentesis (≥2 mL) (extended knee 95% vs. flexed knee 100%, P = 0.12). CONCLUSIONS: The extended knee superolateral approach is superior to the flexed knee for conventional arthrocentesis; however, the extended knee positioning and flexed knee positioning have identical arthrocentesis success when mechanical compression is applied to the superior knee. This new flexed knee technique for arthrocentesis is a useful alternative for patients who are in wheelchairs, have flexion contractures, cannot be supine, or cannot otherwise extend their knee.

Improvement in diagnostic and therapeutic arthrocentesis via constant compression.

We hypothesized that constant compression of the knee would mobilize residual synovial fluid and promote successful arthrocentesis. Two hundred and ten knees with grade II-III osteoarthritis were included in this paired design study: (1) conventional arthrocentesis was performed with manual compression and success and volume (milliliters) determined; and (2) the intra-articular needle was left in place, and a circumferential elastomeric brace was tightened on the knee to provide constant compression. Arthrocentesis was attempted again and additional fluid volume was determined. Diagnostic procedural cost-effectiveness was determined using 2017 US Medicare costs. No serious adverse events were noted in 210 subjects. In the 158 noneffusive (dry) knees, sufficient synovial fluid for diagnostic purposes (≥ 2 ml) was obtained in 5.0% (8/158) without compression and 22.8% (36/158) with compression (p = 0.0001, z for 95% CI = 1.96), and the absolute volume of arthrocentesis fluid obtained without compression was 0.28 ± 0.79 versus 1.10 ± 1.81 ml with compression (293% increase, p = 0.0001). In the 52 effusive knees, diagnostic synovial fluid (≥ 2 ml) was obtained in 75% (39/52) without compression and 100% (52/52) with compression (p = 0.0001, z for 95% CI = 1.96), and the absolute volume of arthrocentesis without compression was 14.7 ± 13.8 versus 25.3 ± 15.5 ml with compression (72.1% increase, p = 0.0002). Diagnostic procedural cost-effectiveness was $655/sample without compression and $387/sample with compression. The new technique of constant compression via circumferential mechanical compression mobilizes residual synovial fluid beyond manual compression improving the success, cost-effectiveness, and yield of diagnostic and therapeutic arthrocentesis in both the effusive and noneffusive knee.

Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD50 of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.

Feasibility of integrating standardized patient-reported outcomes in orthopedic care.

OBJECTIVES: Osteoarthritis of the knee is a chronic disease associated with pain and reduced quality of life. The ability to reliably measure patient-reported symptoms is important for clinical decision making and evaluation of outcomes. Electronic and web-based tools can eliminate much of the labor-intensive aspects of questionnaire administration and enables both real-time evaluation of responses by physicians and integration of data from multiple sites. This article describes the results of implementing a single integrated electronic questionnaire system into routine orthopedic practice at 2 diverse institutions. STUDY DESIGN: Case study. METHODS: A web-based version of a general quality-of-life questionnaire (EuroQol 5-dimension [EQ-5D]) and the pain domain of a disease-specific questionnaire (Knee Osteoarthritis Outcome Score [KOOS]) were administered in the office waiting room to (n = 666) patients at 2 centers over a 9-month period using touchscreen devices. Data were analyzed and descriptive statistics were calculated to assess feasibility of integration into the distinct work flows and to assess the agreement of the results. RESULTS: The electronic questionnaire had a completion rate of 93% to 95%. Average questionnaire completion times were 3 to 5 minutes at each institution. Mean EQ-5D and KOOS scores for patients pre- and postsurgery were also consistent with prior literature studies. CONCLUSIONS: Lessons learned for future adoption of questionnaire systems elsewhere include the need for baseline assessment of clinic work flows to identify the optimal point of administration and the need for IT support. This study demonstrates the feasibility of routinely collecting patient-reported data as part of standard care, which will become increasingly important as the nationwide emphasis on tracking quality and cost-effectiveness of treatments in orthopedics grows.

Atoxic derivative of botulinum neurotoxin A as a prototype molecular vehicle for targeted delivery to the neuronal cytoplasm.

We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.

Basic principles for conducting human research in orthopaedic medicine.

Researchers and clinicians operate in an increasingly complex clinical and regulatory environment in which understanding the principles governing human research is essential. However, most orthopaedic surgeons have not received in-depth training in regulatory requirements and scientific research methods. Ensuring that research is conducted in accordance with state and federal laws and ethical principles is essential to guard compromising patient information and avoid severe penalties for noncompliance. The researcher must understand the regulations for compliance and proper data management, including the requirements of the Health Insurance Portability and Accountability Act, proper application of informed consent, use of the Institutional Review Board, and data protection guidelines. Tools such as a regulatory binder can assist investigators in complying with requirements, maintaining regulatory standards, and ensuring a robust study design and conduct.

The outcomes and cost-effectiveness of intraarticular injection of the rheumatoid knee.

Although intraarticular injections are important to the management of rheumatoid arthritis, there are few studies regarding the cost-effectiveness of alternative injection techniques. This randomized controlled study addressed the cost-effectiveness of two different low-cost, anatomic landmark palpation-directed intraarticular injection techniques. Ninety-six symptomatic rheumatoid knees were randomized to two different low-cost, palpation-guided intraarticular injection techniques utilizing (1) a conventional syringe or (2) a mechanical syringe, the RPD (the reciprocating procedure device). Three milliliters of 1% lidocaine were used to anesthetize the synovial membrane, followed by arthrocentesis and hydrodissection, and injection of 80 mg of triamcinolone acetonide utilizing the one-needle two-syringe technique. Baseline pain, procedural pain, aspirated fluid volume, pain at outcome (2 weeks and 6 months), responders, reinjection rates, cost/patient/year, and cost/responder/year were determined. Pain was measured with the 10 cm Visual Analogue Pain Scale (VAS). Both techniques significantly reduced pain scores at outcome from baseline (P < 0.001). The mechanical syringe technique resulted in a greater volume of aspirated fluid (P < 0.01), a 38% reduction in procedural pain (P < 0.001), a 24% reduction in pain scores at outcome (P < 0.03), an increase in the responder rate (P < 0.025), 33% increase in the time to next injection (P < 0.001), 23% ($35 US) reduction in cost/patient/year for a patient treated in a physician office (P < 0.001), 24% reduction ($26 US) in cost/patient/year for a hospital outpatient (P < 0.001), and 51% ($151 US) reduction in cost/responder/year (P < 0.001). The outcomes and cost-effectiveness of intraarticular injection of the rheumatoid knee can be improved significantly with low-cost alternations in technique.

A randomized controlled trial evaluating the cost-effectiveness of sonographic guidance for intra-articular injection of the osteoarthritic knee.

OBJECTIVE: The present randomized controlled study investigated whether sonographic needle guidance affected the outcomes of intra-articular injection for osteoarthritis of the knee. METHODS: Ninety-four noneffusive knees with osteoarthritis were randomized to injection by conventional palpation-guided anatomic landmark injection or sonographic image-guided injection enhanced with a 1-handed mechanical (the reciprocating procedure device) syringe. After intra-articular placement and synovial space dilation were confirmed by sonography, a syringe exchange was performed, and 80 mg of triamcinolone acetonide was injected with the second syringe through the indwelling intra-articular needle. Baseline pain, procedural pain, pain at outcome (2 weeks and 6 months), responders, therapeutic duration, reinjection rates, total cost, and cost per responder were determined. RESULTS: Relative to conventional palpation-guided anatomic landmark methods, sonographic guidance for injection of the knee resulted in 48% reduction in procedural pain (P < 0.001), a 42% reduction in pain scores at outcome (P < 0.03), 107% increase in the responder rate (P < 0.001), 52% reduction in the nonresponder rate (P < 0.001), a 36% increase in therapeutic duration (P = 0.01), a 13% reduction ($17) in cost per patient per year, and a 58% ($224) reduction in cost per responder per year for a hospital outpatient (P < 0.001). CONCLUSIONS: Sonographic needle guidance reduced procedural pain and improved the clinical outcomes and cost-effectiveness of intra-articular injections of the osteoarthritic knee.

Safety syringes and anti-needlestick devices in orthopaedic surgery.

BACKGROUND: The American Academy of Orthopaedic Surgery (AAOS), The Joint Commission, the Occupational Safety and Health Administration (OSHA), and the Needlestick Safety and Prevention Act encourage the integration of safety-engineered devices to prevent needlestick injuries to health-care workers and patients. We hypothesized that safety syringes and needles could be used in outpatient orthopaedic injection and aspiration procedures. METHODS: The study investigated the orthopaedic uses and procedural idiosyncrasies of safety-engineered devices, including (1) four safety needles (Eclipse, SafetyGlide, SurGuard, and Magellan), (2) a mechanical safety syringe (RPD), (3) two automatic retractable syringes (Integra, VanishPoint), (4) three manual retractable syringes (Procedur-SF, Baksnap, Invirosnap), and (5) three shielded syringes (Safety-Lok, Monoject, and Digitally Activated Shielded [DAS] Syringe). The devices were first tested ex vivo, and then 1300 devices were used for 425 subjects undergoing outpatient arthrocentesis, intra-articular injections, local anesthesia, aspiration biopsy, and ultrasound-guided procedures. RESULTS: During the clinical observation, there were no accidental needlesticks (0 needlesticks per 1300 devices). Safety needles could be successfully used on a Luer syringe but were limited to ≤1.5 in (≤3.81 cm) in length and the shield could interfere with sonography. The mechanical safety syringes functioned well in all orthopaedic procedures. Automatic retractable syringes were too small for arthrocentesis of the knee, and the plunger blew out and prematurely collapsed with high-pressure injections. The manual retractable syringes and shielded syringes could be used with conventional needles for most orthopaedic procedures. CONCLUSIONS: The most effective and reliable safety devices for orthopaedic syringe procedures are shielded safety needles, mechanical syringes, manual retractable syringes, and shielded syringes, but not automatic retractable syringes. Even when adopting safety-engineered devices for an orthopaedic clinic, conventional syringes larger than 20 mL and conventional needles longer than 1.5 in (3.8 cm) are necessary.

The highly accurate anteriolateral portal for injecting the knee.

BACKGROUND: The extended knee lateral midpatellar portal for intraarticular injection of the knee is accurate but is not practical for all patients. We hypothesized that a modified anteriolateral portal where the synovial membrane of the medial femoral condyle is the target would be highly accurate and effective for intraarticular injection of the knee. METHODS: 83 subjects with non-effusive osteoarthritis of the knee were randomized to intraarticular injection using the modified anteriolateral bent knee versus the standard lateral midpatellar portal. After hydrodissection of the synovial membrane with lidocaine using a mechanical syringe (reciprocating procedure device), 80 mg of triamcinolone acetonide were injected into the knee with a 2.0-in (5.1-cm) 21-gauge needle. Baseline pain, procedural pain, and pain at outcome (2 weeks and 6 months) were determined with the 10 cm Visual Analogue Pain Score (VAS). The accuracy of needle placement was determined by sonographic imaging. RESULTS: The lateral midpatellar and anteriolateral portals resulted in equivalent clinical outcomes including procedural pain (VAS midpatellar: 4.6 ± 3.1 cm; anteriolateral: 4.8 ± 3.2 cm; p = 0.77), pain at outcome (VAS midpatellar: 2.6 ± 2.8 cm; anteriolateral: 1.7 ± 2.3 cm; p = 0.11), responders (midpatellar: 45%; anteriolateral: 56%; p = 0.33), duration of therapeutic effect (midpatellar: 3.9 ± 2.4 months; anteriolateral: 4.1 ± 2.2 months; p = 0.69), and time to next procedure (midpatellar: 7.3 ± 3.3 months; anteriolateral: 7.7 ± 3.7 months; p = 0.71). The anteriolateral portal was 97% accurate by real-time ultrasound imaging. CONCLUSION: The modified anteriolateral bent knee portal is an effective, accurate, and equivalent alternative to the standard lateral midpatellar portal for intraarticular injection of the knee. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00651625.

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A.

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.

A randomized controlled trial of the cost-effectiveness of ultrasound-guided intraarticular injection of inflammatory arthritis.

OBJECTIVE: We studied whether sonographic needle guidance affected the outcomes of intraarticular (IA) injection for inflammatory arthritis. METHODS: Joints with inflammatory arthritis (n = 244; 76% rheumatoid arthritis, 3% small joints, 51% intermediate, and 46% large) were randomized to injection by conventional palpation-guided anatomic injection (120 joints) or sonographic image-guided injection enhanced with a 1-handed reciprocating procedure device mechanical syringe (124 joints). A 1-needle, 2-syringe technique was used. After IA placement and synovial space dilation were confirmed by sonography, a syringe exchange was performed, and triamcinolone acetonide was injected with the second syringe through the indwelling IA needle. Baseline pain, procedural pain, pain at outcome (2 weeks and 6 months), responders, therapeutic duration, reinjection rates, total cost, and cost per responder were determined. RESULTS: Relative to conventional palpation-guided methods, sonographic guidance for injection of inflammatory arthritis resulted in an 81% reduction in injection pain (p < 0.001), 35% reduction in pain scores at outcome (p < 0.02), 38% increase in the responder rate (p < 0.003), 34% reduction in the non-responder rate (p < 0.003), 32% increase in therapeutic duration (p = 0.01), 8% reduction ($7) in cost/patient/year, and a 33% ($64) reduction in cost/responder/year for a hospital outpatient (p < 0.001). CONCLUSION: Sonographic needle guidance improves the performance, clinical outcomes, and cost-effectiveness of IA injections for inflammatory arthritis. (Clinical Trial Identifier NCT00651625).

Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery.

Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm.

Does sonographic needle guidance affect the clinical outcome of intraarticular injections?

OBJECTIVE: This randomized controlled study addressed whether sonographic needle guidance affected clinical outcomes of intraarticular (IA) joint injections. METHODS: In total, 148 painful joints were randomized to IA triamcinolone acetonide injection by conventional palpation-guided anatomic injection or sonographic image-guided injection enhanced with a one-handed control syringe (the reciprocating device). A one-needle, 2-syringe technique was used, where the first syringe was used to introduce the needle, aspirate any effusion, and anesthetize and dilate the IA space with lidocaine. After IA placement and synovial space dilation were confirmed, a syringe exchange was performed, and corticosteroid was injected with the second syringe through the indwelling IA needle. Baseline pain, procedural pain, pain at outcome (2 weeks), and changes in pain scores were measured with a 0-10 cm visual analog pain scale (VAS). RESULTS: Relative to conventional palpation-guided methods, sonographic guidance resulted in 43.0% reduction in procedural pain (p < 0.001), 58.5% reduction in absolute pain scores at the 2 week outcome (p < 0.001), 75% reduction in significant pain (VAS pain score > or = 5 cm; p < 0.001), 25.6% increase in the responder rate (reduction in VAS score > or = 50% from baseline; p < 0.01), and 62.0% reduction in the nonresponder rate (reduction in VAS score < 50% from baseline; p < 0.01). Sonography also increased detection of effusion by 200% and volume of aspirated fluid by 337%. CONCLUSION: Sonographic needle guidance significantly improves the performance and outcomes of outpatient IA injections in a clinically significant manner.

Transfer of inter-alpha-inhibitor heavy chains to hyaluronan by surface-linked hyaluronan-TSG-6 complexes.

Inter-alpha-inhibitor, TSG-6, and hyaluronan have important functions in fertility and inflammation. Two subunits of inter-alpha-inhibitor, the heavy chains, form covalent bonds with TSG-6 or hyaluronan in vitro. TSG-6-heavy chain complexes serve as intermediates in the transfer of heavy chains from inter-alpha-inhibitor to hyaluronan. In vivo, in addition to these complexes, stable ternary complexes of hyaluronan with both TSG-6 and heavy chains have been demonstrated in the ovulatory cumulus oophorus. In our ongoing efforts to characterize the multiple interactions between hyaluronan, TSG-6 and inter-alpha-inhibitor, we recently characterized the formation of highly stable complexes of TSG-6 with hyaluronan that had been tethered to a solid surface. Here we show that these hyaluronan-TSG-6 complexes are functionally active and transfer heavy chain subunits from inter-alpha-inhibitor to either free or surface-bound hyaluronan. Transitional hyaluronan-TSG-6-heavy chain complexes do not accumulate in vitro. Our data show the capability for heavy chain transfer by both free TSG-6 and preformed hyaluronan-TSG-6 complexes, suggesting that both might contribute to hyaluronan modification in vivo. Transfer of heavy chains to surface-tethered hyaluronan by either free TSG-6 or surface-tethered hyaluronan-TSG-6 complexes did not affect the CD 44-mediated binding of BW 5147 cells in vitro. We show how TSG-6 and hyaluronan together can deplete inter-alpha-inhibitor and generate bikunin, as has been observed in sepsis, and discuss the role of TSG-6 in the generation of hyaluronan-heavy chain complexes associated with ovulation, arthritis, and sepsis.