Fascial Lengthening at the Time of Surgical Release for de Quervain's Tenosynovitis to Prevent Postsurgical Tendon Subluxation.

BACKGROUND: De Quervain's tenosynovitis is a relatively common, painful condition. Although commonly managed nonoperatively, some patients have recalcitrant disease, necessitating surgical release. Traditional surgical release for de Quervain's tenosynovitis with simple retinacular release can leave patients susceptible to first dorsal extensor compartment tendon subluxation. We present a stair-step flap technique that releases the first dorsal compartment while simultaneously preventing postoperative tendon subluxation via fascial lengthening. METHODS: All patients over the past decade who underwent surgical release with this technique were collected. Preoperative characteristics, surgical details, and postoperative outcomes were reviewed and aggregated as summary statistics. RESULTS: A total of 101 patients were found. Of these, 35 patients had isolated first dorsal compartment release. Tourniquet time for the total group was 1 hour and that for the isolated first dorsal compartment release subgroup was 20 minutes. The average follow-up was 590 days for the total group and only 440 days for the isolated first dorsal compartment release subgroup. No patients who underwent our novel technique experienced subluxation of the tendons postoperatively. One patient required oral antibiotics for a superficial cellulitis, and 1 patient had recurrent symptoms due to excessive scarring that resolved with scar massage and steroid injection. No patient required repeat operations. CONCLUSION: Our study demonstrates that first dorsal compartment release with a stair-step incision allows for closure of the compartment with a very loose sheath without subluxation and simultaneously provides satisfactory decompression. This procedure is safe and efficacious and can be considered a useful modification to traditional retinacular release alone. LEVEL OF EVIDENCE: Level III.

Traumatic Nondissociative Carpal Instability: A Case Series.

PURPOSE: We report 8 cases of acute or subacute unilateral nondissociative carpal instability (CIND) in the context of nondisplaced scaphoid fractures. METHODS: Eight patients from 3 centers developed abnormal volar intercalated segment instability (VISI) or dorsal intercalated segment instability (DISI) following the diagnosis of a nondisplaced scaphoid fracture. An operative inspection in each patient confirmed intact scapholunate and lunotriquetral interosseous ligaments. We outline the demographic characteristics of our patient cohort, radiologic and operative findings of CIND-DISI and CIND-VISI, and the outcomes of acute and delayed treatment. RESULTS: Two patients were diagnosed with CIND-DISI and 6 with CIND-VISI associated with ipsilateral nondisplaced scaphoid fractures. The average time from injury to diagnosis of CIND was 11 weeks, and the mean clinical and radiographic follow-up was 18 months. Rapid healing of the scaphoid fractures was achieved in all patients (4 open reduction internal fixation, 4 cast). All patients underwent surgery to improve proximal carpal row alignment: in 3 of the 4 patients who were diagnosed and treated surgically within 12 weeks of injury, the radiolunate angle (RLA) was successfully restored. A contracture release and ligament repair or reconstruction with tendon graft 12 or more weeks following injury was unsuccessful in restoring proximal row alignment in all 4 patients. Two patients in the delayed treatment group required secondary surgery for partial fusion. CONCLUSIONS: Based on the arthroscopic, imaging, and operative findings, we propose that the ligamentous restraints to CIND-VISI are dorsal at the radiocarpal joint and volar at the midcarpal joint. Conversely, the ligamentous restraints to CIND-DISI are dorsal at the midcarpal joint and volar at both the radiocarpal and midcarpal joints. In our series, a delayed diagnosis and late reconstructive surgery were associated with no improvement in RLA. We recommend early recognition of traumatic CIND and prompt treatment of injured ligaments prior to the development of a fixed deformity. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Intermediate-Term Outcomes Following Operative and Nonoperative Management of Midshaft Clavicle Fractures in Children and Adolescents: Internal Fixation May Improve Outcomes.

Although multiple prospective comparative studies exist for adults with midshaft clavicle fractures, few comparative studies exist in older children and adolescents. This study compares intermediate-term functional, pain, radiographic, and complication outcomes in children and adolescents with midshaft clavicle fractures treated operatively or nonoperatively. METHODS: Children and adolescents who were 10 to 18 years of age with midshaft clavicle fractures treated from 2006 to 2017 were identified through electronic picture archiving and communication system (PACS) radiographic and chart review. Closed injuries with a minimum of 1 year from the time of the injury were included. Patients completed patient-reported outcome measures and a focused questionnaire. The primary outcome was the abbreviated Disabilities of the Arm, Shoulder and Hand Questionnaire (QuickDASH) score; the secondary outcomes included the University of California Los Angeles (UCLA) Activity Scale, Patient-Reported Outcomes Measurement Information System (PROMIS) Pain Interference (PI) and PROMIS Physical Function (PF) Upper Extremity (UE), Hospital for Special Surgery Pediatric Functional Activity Brief Scale (HSS Pedi-FABS), Numeric Rating Scale (NRS) for Pain, and rates of implant removal, nonunion, and refracture. Univariate comparisons between the operatively treated group and the nonoperatively treated group were performed. RESULTS: In this study, 302 patients were identified; of these, 79 patients (43 operatively treated and 36 nonoperatively treated) or their parent or legal guardian consented and the patients completed the study protocol. The mean age (and standard deviation) at the time of the injury was 16.0 ± 1.7 years for the operatively treated group and 13.5 ± 1.8 years for the nonoperatively treated group (p < 0.001). The median follow-up was 3.8 years. The patient-reported outcome scores were similar (p > 0.05 for all comparisons) between the operatively treated group and the nonoperatively treated group, even for patients at or near skeletal maturity and those with shortening of ≥15 mm or ≥100% translation. There were no nonunions. The refracture rate was 5% (2 of 43) for the operatively treated group and 3% (1 of 36) for the nonoperatively treated group (p = 1.00). There were 19% (8 of 43) who underwent implant removal. Responders were similar to nonresponders, other than female patients (p < 0.001) and surgically treated patients (p = 0.049) being more likely to respond. CONCLUSIONS: In the subset of this cohort who responded and completed the study, both operatively and nonoperatively treated patients had excellent functional and pain outcomes, similar refracture rates, and no nonunions. Even in widely displaced and translated fractures, and in those at or near skeletal maturity, outcomes were similar. Operative management should potentially be reserved for special circumstances. A prospective comparative study of older adolescents (female patients who are 14 to 18 years of age and male patients who are 16 to 20 years of age) with displaced midshaft clavicle fractures is warranted. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Exposure of Larval Zebrafish to the Insecticide Propoxur Induced Developmental Delays that Correlate with Behavioral Abnormalities and Altered Expression of hspb9 and hspb11.

Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed.

A CT-based approach with 3D modeling to determine optimal radiographic views of the scaphotrapezial and scaphotrapezoid joints.

PURPOSE: To use a CT-based approach with 3D modeling to determine novel radiographic views of the scaphotrapezial (STl) and scaphotrapezoid (STd) joints. MATERIALS AND METHODS: Consecutive wrist CT scans excluding those with pathology of the distal radius, scaphoid, trapezium, or trapezoid of subjects between ages 18 and 60 years were retrospectively reviewed. Three-dimensional reconstructions of CT scans were created and best-fit planes of the STl and STd joints were generated. Angles of these planes relative to a distal radial coordinate system were calculated to determine tilt of the wrist and the X-ray beam for novel radiographic views of these joints. RESULTS: Fifty eligible wrist CT scans were identified. The mean age was 38 years (range, 18 to 59). For the novel STl PA view, the wrist is supinated 17° from the standard PA view and the X-ray beam is canted 6° caudad. In the STl lateral view, the wrist is pronated 17° from the standard lateral view, and the X-ray beam is canted 20° caudad. In the STd PA tilt view, the wrist is supinated 28° from the standard PA view, and the X-ray beam is canted 13° caudad. In the STd joint lateral tilt view, the wrist is pronated 28° from the standard lateral view, and the X-ray beam is canted 29° caudad. CONCLUSION: We describe novel radiographic views of the STl and STd joints based on 3D modeling of wrist CT scans. Further studies are required to assess the efficacy of these views in detecting joint pathology.

An Initiative to Standardize the Identification of and Acute Response to Postoperative Lower-Extremity Neurological Deficits: Effects on Provider Knowledge, Confidence, and Communication Skills.

BACKGROUND: Although relatively uncommon, neurological deficits following hip and knee arthroplasty can have permanent and debilitating consequences. This study was conducted to quantify the effectiveness of an educational curriculum aimed at standardizing the identification of and acute response to postoperative neurological deficits in the inpatient setting, specifically with respect to improvements in clinician knowledge, confidence levels, and communication skills. METHODS: A multidisciplinary committee at a single, high-volume academic institution created an algorithm delineating the appropriate clinical actions and escalation procedures in the setting of a postoperative neurological deficit for each clinical practitioner involved in care for patients who undergo arthroplasty. An educational curriculum composed of online learning modules and an in-person "boot camp" featuring simulations with standardized patients was developed, along with assessments of clinician knowledge, confidence levels, and communication skills. Nurses, physical therapists, physician assistants, residents, fellows, and attending surgeons were encouraged to participate. The intervention spanned a 5-month period in 2015 with a mean time of 18.4 weeks between baseline assessments and the time of the latest follow-up. RESULTS: Online modules were completed by 322 individuals, boot camp was completed by 70 individuals, and latest assessments were completed by 38 individuals. The percentage correct on the knowledge assessment increased from 74.5% before the learning modules to 89.5% immediately after (p < 0.001) but degraded over time such that there was no significant difference between baseline and the latest follow-up scores (p = 0.11). Over the course of the boot camp, physician assistants and residents successfully performed approximately 91% of the indicated actions on the scoring rubric; physical therapists and nurses successfully performed 78%. Scores on the communication skills assessment showed a significant mean increase (p = 0.02) over the course of the intervention from 30.32 to 32.50, and the mean self-assessed confidence survey scores increased by 16.7%, from 7.2 to 8.4 (p < 0.001). CONCLUSIONS: A multimodality educational curriculum aimed at quality improvement can produce significant knowledge improvements, but these gains may not be maintained over time without further instruction. Gains in confidence and communication skills appear to be more long-lasting.

Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.

A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4) studies. After standardization studies were completed, Good Laboratory Practices (GLP)-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay's precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP) serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.

6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors.

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or ß (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and ß over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

Aligned multilayered electrospun scaffolds for rotator cuff tendon tissue engineering.

The rotator cuff consists of several tendons and muscles that provide stability and force transmission in the shoulder joint. Whereas most rotator cuff tears are amenable to suture repair, the overall success rate of repair is low, and massive tears are prone to re-tear. Extracellular matrix (ECM) patches are used to augment suture repair, but they have limitations. Tissue-engineered approaches provide a promising solution for massive rotator cuff tears. Previous studies have shown that, compared to nonaligned scaffolds, aligned electrospun polymer scaffolds exhibit greater anisotropy and exert a greater tenogenic effect. Nevertheless, achieving rapid cell infiltration through the full thickness of the scaffold is challenging, and scaling to a translationally relevant size may be difficult. Our goal was to evaluate whether a novel method of alignment, combining a multilayered electrospinning technique with a hybrid of several electrospinning alignment techniques, would permit cell infiltration and collagen deposition through the thickness of poly(ε-caprolactone) scaffolds following seeding with human adipose-derived stem cells. Furthermore, we evaluated whether multilayered aligned scaffolds enhanced collagen alignment, tendon-related gene expression, and mechanical properties compared to multilayered nonaligned scaffolds. Both aligned and nonaligned multilayered scaffolds demonstrated cell infiltration and ECM deposition through the full thickness of the scaffold after only 28days of culture. Aligned scaffolds displayed significantly increased expression of tenomodulin compared to nonaligned scaffolds and exhibited aligned collagen fibrils throughout the full thickness, the presence of which may account for the increased yield stress and Young's modulus of cell-seeded aligned scaffolds along the axis of fiber alignment. STATEMENT OF SIGNIFICANCE: Rotator cuff tears are an important clinical problem in the shoulder, with over 300,000 surgical repairs performed annually. Re-tear rates may be high, and current methods used to augment surgical repair have limited evidence to support their clinical use due to inadequate initial mechanical properties and slow cellular infiltration. Tissue engineering approaches such as electrospinning have shown similar challenges in previous studies. In this study, a novel technique to align electrospun fibers while using a multilayered approach demonstrated increased mechanical properties and development of aligned collagen through the full thickness of the scaffolds compared to nonaligned multilayered scaffolds, and both types of scaffolds demonstrated rapid cell infiltration through the full thickness of the scaffold.

Novel 5-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and as potential antitumor agents.

A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.

A comparison of débridement with and without anconeus muscle flap for treatment of refractory lateral epicondylitis.

BACKGROUND: Lateral epicondylitis is a common condition encountered by orthopedic surgeons. Whereas the majority of patients improve with conservative management, a small percentage will require surgery. The purpose of this study was to compare the clinical outcomes of surgical débridement of the common extensor origin alone with débridement combined with rotation of an anconeus muscle flap in patients who failed to respond to conservative management of chronic lateral epicondylitis. METHODS: Fifty-seven patients who failed to respond to a minimum of 5 months of conservative treatment for lateral epicondylitis were retrospectively reviewed. Patients in group 1 were treated with open débridement of the common extensor origin. Patients in group 2 were treated with open débridement combined with rotation of an anconeus muscle flap. Outcome measures included elbow range of motion, grip strength, visual analog scale (VAS) for pain score, and Disabilities of the Arm, Shoulder, and Hand (DASH) score. Statistical analyses were performed by Student's t test with 95% confidence intervals. RESULTS: At final follow-up, average DASH scores were significantly lower in group 2. There were no significant differences between the groups with regard to elbow range of motion or grip strength. VAS pain scores were significantly reduced in both groups. Preoperative VAS pain scores were significantly greater in group 2; however, at final follow-up, there was no significant difference between groups. There were no apparent complications in either group. CONCLUSIONS: In addition to débridement of the common extensor origin, the rotation of an anconeus muscle flap may improve outcomes in cases of lateral epicondylitis that require operative intervention.

Structure-activity profiles of novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with modified amino acids for cellular uptake by folate receptors α and ß and the proton-coupled folate transporter.

Structure-activity relationships for cellular uptake and inhibition of cell proliferation were studied for 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates in which the terminal l-glutamate of the parent structure (7) was replaced by natural or unnatural amino acids. Compounds 7 and 10-13 were selectively inhibitory toward folate receptor (FR) α-expressing Chinese hamster ovary (CHO) cells. Antiproliferative effects of compounds 7 and 9-13 toward FRα- and FRß-expressing CHO cells were only partly reflected in binding affinities to FRα and FRß or in the docking scores with molecular models of FRα and FRß. Compounds 7 and 11 were potent inhibitors of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis in KB human tumor cells. These studies establish for the first time the importance of the α- and γ-carboxylic acid groups, the length of the amino acid, and the conformation of the side chain for transporter binding and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates.

Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine antifolates as dual-acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: implications of inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase to ampk activation and antitumor activity.

We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.

Tumor-targeting with novel non-benzoyl 6-substituted straight chain pyrrolo[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis.

A new series of 6-substituted straight side chain pyrrolo[2,3-d]pyrimidines 3a-d with varying chain lengths (n = 5-8) was designed and synthesized as part of our program to provide targeted antitumor agents with folate receptor (FR) cellular uptake specificity and glycinamide ribonucleotide formyltransferase (GARFTase) inhibition. Carboxylic acids 4a-d were converted to the acid chlorides and reacted with diazomethane, followed by 48% HBr to generate the α-bromomethylketones 5a-d. Condensation of 2,4-diamino-6-hydroxypyrimidine 6 with 5a-d afforded the 6-substituted pyrrolo[2,3-d]pyrimidines 7a-d. Hydrolysis and subsequent coupling with diethyl l-glutamate and saponification afforded target compounds 3a-d. Compounds 3b-d showed selective cellular uptake via FRα and -ß, associated with high affinity binding and inhibition of de novo purine nucleotide biosynthesis via GARFTase, resulting in potent inhibition against FR-expressing Chinese hamster cells and human KB tumor cells in culture. Our studies establish, for the first time, that a side chain benzoyl group is not essential for tumor-selective drug uptake by FRα.

Notch1 receptor regulates AKT protein activation loop (Thr308) dephosphorylation through modulation of the PP2A phosphatase in phosphatase and tensin homolog (PTEN)-null T-cell acute lymphoblastic leukemia cells.

Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

High-frequency alloreactive T cells augment effector function of low-frequency CD8+ T-cell responses under CD28/CD154 blockade.

BACKGROUND: Blockade of costimulatory molecules is a potent method of inducing long-term graft survival. We have previously addressed the issue of donor-reactive T-cell precursor frequency on relative costimulation dependence and found that the presence of a high precursor frequency of donor-reactive CD8 T cells resulted in costimulation blockade-resistant graft rejection, whereas the presence of a low-frequency donor-reactive population did not. To address the mechanisms by which high-frequency T cells obviated the requirement for costimulation, we asked whether a low-frequency population responding concomitantly with a high-frequency response also demonstrated costimulation independence. METHODS: A model system was established in which B6 mice containing a low frequency of anti-membrane bound chicken ovalbumin (mOVA) responders and a high frequency of anti-BALB/c responders received a skin graft from B6.mOVAxBALB/c F1 donors in the presence or absence of cytotoxic T-lymphocyte antigen-4 Ig/anti-CD154 costimulatory blockade. RESULTS: The results revealed that in the presence of costimulation blockade, high-frequency anti-BALB/c T cells augmented the effector activity of low-frequency anti-mOVA T cells, but it did not enhance the accumulation of anti-mOVA T cells capable of mediating graft rejection. CONCLUSIONS: These results demonstrate that both antigen-specific and antigen-independent factors contribute to the relative costimulation independence of high-frequency T-cell responses.

Aerosolized Bacillus anthracis infection in New Zealand white rabbits: natural history and intravenous levofloxacin treatment.

The natural history for inhalational Bacillus anthracis (Ames strain) exposure in New Zealand white rabbits was investigated to better identify potential, early biomarkers of anthrax. Twelve SPF Bordetella-free rabbits were exposed to 150 LD(50) aerosolized B. anthracis spores, and clinical signs, body temperature, complete blood count, bacteremia, and presence of protective antigen in the blood (that is, antigenemia) were examined. The development of antigenemia and bacteremia coincided and preceded both pyrexia and inversion of the heterophil:lymphocyte ratio, an indicator of infection. Antigenemia was determined within 1 h by electrochemiluminescence immunoassay, compared with the 24-h traditional culture needed for bacteremia determination. Rabbits appeared clinically normal until shortly before succumbing to anthrax approximately 47 h after challenge or approximately 22 h after antigenemia, which suggests a relatively narrow therapeutic window of opportunity. To evaluate the therapeutic rabbit model, B. anthracis-exposed rabbits were treated (after determination of antigenemia and later confirmed to be bacteremic) intravenously with the fluoroquinolone antibiotic levofloxacin for 5 d at a total daily dose of 25 or 12.5 mg/kg, resulting in nearly 90% and 70% survival, respectively, to the study end (28 d after challenge). The peak level for 12.5 mg/kg was equivalent to that observed for a 500-mg daily levofloxacin dose in humans. These results suggest that intravenous levofloxacin is an effective therapeutic against inhalational anthrax. Taken together, our findings indicate that antigenemia is a viable and early biomarker for B. anthracis infection that can be used as a treatment trigger to allow for timely intervention against this highly pathogenic disease.

Beyond-use dating of extemporaneously compounded ketamine, acepromazine, and xylazine: safety, stability, and efficacy over time.

Ketamine-acepromazine-xylazine (KAX) has long been a popular combination of injectable anesthetics for use in laboratory rodents. These drugs are compounded extemporaneously at research facilities because a commercial mixture is not available. This study was designed to determine an appropriate period of use for this mixture by examining its safety, stability, and efficacy at 30-d intervals over an aging period of 270 d. For as long as 270 d after compounding, most of the data collected (chemical stability, sterility, pH, particulate formation, times to loss of righting reflex in injected mice and rats, and histopathology from these animals) supported the finding that the component drugs do not change or degrade. However, mice and rats did show significant differences in anesthetic responses after injection with KAX mixtures of different ages. In light of these findings, we suggest that KAX remains safe, stable, and efficacious for at least 180 d after mixing, and that 180 d constitutes an appropriate period of use for this drug combination when stored in a dark, room-temperature environment.