Working memory impairment in calcineurin knock-out mice is associated with alterations in synaptic vesicle cycling and disruption of high-frequency synaptic and network activity in prefrontal cortex.

Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and neuronal mechanisms of working memory dysfunction, we have performed a comprehensive analysis of a mouse model of schizophrenia, the forebrain-specific calcineurin knock-out mouse. Biochemical analyses of cortical tissue from these mice revealed a pronounced hyperphosphorylation of synaptic vesicle cycling proteins known to be necessary for high-frequency synaptic transmission. Examination of the synaptic vesicle cycle in calcineurin-deficient neurons demonstrated an impairment of vesicle release enhancement during periods of intense stimulation. Moreover, brain slice and in vivo electrophysiological analyses showed that loss of calcineurin leads to a gene dose-dependent disruption of high-frequency synaptic transmission and network activity in the PFC, correlating with selective working memory impairment. Finally, we showed that levels of dynamin I, a key presynaptic protein and calcineurin substrate, are significantly reduced in prefrontal cortical samples from schizophrenia patients, extending the disease relevance of our findings. Our data provide support for a model in which impaired synaptic vesicle cycling represents a critical node for disease pathologies underlying the cognitive deficits in schizophrenia.

Primary eosinophilic lung diseases.

Eosinophilic lung diseases typically present as a triad of pulmonary symptoms, an abnormal chest radiograph, and elevated levels of eosinophils in the sputum and lung tissue. This article focuses on primary causes of eosinophilic lung disease including acute eosinophilic pneumonia, chronic eosinophilic pneumonia, Churg-Strauss syndrome, and hypereosinophilic syndromes. In these disorders elevated eosinophil levels in the tissue lead to inflammation and tissue damage. Peripheral eosinophilia can often be measured when tissue levels are elevated but this is not as reliable a marker as tissue biopsy. Because corticosteroids act through a variety of mechanisms to inhibit eosinophil function and induce apoptosis, they are first-line therapy for eosinophilic lung diseases. Targeted immunosuppressive therapies, such as monoclonal antibodies against key regulatory cytokines for eosinophil production and survival, are not formally approved for eosinophilic lung disease but have shown promising results in published research studies.

Expandable intramedullary nails for humeral fractures: a systematic review of clinical and radiological outcomes.

The Fixion(™) system (Disc-O-Tech Medical Technologies, Herzeliya, Israel), which is currently the only expandable nailing system available for use in the humerus, has a number of purported advantages over the standard locked humeral nail, including a reduction in operating and fluoroscopy time since locking screws are not required. A systematic review was undertaken of all published (AMED, CINAHL, EMBASE and Medline via the Ovid platform) and unpublished or grey literature research databases from inception until 1st December 2010. Demographic data, clinical and radiological outcomes, and complications were extracted from each study by two independent investigators, and each study underwent independent critical appraisal using the CASP appraisal tool. Thirteen studies were deemed eligible for review, identified from a total of 154 citations. These included a total of 176 patients with 180 fractured humeri treated with expandable nails. Overall, 7.8% of humeral fractures treated with an expandable nail went on to non-union. Intra- and post-operative device failure rate was found to be 1.1 and 2.8%, respectively. These data compare favourably to published data on the outcome of locked humeral nails. However, there were numerous methodological flaws in the current evidence base; there were no comparative studies, treatment groups were heterogeneous, and there was no blinding of assessors or patients. Initial data indicate that the expandable humeral nail may be an acceptable form of treatment for humeral fracture or impending fracture, but high-quality comparative studies are needed to confirm these findings.

Integrin-associated proteins as potential therapeutic targets.

SUMMARY: Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin-ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin alpha and beta subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the alpha4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with beta2 integrins may participate in adhesive and migratory events in leukocytes. The beta integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates beta1 and beta3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions.

A small molecule that inhibits the interaction of paxillin and alpha 4 integrin inhibits accumulation of mononuclear leukocytes at a site of inflammation.

Extracellular antagonists of alpha 4 integrin are an effective therapy for several autoimmune and inflammatory diseases; however, these agents that directly block ligand binding may exhibit mechanism-based toxicities. Inhibition of alpha 4 integrin signaling by mutations of alpha 4 that block paxillin binding inhibits inflammation while limiting mechanism-based toxicities. Here, we test a pharmacological approach by identifying small molecules that inhibit the alpha 4 integrin-paxillin interaction. By screening a large (approximately 40,000-compound) chemical library, we identified a noncytotoxic inhibitor of this interaction that impaired integrin alpha 4-mediated but not alpha L beta 2-mediated Jurkat T cell migration. The identified compound had no effect on alpha 4-mediated migration in cells bearing the alpha 4(Y991A) mutation that disrupts the alpha 4-paxillin interaction, establishing the specificity of its action. Administration of this compound to mice led to impaired recruitment of mononuclear leukocytes to a site of inflammation in vivo, whereas an isomer that does not inhibit the alpha 4-paxillin interaction had no effect on alpha 4-mediated cell migration, cell spreading, or recruitment of leukocytes to an inflammatory site. Thus, a small molecule inhibitor that interferes with alpha 4 integrin signaling reduces alpha 4-mediated T cell migration in vivo, thus providing proof of principle for inhibition of alpha 4 integrin signaling as a target for the pharmacological reduction of inflammation.

Biomechanical analysis suggests early rehabilitation is possible after single-incision EndoButton distal biceps repair with FiberWire.

PURPOSE: Previous studies that have encouraged early postoperative motion after distal biceps repair shows little agreement on exactly when activity should be resumed after surgery or on the level of weight restriction that should be used. The aim of the current study was to define a service load that would permit, without failure, 2,000 cycles of immediate motion after single-incision EndoButton distal biceps repair with FiberWire. METHODS: In each of 15 cadaveric elbows, the distal biceps tendon was divided at its insertion and then repaired using a single-incision EndoButton technique with FiberWire. The repairs were then challenged according to the "staircase method" by cyclically loading the biceps tendon, so that the forearm flexed between 0° and 90°. RESULTS: The mean failure load of the repair was 166.7 N (95% confidence interval, 132.6-200.8). The data suggested that a 0.9 kg (9-N) weight at the hand was the limit for a 2,000-cycle early rehabilitation protocol after repair of a ruptured distal biceps tendon via a single-incision EndoButton repair technique. CONCLUSION: Early active motion with a 0.9-kg weight restriction may therefore be possible in those patients undergoing distal biceps tendon repair using this technique.

Triceps tendons: a biomechanical comparison of intact and repaired strength.

HYPOTHESIS: Our hypothesis was that the autograft-augmented direct repair of torn triceps tendons would have strength superior than that of direct repair when compared to the strength of intact distal triceps tendons. MATERIALS AND METHODS: The strength of the intact distal triceps tendon in 8 unpaired, fresh frozen cadaver specimens was measured to tendon failure by uniaxial tension in the sagittal plane. The torn triceps tendons were then repaired by direct repair (sutures through drill holes) or an autograft-augmented direct repair. Each tendon repair was biomechanically tested to failure, and load to displacement curves and the site of tendon failure were recorded. Tendon strength after each repair was compared with that of the other repair technique and with that of the intact triceps tendon. Significance was set at P < .05. RESULTS: Average failure loads for intact, direct repair, and augmented repair tendons were 1741, 317, and 593 N, respectively; augmented repairs were significantly stronger than direct repairs. In the intact tendon, failure occurred at the insertion site through a tear at the bone tendon interface or through a small cortical avulsion. In the repaired tendons, all but 1 failure occurred through the suture; 1 augmented repair failed first at the tendon and then through the suture. DISCUSSION: There is a paucity of clinical data regarding the optimal repair for distal triceps avulsion. We found that triceps repair affords less strength than the intact tendon, but augmented repair was nearly twice as strong as that of direct repair. Augmented repair may allow earlier range of motion, weightbearing, and rehabilitation, theoretically decreasing complications associated with the procedure. CONCLUSIONS: Augmented triceps repair is superior to direct triceps repair for a distal triceps avulsion produced in a cadaver model.

Evidence that cathelicidin peptide LL-37 may act as a functional ligand for CXCR2 on human neutrophils.

LL-37, derived from human cathelicidin, stimulates immune responses in neutrophils. Although FPR2 and P2X7 were proposed as LL-37 receptors, we have shown that among 21 neutrophil receptors only CXCR2 was down-regulated by LL-37. LL-37 functions similarly to CXCR2-specific chemokines CXCL1 and CXCL7 in terms of receptor down-regulation and intracellular calcium mobilization on freshly isolated neutrophils. Neutrophils pretreated with CXCL8, a chemokine that binds both CXCR1/2, completely blocked the calcium mobilization in response to LL-37, while LL-37 also partially inhibited (125)I-CXCL8 binding to neutrophils. SB225002, a selective CXCR2 antagonist, blocked LL-37-induced calcium mobilization and migration of neutrophils. LL-37 stimulates calcium mobilization in CXCR2-transfected HEK293 cells, CXCR2(+) THP-1 cells and monocytes, but not in CXCR1-transfected HEK293 cells. WKYMVm peptide (ligand for FPR2) does not block LL-37-stimulated calcium flux in either THP-1 (FPR2(-)) or monocytes (FPR2(high)), further confirming the specificity of LL-37 for CXCR2 and not FPR2. Among all ligands tested (ATP, BzATP, WKYMVm, CXCL1, and LL-37), only LL-37 stimulated migration of monocytes (CXCR2(+) and FPR2(+)) and migration was inhibited by the CXCR2 inhibitor SB225002. Moreover, CXCR2 but not CXCR1 was internalized in LL-37-treated neutrophils. Thus, our data provide evidence that LL-37 may act as a functional ligand for CXCR2 on human neutrophils.

Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

Blocking the alpha 4 integrin-paxillin interaction selectively impairs mononuclear leukocyte recruitment to an inflammatory site.

Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer's patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.

The role of the alpha4 integrin-paxillin interaction in regulating leukocyte trafficking.

The movement of leukocytes from the blood into peripheral tissues is a central feature of immune surveillance, but also contributes to the pathogenesis of inflammatory and autoimmune diseases. Integrins are a family of adhesion and signaling molecules made up of paired a and beta subunits, and the integrin alpha4beta1 plays a prominent role in the trafficking of mononuclear leukocytes. We have previously described the direct interaction of the signaling adaptor molecule paxillin with the cytoplasmic domain of the alpha4 integrin subunit. This interaction is critical for alpha4beta1 integrin dependent cell adhesion under shear flow conditions as it provides a needed connection to the actin cytoskeleton. Furthermore, the alpha4-paxillin interaction is required for effective alpha4beta1 dependent leukocyte migration and does so through the temporal and spatial regulation of the small GTPase Rac. These findings make the alpha4-paxillin interaction a potentially attractive therapeutic target in controlling leukocyte trafficking.

Fine-tuning Tumor Immunity with Integrin Trans-regulation.

Inefficient T-cell homing to tissues limits adoptive T-cell immunotherapy of solid tumors. αLß2 and α4ß1 integrins mediate trafficking of T cells into tissues via engagement of ICAM-1 and VCAM-1, respectively. Inhibiting protein kinase A (PKA)-mediated phosphorylation of α4 integrin in cells results in an increase in αLß2-mediated migration on mixed ICAM-1-VCAM-1 substrates in vitro, a phenomenon termed "integrin trans-regulation." Here, we created an α4(S988A)-bearing mouse, which precludes PKA-mediated α4 phosphorylation, to examine the effect of integrin trans-regulation in vivo. The α4(S988A) mouse exhibited a dramatic and selective increase in migration of lymphocytes, but not myeloid cells, to sites of inflammation. Importantly, we found that the α4(S988A) mice exhibited a marked increase in T-cell entry into and reduced growth of B16 melanomas, consistent with antitumor roles of infiltrating T cells and progrowth functions of tumor-associated macrophages. Thus, increased α4 trans-regulation of αLß2 integrin function biases leukocyte emigration toward lymphocytes relative to myeloid cells and enhances tumor immunity.

Expandable intramedullary nails in lower limb trauma: a systematic review of clinical and radiological outcomes.

This study systematically reviews the evidence-base for the use of expandable nails in the treatment of acute diaphyseal fractures of the lower limb. Both electronic and hand searches were undertaken of the published and grey literature to 1 December 2011. A total of 154 citations were identified, of which 15 were deemed suitable and assessed with the Critical Appraisals Skills Programme tool. A total of 625 nailing procedures were performed in 620 patients: 279 femoral and 346 tibial nails. The expandable nail was found to be significantly quicker to insert than interlocked nails (p < 0.05), and the total incidence of non-union or other complication was 13 and 14 % for expandable femoral and tibial nails, respectively. Notable complications with the expandable nail included fracture propagation on nail inflation in 2.5 % and post-operative shortening in 3.3 %. Device failure secondary to problems with the expansion mechanism was seen in 2.9 %. The rate of non-union and infection following expandable nailing was 3.1 and 1.4 %, respectively. Despite promising initial results, there remains a paucity of good quality studies to support the use of expandable nails over interlocked nails for the treatment of acute diaphyseal fractures of the lower limb.

Effect of anterior versus posterior in situ decompression on ulnar nerve subluxation.

We sought to determine the effect anterior versus posterior in situ decompression with 360° external neurolysis on ulnar nerve subluxation. Ten cadaveric specimens were used, with anterior release performed on 5 specimens and posterior release the other 5 specimens. Each specimen was released for 4 cm centered over the cubital tunnel followed by 12 cm, 20 cm, and 20 cm with 360° external neurolysis. After release, the elbow was brought through a range of motion from 0° to 140° of flexion. Compared with posterior release, anterior release demonstrated significantly more total subluxation of the ulnar nerve for all release types from 80° to 120° of flexion (P<.05). At 140° of flexion, the 4-cm release, the 12-cm release, and the 20-cm release with 360° external neurolysis also demonstrated significantly more total subluxation with anterior release (P<.05). Ulnar nerve subluxation was significantly lower with posterior release, compared with anterior release for limited and complete in situ decompression.

The role of axial compressive and quadriceps forces in noncontact anterior cruciate ligament injury: a cadaveric study.

BACKGROUND: Compressive and quadriceps forces have been associated with noncontact anterior cruciate ligament (ACL) injury. The purpose of this study was to quantify the relative importance of each load component during noncontact ACL injury. HYPOTHESIS: We hypothesized that the introduction of a quadriceps force lowers the axial compressive force threshold to produce ACL injury. STUDY DESIGN: Controlled laboratory study. METHODS: Six pairs of fresh-frozen cadaveric knees, flexed to 15°, were loaded with axial compression (group A) or compression with a quadriceps force (group B) until failure. All specimens underwent axial compressive loading under displacement control with a time to peak load of 50 msec. The initial displacement of the MTS actuator was 8 mm and was increased in 2-mm increments with successive tests until catastrophic damage of the joint occurred. Failure was determined by a combination of clinical specimen examination and force-displacement data analysis and by dissection and direct visualization after failure was recognized. Differences in failure load between groups were examined using a paired t test (significance, P ≤ .05). RESULTS: In group A, there were 2 isolated ACL injuries, 2 ACL ruptures combined with a tibial plateau fracture, and 2 isolated tibial plateau fractures. In group B, there were 5 isolated ACL ruptures and 1 tibial plateau fracture. There was a significant difference in the average failure load between groups A and B: 10 832 N (95% confidence interval [CI], 9743-11,604 N) and 6119 N (95% CI, 4335-7903 N), respectively. CONCLUSION: Isolated compressive forces displayed an ability to produce an ACL injury in this cadaveric model, but the addition of a quadriceps load significantly reduced the compressive force required for ACL injury. CLINICAL RELEVANCE: Compressive and quadriceps forces contribute to noncontact ACL injury and should be taken into account when developing ACL injury prevention programs and rehabilitation after ACL reconstruction.

A system for performing high throughput assays of synaptic function.

Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders.

Proximal humeral fractures: a biomechanical comparison of locking plate constructs in a cadaveric 3-part fracture model.

The purpose of our study was to biomechanically compare, under cyclic loading conditions, fracture site motion, humeral head collapse, and intra-articular hardware penetration in simulated 3-part osteoporotic proximal humeral fractures stabilized with 1 of 2 locking-plate constructs. We performed fixation on simulated 3-part proximal humeral fractures in 10 pairs of cadaveric osteoporotic humeri with a Hand Innovations S3 Proximal Humerus Plate (S3 plate) or an LCP Proximal Humerus Plate (LCP plate; 1 each for each pair). The specimens were potted, mounted on a materials testing machine, and subjected to 5000 cycles of abduction in the scapular plane, loading through the supraspinatus tendon. Interfragmentary displacement at 2 virtual points (the most medial aspect of the calcar and the most superior aspect of the osteotomy line between the greater tuberosity and humeral head) was measured using an optical tracking system. Humeral head rotation was also measured. We used a generalized linear latent and mixed model to check for an effect of cyclic loading and treatment on the parameters of interest (significance, P < .05). After cyclic loading, the S3 plate humeri showed significantly greater displacement of the greater tuberosity fragment and rotation of the humeral head and a trend (not a significant difference) toward greater displacement at the calcar. No hardware penetration was noted for either repair. Although the S3 plate repairs resulted in significantly more fracture site motion, it is unknown whether the magnitude of the motion is clinically significant.