Development of a multigenomic liquid biopsy (PROSTest) for prostate cancer in whole blood.

INTRODUCTION: We describe the development of a molecular assay from publicly available tumor tissue mRNA databases using machine learning and present preliminary evidence of functionality as a diagnostic and monitoring tool for prostate cancer (PCa) in whole blood. MATERIALS AND METHODS: We assessed 1055 PCas (public microarray data sets) to identify putative mRNA biomarkers. Specificity was confirmed against 32 different solid and hematological cancers from The Cancer Genome Atlas (n = 10,990). This defined a 27-gene panel which was validated by qPCR in 50 histologically confirmed PCa surgical specimens and matched blood. An ensemble classifier (Random Forest, Support Vector Machines, XGBoost) was trained in age-matched PCas (n = 294), and in 72 controls and 64 BPH. Classifier performance was validated in two independent sets (n = 263 PCas; n = 99 controls). We assessed the panel as a postoperative disease monitor in a radical prostatectomy cohort (RPC: n = 47). RESULTS: A PCa-specific 27-gene panel was identified. Matched blood and tumor gene expression levels were concordant (r = 0.72, p < 0.0001). The ensemble classifier ("PROSTest") was scaled 0%-100% and the industry-standard operating point of ≥50% used to define a PCa. Using this, the PROSTest exhibited an 85% sensitivity and 95% specificity for PCa versus controls. In two independent sets, the metrics were 92%-95% sensitivity and 100% specificity. In the RPCs (n = 47), PROSTest scores decreased from 72% ± 7% to 33% ± 16% (p < 0.0001, Mann-Whitney test). PROSTest was 26% ± 8% in 37 with normal postoperative PSA levels (<0.1 ng/mL). In 10 with elevated postoperative PSA, PROSTest was 60% ± 4%. CONCLUSION: A 27-gene whole blood signature for PCa is concordant with tissue mRNA levels. Measuring blood expression provides a minimally invasive genomic tool that may facilitate prostate cancer management.

Vascular persistence following precision micropuncture.

OBJECTIVE: The success of engineered tissues continues to be limited by time to vascularization and perfusion. Recently, we described a simple microsurgical approach, termed micropuncture (MP), which could be used to rapidly vascularize an adjacently placed scaffold from the recipient macrovasculature. Here we studied the long-term persistence of the MP-induced microvasculature. METHODS: Segmental 60 µm diameter MPs were created in the recipient rat femoral artery and vein followed by coverage with a simple Type 1 collagen scaffold. The recipient vasculature and scaffold were then wrapped en bloc with a silicone sheet to isolate intrinsic vascularization. Scaffolds were harvested at 28 days post-implantation for detailed analysis, including using a novel artificial intelligence (AI) approach. RESULTS: MP scaffolds demonstrated a sustained increase of vascular density compared to internal non-MP control scaffolds (p < 0.05) secondary to increases in both vessel diameters (p < 0.05) and branch counts (p < 0.05). MP scaffolds also demonstrated statistically significant increases in red blood cell (RBC) perfused lumens. CONCLUSIONS: This study further highlights that the intrinsic MP-induced vasculature continues to persist long-term. Its combination of rapid and stable angiogenesis represents a novel surgical platform for engineered scaffold and graft perfusion.

Accelerating Patterned Vascularization Using Granular Hydrogel Scaffolds and Surgical Micropuncture.

Bulk hydrogel scaffolds are common in reconstructive surgery. They allow for the staged repair of soft tissue loss by providing a base for revascularization. Unfortunately, they are limited by both slow and random vascularization, which may manifest as treatment failure or suboptimal repair. Rapidly inducing patterned vascularization within biomaterials has profound translational implications for current clinical treatment paradigms and the scaleup of regenerative engineering platforms. To address this long-standing challenge, a novel microsurgical approach and granular hydrogel scaffold (GHS) technology are co-developed to hasten and pattern microvascular network formation. In surgical micropuncture (MP), targeted recipient blood vessels are perforated using a microneedle to accelerate cell extravasation and angiogenic outgrowth. By combining MP with an adjacent GHS with precisely tailored void space architecture, microvascular pattern formation as assessed by density, diameter, length, and intercapillary distance is rapidly guided. This work opens new translational opportunities for microvascular engineering, advancing reconstructive surgery, and regenerative medicine.

Identification of a Metabolic, Transcriptomic, and Molecular Signature of Patatin-Like Phospholipase Domain Containing 3-Mediated Acceleration of Steatohepatitis.

BACKGROUND AND AIMS: The mechanisms by which the I148M mutant variant of the patatin-like phospholipase domain-containing 3 (PNPLA3I148M ) drives development of nonalcoholic steatohepatitis (NASH) are not known. The aim of this study was to obtain insights on mechanisms underlying PNPLA3I148M -induced acceleration of NASH. APPROACH AND RESULTS: Hepatocyte-specific overexpression of empty vector (luciferase), human wild-type PNPLA3, or PNPLA3I148M was achieved using adeno-associated virus 8 in a diet-induced mouse model of nonalcoholic fatty liver disease followed by chow diet or high-fat Western diet with ad libitum administration of sugar in drinking water (WDSW) for 8 weeks. Under WDSW, PNPLA3I148M overexpression accelerated steatohepatitis with increased steatosis, inflammation ballooning, and fibrosis (P < 0.001 versus other groups for all). Silencing PNPLA3I148M after its initial overexpression abrogated these findings. PNPLA3I148M caused 22:6n3 docosahexanoic acid depletion and increased ceramides under WDSW in addition to increasing triglycerides and diglycerides, especially enriched with unsaturated fatty acids. It also increased oxidative stress and endoplasmic reticulum stress. Increased total ceramides was associated with signature of transducer and activator of transcription 3 (STAT3) activation with downstream activation of multiple immune-inflammatory pathways at a transcriptomic level by network analyses. Silencing PNPLA3I148M reversed STAT3 activation. Conditioned media from HepG2 cells overexpressing PNPLA3I148M increased procollagen mRNA expression in LX2 cells; this was abrogated by hepatocyte STAT3 inhibition. CONCLUSIONS: Under WDSW, PNPLA3I148M overexpression promotes steatosis and NASH by metabolic reprogramming characterized by increased triglycerides and diglycerides, n3 polyunsaturated fatty acid depletion, and increased ceramides with resultant STAT3 phosphorylation and downstream inflammatory pathway activation driving increased stellate cell fibrogenic activity.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

Induction of scaffold angiogenesis by recipient vasculature precision micropuncture.

The success of engineered tissues continues to be limited by time to vascularization and perfusion. Here, we studied the effects of precision injury to a recipient macrovasculature in promoting neovessel formation in an adjacently placed scaffold. Segmental 60 µm diameter micropunctures (MP) were created in the recipient rat femoral artery and vein followed by coverage with a simple collagen scaffold. Scaffolds were harvested at 24, 48, 72, and 96 h post-implantation for detailed analysis. Those placed on top of an MP segment showed an earlier and more robust cellular infiltration, including both endothelial cells (CD31) and macrophages (F4/80), compared to internal non-micropunctured control limbs (p < 0.05). At the 96-hour timepoint, MP scaffolds demonstrated an increase in physiologic perfusion (p < 0.003) and a 2.5-fold increase in capillary network formation (p < 0.001). These were attributed to an overall upsurge in small vessel quantity. Furthermore, MP positioned scaffolds demonstrated significant increases in many modulators of angiogenesis, including VEGFR2 and Tie-2 despite a decrease in HIF-1α at all timepoints. This study highlights a novel microsurgical approach that can be used to rapidly vascularize or inosculate contiguously placed scaffolds and grafts. Thereby, offering an easily translatable route towards the creation of thicker and more clinically relevant engineered tissues.

Item analysis of multiple choice questions: A quality assurance test for an assessment tool.

BACKGROUND: The item analysis of multiple choice questions (MCQs) is an essential tool that can provide input on validity and reliability of items. It helps to identify items which can be revised or discarded, thus building a quality MCQ bank. METHODS: The study focussed on item analysis of 90 MCQs of three tests conducted for 150 first year Bachelor of Medicine and Bachelor of Surgery (MBBS) physiology students. The item analysis explored the difficulty index (DIF I) and discrimination index (DI) with distractor effectiveness (DE). Statistical analysis was performed by using MS Excel 2010 and SPSS, version 20.0. RESULTS: Of total 90 MCQs, the majority, that is, 74 (82%) MCQs had a good/acceptable level of difficulty with a mean DIF I of 55.32 ± 7.4 (mean ± SD), whereas seven (8%) were too difficult and nine (10%) were too easy. A total of 72 (80%) items had an excellent to acceptable DI and 18 (20%) had a poor DI with an overall mean DI of 0.31 ± 0.12. There was significant weak correlation between DIF I and DI (r = 0.140, p < .0001). The mean DE was 32.35 ± 31.3 with 73% functional distractors in all. The reliability measure of test items by Cronbach alpha was 0.85 and Kuder-Richardson Formula 20 was 0.71, which is good. The standard error of measurement was 1.22. CONCLUSION: Our study helped teachers identify good and ideal MCQs which can be part of the question bank for future and those MCQs which needed revision. We recommend that item analysis must be performed for all MCQ-based assessments to determine validity and reliability of the assessment.

Stimulation with FITC-labeled antigens confers B cells with regulatory properties.

B cells with regulatory properties (Bregs) were identified in human and in mice among different B-cell subsets. Their regulatory properties rely mainly on the production of anti-inflammatory cytokines, in particular IL10, IL-35 and TGFß, and were extensively studied in mouse models of autoimmune and inflammatory diseases. However, the exact nature of the stimulatory signals conferring regulatory properties to B cells is still not clear. We serendipitously observed that fluorescein isothiocyanate (FITC) binds to a significant proportion of naïve mouse B cells. Binding of FITC to the B-cell surface implicated at least in part the B-cell receptor. It triggered IL-10 production and allowed the endocytosis of FITC-coupled antigens followed by their presentation to CD4+ T cells. In particular, B cells incubated with FITC-OVA polarized OTII T cells towards a Tr1/Th2 phenotype in vitro. Further, the adoptive transfer of B cells incubated with FITC-labeled myelin oligodendrocyte glycoprotein peptide protected mice from experimental autoimmune encephalomyelitis, a T-cell-dependent autoimmune model. Together, the data show that FITC-stimulated B cells polarize immune responses towards Tr1/Th2 and acquire immuno-modulatory properties.

Correction of bleeding in experimental severe hemophilia A by systemic delivery of factor VIII-encoding mRNA.

The treatment or prevention of bleeding in patients with hemophilia A relies on replacement therapy with different factor VIII (FVIII)-containing products or on the use of by-passing agents, i.e., activated prothrombin complex concentrates or recombinant activated factor VII. Emerging approaches include the use of bispecific anti-factor IXa/factor X antibodies, anti-tissue factor pathway inhibitor antibodies, interfering RNA to antithrombin, and activated protein C-specific serpins or gene therapy. The latter strategies are, however, hampered by the short clinical experience and potential adverse effects including the absence of tight temporal and spatial control of coagulation and the risk of uncontrolled insertional mutagenesis. Systemic delivery of mRNA allows endogenous production of the corresponding encoded protein. Thus, injection of erythropoietin-encoding mRNA in a lipid nanoparticle formulation resulted in increased erythropoiesis in mice and macaques. Here, we demonstrate that a single injection of in vitro transcribed B domain-deleted FVIII-encoding mRNA to FVIII-deficient mice enables endogenous production of pro-coagulant FVIII. Circulating FVIII:C levels above 5% of normal levels were maintained for up to 72 h, with an estimated half-life of FVIII production of 17.9 h, and corrected the bleeding phenotype in a tail clipping assay. The endogenously produced FVIII did however exhibit low specific activity and induced a potent neutralizing IgG response upon repeated administration of the mRNA. Our results suggest that the administration of mRNA is a plausible strategy for the endogenous production of proteins characterized by poor translational efficacy. The use of alternative mRNA delivery systems and improved FVIII-encoding mRNA should foster the production of functional molecules and reduce their immunogenicity.

Serous ovarian tumors: Immunohistochemical profiling as an aid to grading and understanding tumorigenesis.

BACKGROUND: The aim of this study was to evaluate the expression of p53, p16, Wilms tumor gene (WT1), and Mindbomb E3 Ubiquitin Protein Ligase 1 (MIB-1) index by immunohistochemical (IHC) staining in benign, low-grade, and high-grade serous ovarian tumors. METHODS: Forty-one cases of ovarian serous tumors were included in the study (benign serous tumor [n = 10], low-grade ovarian serous carcinoma [n = 8], and high-grade ovarian serous carcinoma [n = 23]). Expression of p53, p16, WT1, and MIB-1 by IHC was evaluated statistically with the grade of tumor. Semiquantitative scoring system for percentage (0-5) and intensity (1-3) of staining pattern was used to bring about objectivity. RESULTS: p53, p16, and WT1 showed significantly higher staining scores in ovarian serous carcinoma group than in the benign group (p < 0.05). However, p16 score was not significant in benign versus low-grade tumors. In the carcinoma group, the high-grade serous tumors showed significantly higher staining scores of p53, p16, and WT1 than the low-grade serous tumors (p < 0.05). Papillary serous tumors had comparatively lower p53 and WT1 scores for the same grade of tumor. MIB-1 scores were not significant. CONCLUSION: p53, p16, and WT1 are helpful for the subtyping of serous ovarian tumors as low grade and high grade. WT1 is helpful in establishing primary ovarian serous tumors. The combination of moderate-to-high p53 and WT1 scores provides a robust way of confirming high-grade tumors.

The contribution of cross-talk between the cell-surface proteins CD36 and CD47-TSP-1 in osteoclast formation and function.

Antibody-mediated blockade of cluster of differentiation 47 (CD47)-thrombospondin-1 (TSP-1) interactions blocks osteoclast formation in vitro and attenuates parathyroid hormone (PTH)-induced hypercalcemia in vivo in mice. Hypercalcemia in this model reflects increased bone resorption. TSP-1 has two cell-associated binding partners, CD47 and CD36. The roles of these two molecules in mediating the effects of TSP1 in osteoclasts are unclear. Osteoclast formation was attenuated but not absent when preosteoclasts isolated from CD47-/- mice were cocultured with WT osteoblasts. Suppressing CD36 in osteoclast progenitors also attenuated osteoclast formation. The hypercalcemic response to a PTH infusion was blunted in CD47-/-/CD36-/- (double knockout (DKO)) female mice but not CD47-/- mice or CD36-/- animals, supporting a role for both CD47 and CD36 in mediating this effect. Consistent with this, DKO osteoclasts had impaired resorptive activity when analyzed in vitro Inhibition of nitric oxide (NO) signaling is known to promote osteoclastogenesis, and TSP-1 suppresses NO production and signaling. An anti-TSP-1 antibody increased NO production in osteoclasts, and the inhibitory effect of anti-TSP-1 on osteoclastogenesis was completely rescued by l-nitroarginine methyl ester (l-NAME), a competitive NO synthase inhibitor. Supportive of an important role for CD36 in mediating the pro-osteoclastogenic effects of TSP-1, engaging CD36 with a synthetic agonist, p907, suppressed NO production in anti-TSP-1-treated cultures, allowing osteoclast maturation to occur. These results establish that CD36 and CD47 both participate in mediating the actions of TSP-1 in osteoclasts and establish a physiologically relevant cross-talk in bone tissue between these two molecules.

Prevention of the anti-factor VIII memory B-cell response by inhibition of Bruton tyrosine kinase in experimental hemophilia A.

Hemophilia A is a rare hemorrhagic disorder caused by the lack of functional pro-coagulant factor VIII. Factor VIII replacement therapy in patients with severe hemophilia A results in the development of inhibitory anti-factor VIII IgG in up to 30% of cases. To date, immune tolerance induction, with daily injection of large amounts of factor VIII, is the only strategy to eradicate factor VIII inhibitors. This strategy is, however, efficient in only 60-80% of patients. We investigated whether blocking B-cell receptor signaling upon inhibition of Bruton tyrosine kinase prevents anti-factor VIII immune responses in a mouse model of severe hemophilia A. Factor VIII-naïve and factor VIII-sensitized factor VIII-deficient mice were fed with the selective inhibitor of Bruton tyrosine kinase, (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)-1H pyrazole-4-carboxamide (PF-06250112), to inhibit B-cell receptor signaling prior to challenge with exogenous factor VIII. The consequences on the anti-factor VIII immune response were studied. Inhibition of Bruton tyrosine kinase during the primary anti-factor VIII immune response in factor VIII-naïve mice did not prevent the development of inhibitory anti-factor VIII IgG. In contrast, the anti-factor VIII memory B-cell response was consistently reduced upon treatment of factor VIII-sensitized mice with the Bruton tyrosine kinase inhibitor. The Bruton tyrosine kinase inhibitor reduced the differentiation of memory B cells ex vivo and in vivo following adoptive transfer to factor VIII-naïve animals. Taken together, our data identify inhibition of Bruton tyrosine kinase using PF-06250112 as a strategy to limit the reactivation of factor VIII-specific memory B cells upon re-challenge with therapeutic factor VIII.

Atomic-scale patterning in two-dimensional van der Waals superlattices.

Two-dimensional (2D) van der Waals superlattices comprised of two stacked monolayer materials have attracted significant interest as platforms for novel optoelectronic and structural behavior. Although studies are focused on superlattice fabrication, less effort has been given to the nanoscale patterning and structural modification of these systems. In this report, we demonstrate the localized layer-by-layer thinning and formation of nanopores/defects in 2D superlattices, such as stacked MoS2-WS2 van der Waals heterostructures and chemical vapor deposited bilayer WSe2, using aberration-corrected scanning transmission electron microscopy (STEM). Controlled electron beam irradiation is used to locally thin superlattices by removing the bottom layer of atoms, followed by defect formation through ablation of the second layer of atoms. The resulting defects exhibit atomically-sharp pore edges with tunable diameters down to 0.6 nm. Structural periodicities and focused STEM irradiation are also utilized to form close-packed nanopore arrays in superlattices with varying twist angles and commensurability. Applying these methods and mechanisms provides a forward approach in the atomic-scale patterning of stacked 2D nanodevices.

Thermodynamic stability contributes to immunoglobulin specificity.

Antigen-binding specificity of immunoglobulins is important for their function in immune defense. However, immune repertoires contain a considerable fraction of immunoglobulins with promiscuous binding behavior, the physicochemical basis of which is not well understood. Evolution of immunoglobulin specificity occurs through iterative processes of mutation and selection, referred to as affinity maturation. Recent studies reveal that some somatic mutations could compromise the thermodynamic stability of the variable regions of immunoglobulins. By integrating this observation with the wealth of data on the evolution of novel enzyme activities, we propose that antibody specificity is linked to the thermodynamic stability of the antigen-binding regions, which provides a quantitative distinction between highly specific and promiscuous antibodies.

Oxidation of factor VIII increases its immunogenicity in mice with severe hemophilia A.

The development of antibodies against therapeutic factor VIII (FVIII) represents the major complication of replacement therapy in patients with severe hemophilia A. Amongst the environmental risk factors that influence the anti-FVIII immune response, the presence of active bleeding or hemarthrosis has been evoked. Endothelium damage is typically associated with the release of oxidative compounds. Here, we addressed whether oxidation contributes to FVIII immunogenicity. The control with N-acetyl cysteine of the oxidative status in FVIII-deficient mice, a model of severe hemophilia A, reduced the immune response to exogenous FVIII. Ex vivo exposure of therapeutic FVIII to HOCl induced a mild oxidation of the molecule as evidenced by the loss of free amines and resulted in increased FVIII immunogenicity in vivo when compared to native FVIII. The increased immunogenicity of oxidized FVIII was not reverted by treatment of mice with N-acetyl cysteine, and did not implicate an increased maturation of professional antigen-presenting cells. Our data document that oxidation influences the immunogenicity of therapeutic FVIII.

Inhibitor Formation in Congenital Hemophilia A: an Immunological Perspective.

The immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A has been puzzling scientific and clinical communities for more than 3 decades. Indeed, the development of inhibitory antibodies to FVIII remains a major clinical challenge and is associated with enormous societal costs. Thus, the reasons for which a presumably innocuous, short-lived, intravenously administered glycoprotein triggers such a deleterious, long-lasting neutralizing immune response is an enigma. This review does not pretend to bring an answer to this challenging question. It will however summarize the latest findings regarding the molecular interactions at play in the recognition of FVIII by the immune cells, the validity of the proposed risk factors for FVIII alloimmunization, and the different solutions that allow induction of FVIII-specific tolerance in preclinical models of hemophilia A.

Complement C3 is a novel modulator of the anti-factor VIII immune response.

Development of neutralizing antibodies against therapeutic Factor VIII (FVIII) is the most serious complication of the treatment of hemophilia A. There is growing evidence to show the multifactorial origin of the anti-FVIII immune response, combining both genetic and environmental factors. While a role for the complement system on innate as well as adaptive immunity has been documented, the implication of complement activation on the onset of the anti-FVIII immune response is unknown. Here, using in vitro assays for FVIII endocytosis by human monocyte-derived dendritic cells and presentation to T cells, as well as in vivo complement depletion in FVIII-deficient mice, we show a novel role for complement C3 in enhancing the immune response against therapeutic FVIII. In vitro, complement C3 and its cleavage product C3b enhanced FVIII endocytosis by dendritic cells and presentation to a FVIII-specific CD4+ T-cell hybridoma. The C1 domain of FVIII had previously been shown to play an important role in FVIII endocytosis, and alanine substitutions of the K2092, F2093 and R2090 C1 residues drastically reduce FVIII uptake in vitro Interestingly, complement activation rescued the endocytosis of the FVIII C1 domain triple mutant. In a mouse model of severe hemophilia A, transient complement C3 depletion by humanized cobra venom factor, which does not generate anaphylatoxin C5a, significantly reduced the primary anti-FVIII immune response, but did not affect anti-FVIII recall immune responses. Taken together, our results suggest an important adjuvant role for the complement cascade in the initiation of the immune response to therapeutic FVIII.

Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.

The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells.

The development of inhibitory antibodies to therapeutic factor VIII is the major complication of replacement therapy in patients with hemophilia A. The first step in the initiation of the anti-factor VIII immune response is factor VIII interaction with receptor(s) on antigen-presenting cells, followed by endocytosis and presentation to naïve CD4+ T cells. Recent studies indicate a role for the C1 domain in factor VIII uptake. We investigated whether charged residues in the C2 domain participate in immunogenic factor VIII uptake. Co-incubation of factor VIII with BO2C11, a monoclonal C2-specific immunoglobulin G, reduced factor VIII endocytosis by dendritic cells and presentation to CD4+ T cells, and diminished factor VIII immunogenicity in factor VIII-deficient mice. The mutation of basic residues within the BO2C11 epitope of C2 replicated reduced in vitro immunogenic uptake, but failed to prevent factor VIII immunogenicity in mice. BO2C11 prevents factor VIII binding to von Willebrand factor, thus potentially biasing factor VIII immunogenicity by perturbing its half-life. Interestingly, a factor VIIIY1680C mutant, that does not bind von Willebrand factor, demonstrated unaltered endocytosis by dendritic cells as well as immunogenicity in factor VIII-deficient mice. Co-incubation of factor VIIIY1680C with BO2C11, however, resulted in decreased factor VIII immunogenicity in vivo In addition, a previously described triple C1 mutant showed decreased uptake in vitro, and reduced immunogenicity in vivo, but only in the absence of endogenous von Willebrand factor. Taken together, the results indicate that residues in the C1 and/or C2 domains of factor VIII are implicated in immunogenic factor VIII uptake, at least in vitro Conversely, in vivo, the binding to endogenous von Willebrand factor masks the reducing effect of mutations in the C domains on factor VIII immunogenicity.

Topical vanadate enhances the repair of median laparotomy incisions.

BACKGROUND: There are over two million laparotomies performed in the United States each year with an incisional hernia rate between 2% and 11%. A total of 100,000 ventral hernia repairs are undertaken each year with recurrences as high as 50%. MATERIALS AND METHODS: Full thickness midline fascia incisions from the xiphoid to the pubic symphysis were made in rats. The fascia and/or muscular layer was sutured closed and a gel with 300 µM of sodium orthovanadate or saline was placed over the suture line with the skin closed over it. On day 10, 1-cm strips from the superior, middle, and inferior regions of the abdominal wall were tested for breaking strength and processed for histology. RESULTS: The mean wound breaking strength of vanadate-treated wounds was 18.6 ± 2.7 N compared with 9.4 ± 3.6 N for controls (P < 0.0001). Similar quantities of granulation tissue were deposited in treated and control wounds. Fine green birefringence patterns, characteristic of immature connective tissue, were seen in control samples viewed with polarized light. In contrast, vanadate-treated wounds showed thick yellow-orange birefringence patterns characteristics of more mature connective tissue. Using α-smooth muscle actin immunostaining, myofibroblasts were prominent in control incisions, but few were identified in vanadate-treated incisions. CONCLUSIONS: In rat laparotomy wounds, a single application of vanadate increases wound breaking strength, through enhanced connective tissue organization. These combined data suggest topical application of vanadate immediately after fascial closure will increase wound strength, possibly reducing hernia recurrences in the repaired abdominal wall.