An Update on Orthobiologics: Cautious Optimism.

Orthobiologics are rapidly growing in use given their potential to augment healing for multiple musculoskeletal conditions. Orthobiologics consist of a variety of treatments including platelet-rich plasma and stem cells that provide conceptual appeal in providing local delivery of growth factors and inflammation modulation. The lack of standardization in nomenclature and applications within the literature has led to a paucity of high-quality evidence to support their frequent use. The purpose of this review was to describe the current landscape of orthobiologics and the most recent evidence regarding their use.

Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure.

Background: The Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses. Methods: Characterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis. Results: We observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C). Conclusions: Our findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases.

CGM Metrics Identify Dysglycemic States in Participants From the TrialNet Pathway to Prevention Study.

OBJECTIVE: Continuous glucose monitoring (CGM) parameters may identify individuals at risk for progression to overt type 1 diabetes. We aimed to determine whether CGM metrics provide additional insights into progression to clinical stage 3 type 1 diabetes. RESEARCH DESIGN AND METHODS: One hundred five relatives of individuals in type 1 diabetes probands (median age 16.8 years; 89% non-Hispanic White; 43.8% female) from the TrialNet Pathway to Prevention study underwent 7-day CGM assessments and oral glucose tolerance tests (OGTTs) at 6-month intervals. The baseline data are reported here. Three groups were evaluated: individuals with 1) stage 2 type 1 diabetes (n = 42) with two or more diabetes-related autoantibodies and abnormal OGTT; 2) stage 1 type 1 diabetes (n = 53) with two or more diabetes-related autoantibodies and normal OGTT; and 3) negative test for all diabetes-related autoantibodies and normal OGTT (n = 10). RESULTS: Multiple CGM metrics were associated with progression to stage 3 type 1 diabetes. Specifically, spending ≥5% time with glucose levels ≥140 mg/dL (P = 0.01), ≥8% time with glucose levels ≥140 mg/dL (P = 0.02), ≥5% time with glucose levels ≥160 mg/dL (P = 0.0001), and ≥8% time with glucose levels ≥160 mg/dL (P = 0.02) were all associated with progression to stage 3 disease. Stage 2 participants and those who progressed to stage 3 also exhibited higher mean daytime glucose values; spent more time with glucose values over 120, 140, and 160 mg/dL; and had greater variability. CONCLUSIONS: CGM could aid in the identification of individuals, including those with a normal OGTT, who are likely to rapidly progress to stage 3 type 1 diabetes.

A data-validated temporary immunity model of COVID-19 spread in Michigan.

We introduce a distributed-delay differential equation disease spread model for COVID-19 spread. The model explicitly incorporates the population's time-dependent vaccine uptake and incorporates a gamma-distributed temporary immunity period for both vaccination and previous infection. We validate the model on COVID-19 cases and deaths data from the state of Michigan and use the calibrated model to forecast the spread and impact of the disease under a variety of realistic booster vaccine strategies. The model suggests that the mean immunity duration for individuals after vaccination is 350 days and after a prior infection is 242 days. Simulations suggest that both high population-wide adherence to vaccination mandates and a more-than-annually frequency of booster doses will be required to contain outbreaks in the future.

Isolated Acetabuloplasty for Femoroacetabular Impingement: Favorable Patient-Reported Outcomes and Sustained Survivorship at Minimum 5-Year Follow-Up.

PURPOSE: To evaluate minimum 5-year outcomes and conversion rate to total hip arthroplasty (THA) for the treatment of femoroacetabular impingement (FAI) syndrome with an isolated acetabular osteoplasty. METHODS: Patients undergoing hip arthroscopy with an isolated acetabular osteoplasty from March 2009 to June 2014 for FAI syndrome with pincer and/or cam morphology and a labral tear were identified. Those who underwent femoroplasty or prior ipsilateral hip surgery or who had previous hip conditions, ipsilateral hip dysplasia, or a Tönnis grade higher than 2 were excluded. Patient-reported outcomes (PROs) collected included Patient-Reported Outcomes Measurement Information System (PROMIS) scores specific to physical functioning and pain interference, modified Harris Hip Score, International Hip Outcome Tool 12, Hip Outcome Score-Activities of Daily Living, Hip Outcome Score-Sport-Specific Subscale, and Numeric Pain Rating Scale. Patients were also queried about secondary surgical procedures and conversion to THA. RESULTS: We identified 86 patients at minimum 5-year follow-up (average, 7.4 years). The average patient age was 39.8 ± 12.3 years, 70.9% of patients were female, and 7% of patients had Tönnis grade 2. The mean PRO scores were 52.0 ± 8.9 for the PROMIS physical functioning score, 39.6 ± 7.5 for the PROMIS pain interference score, 78.7 ± 12.0 for the modified Harris Hip Score, 73.3 ± 23.1 for the International Hip Outcome Tool 12 score, 89.9 ± 12.0 for the Hip Outcome Score-Activities of Daily Living, and 81.4 ± 21.0 for the Hip Outcome Score-Sport-Specific Subscale. Of the patients, 72.1% achieved the patient acceptable symptomatic state (PASS) according to previously established PASS scores for FAI syndrome treated with hip arthroscopy at minimum 5-year follow-up. The overall rate of revision arthroscopy was 3.5%, and the rate of conversion to THA was 5.8%. CONCLUSIONS: An isolated acetabular osteoplasty can provide sustained clinical benefits for the treatment of FAI syndrome with labral tears, with good to excellent PROs and PASS rates and a low rate of conversion to THA at minimum 5-year follow-up. LEVEL OF EVIDENCE: Level IV, case series.

Arthroscopic Evaluation of the Subtalar Joint: A Review and Survey of Pathology.

BACKGROUND: A survey of pathology present in the subtalar joint by means of subtalar arthroscopy with anterolateral and middle portals has not been extensively explored in the literature. The focus of this study was to identify pathology in the subtalar joint with subtalar joint arthroscopy using this approach. We also compared these arthroscopic findings with those reported in the literature. METHODS: We performed a retrospective review of 49 consecutive patients who had undergone 53 subtalar arthroscopic procedures. Data were obtained from intraoperative arthroscopic findings that were documented in the operative note or with arthroscopic photography. Additional procedures, including ankle arthroscopy, lateral ankle stabilization, and peroneal tendon repair, were recorded. Descriptive statistics were calculated and reported. RESULTS: Subtalar arthroscopic examination revealed that all of the patients had intra-articular synovitis or adhesions present. Twenty-two procedures (42%) demonstrated subtalar joint instability, seven (13%) revealed chondromalacia, and one (2%) had an exostosis present. These observations are consistent with other reported findings in the literature. CONCLUSIONS: This study found that the subtalar joint was most often affected by synovitis, adhesions, and instability in patients with symptomatic pathologies requiring subtalar arthroscopy. There was a relatively low incidence of chondromalacia or exostosis formation in the survey.

The Proteocephalus species-aggregate (Cestoda) in sticklebacks (Gasterosteidae) of the Nearctic Region, including description of a new species from brook stickleback, Culaea inconstans.

A survey of the species of the Proteocephalus-aggregate from sticklebacks (Actinopterygii: Gasterosteidae) is provided. The occurrence of three species in North America is confirmed: (i) Proteocephalus filicollis (Rudolphi, 1802), which has been reported from the three-spined stickleback, Gasterosteus aculeatus Linnaeus, in the northeastern part of North America (Newfoundland); (ii) Proteocephalus pugetensis Hoff et Hoff, 1929 occurs also in G. aculeatus, but in northwestern North America (British Columbia and Washington); and (iii) Proteocephalus culaeae sp. n., which is described from the brook stickleback, Culaea inconstans (Kirtland), in Manitoba (Canada). Another species, Proteocephalus ambiguus (Dujardin, 1845), a specific parasite of the nine-spined stickleback, Pungitius pungitius (Linnaeus), and type species of the genus, has also been found in North America (Alberta, Canada), but its vouchers are in poor condition and cannot be reliable assigned to this species. Both species reported from three-spined stickleback differ from each other by the shape of the scolex (rounded in P. filicollis versus continuously tapered towards the anterior extremity in P. pugetensis) and the apical sucker (widely oval to subspherical in frontal view in P. filicollis versus flattened in P. pugetensis). Proteocephalus culaeae sp. n. is characterised by a short body composed of a few, continuously widened proglottids, a short scolex narrower than the strobila and devoid of an apical sucker, a short, pyriform cirrus sac, no vaginal sphincter, and few testes. A key to species of the Proteocephalus-aggregate from sticklebacks is provided.

Surface-Enhanced Raman Spectroscopy for Rapid Screening of Cinnamon Essential Oils.

Cinnamon essential oil is used in food flavoring, food preservation, and for complementary medicine. The most common types of cinnamon used in essential oils are true cinnamon (Cinnamomum verum) and cassia cinnamon (Cinnamomum cassia). True cinnamon is commonly adulterated with cassia cinnamon because it is cheaper. However, cassia cinnamon contains higher concentrations of coumarin which has been shown to have adverse health effects. There is a need to develop simple, nondestructive, rapid screening methods for quality control and food authentication and to identify adulteration of cinnamon essential oil. Currently, the most common methods to screen for coumarin in cinnamon include high-performance liquid chromatography (HPLC) and gas chromatography (GC). However, these methods require time-consuming sample preparation and detection. Vibrational spectroscopy methods are emerging as a promising alternative for rapid, nondestructive screening for food safety applications. In this study, a rapid screening method has been developed to examine cinnamon essential oils using surface-enhanced Raman spectroscopy (SERS). The experimental spectra were compared to theoretical calculations using the DFT method BP86/6-311++G(d,p) basis set. The limit of detection of coumarin was determined to be 1 × 10-6 M or 1.46 mg/L using SERS with colloid paste substrates. Furthermore, 1:16 dilutions of cinnamaldehyde and 1:8 dilutions of eugenol were detected using SERS which can help determine if the cinnamon essential oil was made from bark or from leaves. Seven commercially available cinnamon essential oils were also analyzed and compared to reference solutions. SERS was able to discriminate between essential oils primarily composed of cinnamaldehyde and those composed of eugenol. Furthermore, the SERS method detected peaks that are attributed to coumarin in two of the commercially available samples. To date, this is the first time SERS has been used to rapidly screen cinnamon essential oils.

Assessment of Flexion Strength Following Single- Versus Double-Hamstring Tendon Harvest for Anterior Cruciate Ligament Reconstruction.

PURPOSE: To compare isometric hamstring strength deficits, knee laxity, functional outcomes, and patient-reported outcomes between patients who underwent anterior cruciate ligament (ACL) reconstruction with doubled semitendinosus and gracilis tendon autograft (ST/G) versus quadrupled semitendinosus autograft (ST), at a minimum follow-up of 1-year postoperatively. METHODS: Patients who underwent ACL reconstruction with ST/G or ST hamstring autografts were retrospectively identified. Isometric hamstring strength was tested with a hand-held dynamometer at 30, 60, and 90° of knee flexion. Anterior knee laxity was assessed using a KT-1000 arthrometer. Functional outcomes were collected using the single-leg hop test and single-leg squat test. Side-to-side differences were determined and compared between the ST/G and ST groups. Patient-reported outcomes were collected on all patients. RESULTS: Eighty-four patients who underwent ST/G (n = 34) or ST (n = 50) autograft ACL reconstruction were recruited to participate in this study. There was no difference in knee laxity between the groups. Side-to-side hamstring strength deficits increased with increased flexion angles. At 90° of flexion, the ST/G group had a significantly greater flexion strength deficit compared with the ST group (37.8 ± 15.1% vs 24.7 ± 12.5%, P < .001). Aside from a significant difference in the KOOS pain Score (P .045), no other significant differences in functional or patient reported outcomes between the groups were identified. CONCLUSIONS: Patients who underwent ACL reconstruction with ST/G compared with ST autograft have a significantly greater isometric flexion strength deficit at 90° of flexion. Future investigations are required to determine the clinical relevance of this difference and whether specialized therapy protocols can mitigate this deficit. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Primary Versus Salvage Reverse Total Shoulder Arthroplasty for Displaced Proximal Humerus Fractures in the Elderly: A Systematic Review and Meta-analysis.

BACKGROUND: There is currently no established consensus on best treatment for complex proximal humerus fractures (PHFs) in the elderly. Reverse total shoulder arthroplasty (RTSA) is a viable option in this population but many times is used as a salvage procedure. METHODS: A systematic review of studies comparing RTSA as a primary treatment for PHF versus as a salvage procedure following failed open reduction internal fixation (ORIF), humeral intramedullary nailing, hemiarthroplasty (HA) or non-operative treatment was conducted using PRISMA guidelines. Pooled outcomes and sub-group analyses assessing range of motion, patient reported outcomes and complications were examined using RevMan. RESULTS: Five articles were included in final analysis with 104 patients in the primary RTSA group and 147 in the salvage RTSA group compromising 251 total patients. Primary RTSA had a statistically significant advantage in range of motion (forward flexion and external rotation), patient reported outcomes, and complications compared to salvage RTSA. CONCLUSIONS: Based on the best available evidence, primary RTSA may result in slightly better patient reported outcomes, range of motion and a lower rate of complication when compared to salvage RTSA. Further high-quality prospective studies are needed to confirm the findings of the current review.

Report of the 2017-2018 Strategic Engagement Standing Committee.

EXECUTIVE SUMMARY Student engagement is key to the success of schools and colleges of pharmacies in meeting their mission and programmatic needs. Student engagement in the pharmacy profession often occurs before acceptance to pharmacy school and is essential during students' formal period of study both for the student's professional growth and in meeting the mission of the school. Alumni engagement is vital to a school's continued success in regard to engaging with current students and support of their alma mater. The committee offers best practice recommendations for engaging students in service, scholarship, education, professional practice and continuing professional development.

Modeling the Emergence of Antibiotic Resistance in the Environment: an Analytical Solution for the Minimum Selection Concentration.

Environmental antibiotic risk management requires an understanding of how subinhibitory antibiotic concentrations contribute to the spread of resistance. We develop a simple model of competition between sensitive and resistant bacterial strains to predict the minimum selection concentration (MSC), the lowest level of antibiotic at which resistant bacteria are selected. We present an analytical solution for the MSC based on the routinely measured MIC, the selection coefficient (sc) that expresses fitness differences between strains, the intrinsic net growth rate, and the shape of the bacterial growth dose-response curve with antibiotic or metal exposure (the Hill coefficient [κ]). We calibrated the model by optimizing the Hill coefficient to fit previously reported experimental growth rate difference data. The model fit varied among nine compound-taxon combinations examined but predicted the experimentally observed MSC/MIC ratio well (R2 ≥ 0.95). The shape of the antibiotic response curve varied among compounds (0.7 ≤ κ ≤ 10.5), with the steepest curve being found for the aminoglycosides streptomycin and kanamycin. The model was sensitive to this antibiotic response curve shape and to the sc, indicating the importance of fitness differences between strains for determining the MSC. The MSC can be >1 order of magnitude lower than the MIC, typically by the factor scκ This study provides an initial quantitative depiction and a framework for a research agenda to examine the growing evidence of selection for resistant bacterial communities at low environmental antibiotic concentrations.

Rethinking the influence of hydroelectric development on gene flow in a long-lived fish, the Lake Sturgeon Acipenser fulvescens.

Many hydroelectric dams have been in place for 50 - >100 years, which for most fish species means that enough generations have passed for fragmentation induced divergence to have accumulated. However, for long-lived species such as Lake Sturgeon, Acipenser fulvescens, it should be possible to discriminate between historical population structuring and contemporary gene flow and improve the broader understanding of anthropogenic influence. On the Winnipeg River, Manitoba, two hypotheses were tested: 1) Measureable quantities of former reservoir dwelling Lake Sturgeon now reside downstream of the Slave Falls Generating Station, and 2) genetically differentiated populations of Lake Sturgeon occur upstream and downstream, a result of historical structuring. Genetic methods based on ten microsatellite markers were employed, and simulations were conducted to provide context. With regards to contemporary upstream to downstream contributions, the inclusion of length-at-age data proved informative. Both pairwise relatedness and Bayesian clustering analysis substantiated that fast-growing outliers, apparently entrained after residing in the upstream reservoir for several years, accounted for ~15% of the Lake Sturgeon 525-750 mm fork length captured downstream. With regards to historical structuring, upstream and downstream populations were found to be differentiated (FST = 0.011, and 0.013-0.014 when fast-growing outliers were excluded), and heterozygosity metrics were higher for downstream versus upstream juveniles. Historical asymmetric (downstream) gene flow in the vicinity of the generating station was the most logical explanation for the observed genetic structuring. In this section of the Winnipeg River, construction of a major dam does not appear to have fragmented a previously panmictic Lake Sturgeon population, but alterations to habitat may be influencing upstream to downstream contributions in unexpected ways.

Association between impaired cardiovascular autonomic function and hypoglycemia in patients with type 1 diabetes.

OBJECTIVE: We studied the association between glycemic variability (GV) reflecting hypoglycemic stress and cardiovascular autonomic function in subjects with type 1 diabetes. RESEARCH DESIGN AND METHODS: Forty-four type 1 diabetic patients (mean age 34 ± 13 years, 40% male, 86% Caucasian, mean diabetes duration 13 ± 6 years, mean hemoglobin A1c [HbA1c] 8.0 ± 1.2% [64 ± 5 mmol/mol]) without cardiovascular disease, dyslipidemia, or hypertension participated in this pilot study. Indices of GV reflective of hypoglycemic stress (low blood glucose index [LBGI] and area under the curve [AUC] for hypoglycemia) were computed using data obtained during 5-day continuous glucose monitoring. Cardiovascular autonomic neuropathy (CAN) was assessed using standardized cardiovascular reflex testing and measures of heart rate variability (HRV), which were analyzed as time and frequency domain measures. RESULTS: Both LBGI and AUC hypoglycemia had a significant negative association with the low-frequency power of HRV (r = -0.47, P = 0.002; r = -0.43, P = 0.005, respectively) and with the high-frequency power of HRV (r = -0.37, P = 0.018; r = -0.38, P = 0.015, respectively). These inverse associations persisted after adjusting for HbA1c, although they were attenuated in multivariable analysis after adjustment for age, diabetes duration, and several other covariates. CONCLUSIONS: Increased GV promoting hypoglycemic stress was associated with reduced HRV independent of glycemic control as assessed by HbA1c. These pilot data suggest that glucose variability may contribute to cardiovascular autonomic dysfunction among adults with type 1 diabetes.

Modeling adaptive regulatory T-cell dynamics during early HIV infection.

Regulatory T-cells (Tregs) are a subset of CD4(+) T-cells that have been found to suppress the immune response. During HIV viral infection, Treg activity has been observed to have both beneficial and deleterious effects on patient recovery; however, the extent to which this is regulated is poorly understood. We hypothesize that this dichotomy in behavior is attributed to Treg dynamics changing over the course of infection through the proliferation of an 'adaptive' Treg population which targets HIV-specific immune responses. To investigate the role Tregs play in HIV infection, a delay differatial equation model was constructed to examine (1) the possible existence of two distinct Treg populations, normal (nTregs) and adaptive (aTregs), and (2) their respective effects in limiting viral load. Sensitivity analysis was performed to test parameter regimes that show the proportionality of viral load with adaptive regulatory populations and also gave insight into the importance of downregulation of CD4(+) cells by normal Tregs on viral loads. Through the inclusion of Treg populations in the model, a diverse array of viral dynamics was found. Specifically, oscillatory and steady state behaviors were both witnessed and it was seen that the model provided a more accurate depiction of the effector cell population as compared with previous models. Through further studies of adaptive and normal Tregs, improved treatments for HIV can be constructed for patients and the viral mechanisms of infection can be further elucidated.

Global gene expression profiling in mouse plasma cell tumor precursor and bystander cells reveals potential intervention targets for plasma cell neoplasia.

Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eµ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro.

Translating glucose variability metrics into the clinic via Continuous Glucose Monitoring: a Graphical User Interface for Diabetes Evaluation (CGM-GUIDE©).

BACKGROUND: Several metrics of glucose variability have been proposed to date, but an integrated approach that provides a complete and consistent assessment of glycemic variation is missing. As a consequence, and because of the tedious coding necessary during quantification, most investigators and clinicians have not yet adopted the use of multiple glucose variability metrics to evaluate glycemic variation. METHODS: We compiled the most extensively used statistical techniques and glucose variability metrics, with adjustable hyper- and hypoglycemic limits and metric parameters, to create a user-friendly Continuous Glucose Monitoring Graphical User Interface for Diabetes Evaluation (CGM-GUIDE©). In addition, we introduce and demonstrate a novel transition density profile that emphasizes the dynamics of transitions between defined glucose states. RESULTS: Our combined dashboard of numerical statistics and graphical plots support the task of providing an integrated approach to describing glycemic variability. We integrated existing metrics, such as SD, area under the curve, and mean amplitude of glycemic excursion, with novel metrics such as the slopes across critical transitions and the transition density profile to assess the severity and frequency of glucose transitions per day as they move between critical glycemic zones. CONCLUSIONS: By presenting the above-mentioned metrics and graphics in a concise aggregate format, CGM-GUIDE provides an easy to use tool to compare quantitative measures of glucose variability. This tool can be used by researchers and clinicians to develop new algorithms of insulin delivery for patients with diabetes and to better explore the link between glucose variability and chronic diabetes complications.

Phosphorylation of paxillin at threonine 538 by PKCdelta regulates LFA1-mediated adhesion of lymphoid cells.

We investigated the PKCdelta-mediated phosphorylation of paxillin within its LIM4 domain and the involvement of this phosphorylation in activation of LFA-1 integrins of the Baf3 pro-B lymphocytic cell line. Using phosphorylated-threonine-specific antibodies, phosphorylated amino acid analysis and paxillin phosphorylation mutants, we demonstrated that TPA, the pharmacological analog of the endogenous second messenger diacyl glycerol, stimulates paxillin phosphorylation at threonine 538 (T538). The TPA-responsive PKC isoform PKCdelta directly binds paxillin in a yeast two-hybrid assay and phosphorylates paxillin at T538 in vitro and also co-immunoprecipitates with paxillin and mediates phosphorylation of this residue in vivo. Recombinant wild-type paxillin, its phospho-inhibitory T538A or phospho-mimetic T538E mutants were expressed in the cells simultaneously with siRNA silencing of the endogenous paxillin. These experiments suggest that phosphorylation of paxillin T538 contributes to dissolution of the actin cytoskeleton, redistribution of LFA-1 integrins and an increase in their affinity. We also show that phosphorylation of T538 is involved in the activation of LFA-1 integrins by TPA.

Humoral autoimmunity against the extracellular domain of the neuroendocrine autoantigen IA-2 heightens the risk of type 1 diabetes.

The objective of this study was to determine whether antigenic determinants localized within the extracellular domain of the neuroendocrine autoantigen tyrosine phosphatase-like protein IA-2 are targets of humoral responses in type 1 diabetes (T1DM). Previous studies indicated that the immunodominant region of IA-2 is localized within its intracellular domain (IA-2ic; amino acids 601-979). We analyzed 333 subjects from the Children's Hospital of Pittsburgh study, 102 of whom progressed to insulin-requiring diabetes (prediabetics). Autoantibodies from these individuals were initially assayed for ICA512bdc (Barbara Davis Center amino acids 257-556; 630-979), IA-2ic (amino acids 601-979), and IA-2 full-length (amino acids 1-979) in addition to islet cell antibody (ICA), glutamic acid decarboxylase, 65-kDa isoform, and insulin autoantibodies. We identified an autoantibody response reactive with the extracellular domain of IA-2 that is associated with very high risk of T1DM progression. Relatives with no detectable autoantibodies against ICA512bdc (or IA-2ic) exhibited antibody responses against the IA-2 full-length peptide (log rank, P = 0.008). This effect was also observed in first-degree relatives who were positive for glutamic acid decarboxylase, 65-kDa isoform (log rank, P = 0.026) or at least two islet autoantibodies but were negative for ICA512bdc (log rank, P = 0.022). Competitive binding experiments and immunoprecipitation of the IA-2 extracellular domain (amino acid residues 26-577) further lend support for the presence of autoantibodies reactive with new antigenic determinants within the extracellular domain of IA-2. In summary, the addition of measurements of autoantibodies reactive with the IA-2 extracellular domain to assays geared to assess the progression of autoimmunity to clinical T1DM may more accurately characterize this risk. This has considerable implications not only for stratifying high diabetes risk but also facilitating the search for pathogenic epitopes to enable the design of peptide-based immunotherapies that may prevent the progression to overt T1DM at its preclinical stages.

Dynamics of human complement-mediated killing of Klebsiella pneumoniae.

With an in vitro system that used a luminescent strain of Klebsiella pneumoniae to assess bacterial metabolic activity in near-real-time, we investigated the dynamics of complement-mediated attack in healthy individuals and in patients presenting to the emergency department with community-acquired severe sepsis. A novel mathematical/statistical model was developed to simplify light output trajectories over time into two fitted parameters, the rate of complement activation and the delay from activation to the onset of killing. Using Factor B-depleted serum, the alternative pathway was found to be the primary bactericidal effector: In the absence of B, C3 opsonization as measured by flow cytometry did not progress and bacteria proliferated near exponentially. Defects in bacterial killing were easily demonstrable in patients with severe sepsis compared with healthy volunteers. In most patients with sepsis, the rate of activation was higher than in normal subjects but was associated with a prolonged delay between activation and bacterial killing (P < 0.05 for both). Theoretical modeling suggested that this combination of accentuated but delayed function should allow successful bacterial killing but with significantly greater complement activation. The use of luminescent bacteria allowed for the development of a novel and powerful tool for assessing complement immunology for the purposes of mechanistic study and patient evaluation.