In Vivo Optical Characterization of Middle Ear Effusions and Biofilms During Otitis Media.

Otitis media (OM), a common ear infection, is characterized by the presence of an accumulated middle ear effusion (MEE) in a normally air-filled middle ear cavity. While assessing the MEE plays a critical role in the overall management of OM, identifying and examining the MEE is challenging with the current diagnostic tools since the MEE is located behind the semi-opaque eardrum. The objective of this cross-sectional, observational study is to non-invasively visualize and characterize MEEs and bacterial biofilms in the middle ear. A portable, handheld, otoscope-integrated optical coherence tomography (OCT) system combined with novel analytical methods has been developed. In vivo middle ear OCT images were acquired from 53 pediatric subjects (average age of 3.9 years; all awake during OCT imaging) diagnosed with OM and undergoing a surgical procedure (ear tube surgery) to aspirate the MEE and aerate the middle ear. In vivo middle ear OCT acquired prior to the surgery was compared with OCT of the freshly extracted MEEs, clinical diagnosis, and post-operative evaluations. Among the subjects who were identified with the presence of MEEs, 89.6% showed the presence of the TM-adherent biofilm in in vivo OCT. This study provides an atlas of middle ear OCT images exhibiting a range of depth-resolved MEE features, which can only be visualized and assessed non-invasively through OCT. Quantitative metrics of OCT images acquired prior to the surgery were statistically correlated with surgical evaluations of MEEs. Measurements of MEE characteristics will provide new readily available information that can lead to improved diagnosis and management strategies for the highly prevalent OM in children.

Versatility of mesenchymal stem cell-derived extracellular vesicles in tissue repair and regenerative applications.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic cells that have been widely explored in the field of regenerative medicine. MSCs possess the ability to secrete soluble factors as well as lipid bound extracellular vesicles (EVs). MSCs have gained increased interest and attention as a result of their therapeutic properties, which are thought to be attributed to their secretome. However, while the use of MSCs as whole cells pose heterogeneity concerns and survival issues post-transplantation, such limitations are absent in cell-free EV-based treatments. EVs derived from MSCs are promising therapeutic agents for a range of clinical conditions and disorders owing to their immunomodulatory, pro-regenerative, anti-inflammatory, and antifibrotic activity. Recent successes with preclinical studies using EVs for repair and regeneration of damaged tissues such as cardiac tissue, lung, liver, pancreas, bone, skin, cornea, and blood diseases are discussed in this review. We also discuss delivery strategies of EVs using biomaterials as delivery vehicles through systemic or local administration. Despite its effectiveness in preclinical investigations, the application of MSC-EV in clinical settings will necessitate careful consideration surrounding issues such as: i) scalability and isolation, ii) biodistribution, iii) targeting specific tissues, iv) quantification and characterization, and v) safety and efficacy of dosage. The future of EVs in regenerative medicine is promising yet still needs further investigation on enhancing the efficacy, scalability, and potency for clinical applications.

Zanubrutinib in patients with previously treated B-cell malignancies intolerant of previous Bruton tyrosine kinase inhibitors in the USA: a phase 2, open-label, single-arm study.

BACKGROUND: We hypothesised that zanubrutinib, a highly selective next-generation Bruton tyrosine kinase (BTK) inhibitor, would be a safe and active treatment for patients intolerant of ibrutinib, acalabrutinib, or both. We aimed to assess whether zanubrutinib would prolong treatment duration by minimising treatment-related toxicities and discontinuations in patients with previously treated B-cell malignancies. METHODS: This ongoing, phase 2, multicentre, open-label, single-arm study was done in 20 centres in the USA. Patients aged 18 or older with previously treated B-cell malignancies (chronic lymphocytic leukaemia, small lymphocytic lymphoma, mantle cell lymphoma, Waldenström macroglobulinaemia, or marginal zone lymphoma) who became intolerant of ibrutinib, acalabrutinib, or both, were orally administered zanubrutinib 160 mg twice daily or 320 mg once daily per investigator. The primary endpoint was recurrence and change in severity of ibrutinib or acalabrutinib intolerance events based on investigator-assessed adverse events. Secondary endpoints were investigator-assessed overall response rate; duration of response; disease control rate; and progression-free survival. Analyses included all patients who received any dose of the study drug. This study is registered with ClinicalTrials.gov, NCT04116437. FINDINGS: Between Oct 14, 2019, and Sept 8, 2021, 67 patients (36 [54%] men and 31 [46%] women) who were intolerant of ibrutinib (n=57; cohort 1) or of acalabrutinib or acalabrutinib and ibrutinib (n=10; cohort 2) were enrolled. 63 (94%) patients were White, one (2%) had multiple ethnicities, and three (5%) had unreported or unknown ethnicity. Most intolerance events (81 [70%] of 115 for ibrutinib; 15 [83%] of 18 for acalabrutinib) did not recur with zanubrutinib. Of the recurring events, seven (21%) of 34 ibrutinib intolerance events and two (67%) of three acalabrutinib intolerance events recurred at the same severity with zanubrutinib; 27 (79%) ibrutinib intolerance events and one (33%) acalabrutinib intolerance event recurred at a lower severity with zanubrutinib. No events recurred at higher severity. No grade 4 intolerance events recurred. 64 (96%) of 67 patients had one or more adverse events with zanubrutinib; the most common adverse events were contusion (in 15 [22%] of 67 patients), fatigue (14 [21%]), myalgia (ten [15%]), arthralgia (nine [13%]), and diarrhoea (nine [13%]). Atrial fibrillation occurred in three (4%) patients (all grade 2). Eight (12%) of 67 patients had serious adverse events (anaemia, atrial fibrillation, bronchitis, COVID-19, COVID-19 pneumonia, febrile neutropenia, salmonella gastroenteritis, transfusion reaction, trigeminal nerve disorder, and urinary tract infection). No treatment-related deaths occurred. The median follow-up time was 12·0 months (IQR 8·2-15·6). Among the 64 efficacy-evaluable patients, disease control rate was 93·8% (60; 95% CI 84·8-98·3) and overall response rate was 64·1% (41; 95% CI 51·1-75·7). The median duration of response was not reached; the 12-month event-free duration of response rate was 95·0% (95% CI 69·5-99·3). Similarly, median progression-free survival was not reached; 18-month progression-free survival was 83·8% (95% CI 62·6-93·6). INTERPRETATION: Patients intolerant of previous BTK inhibitors have limited treatment options. These results suggest that zanubrutinib, a safe and viable treatment for patients with B-cell malignancies, might fill that unmet need for those who exhibit intolerance to ibrutinib or acalabrutinib. FUNDING: BeiGene.

Specific, Sensitive, and Stable Reporting of Human Mesenchymal Stromal Cell Chondrogenesis.

IMPACT STATEMENT: The promoter characterized in this study has been made accessible as a resource for the skeletal tissue engineering and regenerative medicine community. When combined with suitable reporter vectors, the resulting tools can be used for noninvasive and/or high-throughput screening of test conditions for stimulating chondrogenesis by candidate stem/progenitor cells. As demonstrated in this study, they can also be used with small animal imaging platforms to monitor the chondrogenic activity of implanted progenitors within orthotopic models of bone and cartilage repair.

A Phase II Study of Pemetrexed in Patients with Recurrent Thymoma and Thymic Carcinoma.

INTRODUCTION: Thymoma and thymic carcinoma (TC) are neoplastic diseases with reported chemosensitivity to a broad range of agents. However, because of the rarity of these diseases, few prospective trials have been conducted in patients with advanced thymic malignancies. We conducted a prospective phase II trial to evaluate the clinical activity of pemetrexed, a multitargeted antifolate agent, in previously treated patients with thymoma and TC. METHODS: A total of 27 previously treated patients (16 with thymoma and 11 with TC) with advanced, unresectable disease were treated with pemetrexed, 500 mg/m2, intravenously every 3 weeks for a maximum of six cycles or until undue toxicity or progressive disease. All patients received folic acid, vitamin B12, and steroid prophylaxis. RESULTS: The median number of cycles administered was 6 (range 1-6). Nine patients with a total of 14 events had grade 3 toxicities; no grade 4 toxicities were noted. In 26 fully evaluable patients, two complete and three partial responses (according to the Response Evaluation Criteria in Solid Tumors) were documented (all in patients with stage IVA thymoma, except for one partial response with stage IVA TC). A total of 14 patients completed the full six cycles of treatment, 7 patients progressed while undergoing therapy, 5 patients discontinued therapy because of intolerance, and 1 patient discontinued therapy because of progressive Morvan syndrome. The median progression-free survival time for all patients was 10.6 months (12.1 months for those with thymoma versus 2.9 months for those with TC). With 23 deaths at data cutoff, the median overall survival time was 28.7 months (46.4 months for those with thymoma versus 9.8 months for those with TC). CONCLUSIONS: Pemetrexed is an active agent in this heavily pretreated population of patients with recurrent thymic malignancies, especially thymoma.

Direct Analysis of Pathogenic Structures Affixed to the Tympanic Membrane during Chronic Otitis Media.

Objective To characterize otitis media-associated structures affixed to the mucosal surface of the tympanic membrane (TM) in vivo and in surgically recovered in vitro samples. Study Design Prospective case series without comparison. Setting Outpatient surgical care center. Subjects and Methods Forty pediatric subjects scheduled for tympanostomy tube placement surgery were imaged intraoperatively under general anesthesia. Postmyringotomy, a portable optical coherence tomography (OCT) imaging system assessed for the presence of any biofilm affixed to the mucosal surface of the TM. Samples of suspected microbial infection-related structures were collected through the myringotomy incision. The sampled site was subsequently reimaged with OCT to confirm collection from the original image site on the TM. In vitro analysis based on confocal laser scanning microscope (CLSM) images of fluorescence in situ hybridization-tagged samples and polymerase chain reaction (PCR) provided microbiological characterization and verification of biofilm activity. Results OCT imaging was achieved for 38 of 40 subjects (95%). Images from 38 of 38 (100%) of subjects observed with OCT showed the presence of additional microbial infection-related structures. Thirty-four samples were collected from these 38 subjects. CLSM images provided evidence of clustered bacteria in 32 of 33 (97%) of samples. PCR detected the presence of active bacterial DNA signatures in 20 of 31 (65%) of samples. Conclusion PCR and CLSM analysis of fluorescence in situ hybridization-stained samples validates the presence of active bacteria that have formed into a middle ear biofilm that extends across the mucosal layer of the TM. OCT can rapidly and noninvasively identify middle ear biofilms in subjects with severe and persistent cases of otitis media.

Contribution of Implanted, Genetically Modified Muscle Progenitor Cells Expressing BMP-2 to New Bone Formation in a Rat Osseous Defect.

Because muscle contains osteoprogenitor cells and has a propensity to form bone, we have explored its utility in healing large osseous defects. Healing is achieved by the insertion of muscle fragments transduced with adenovirus encoding BMP-2 (Ad.BMP-2). However, it is not known whether the genetically modified muscle contributes osteoprogenitor cells to healing defects or merely serves as a local source of BMP-2. This question is part of the larger debate on the fate of progenitor cells introduced into sites of tissue damage to promote regeneration. To address this issue, we harvested fragments of muscle from rats constitutively expressing GFP, transduced them with Ad.BMP-2, and implanted them into femoral defects in wild-type rats under various conditions. GFP+ cells persisted within defects for the entire 8 weeks of the experiments. In the absence of bone formation, these cells presented as fibroblasts. When bone was formed, GFP+ cells were present as osteoblasts and osteocytes and also among the lining cells of new blood vessels. The genetically modified muscle thus contributed progenitor cells as well as BMP-2 to the healing defect, a property of great significance in light of the extensive damage to soft tissue and consequent loss of endogenous progenitors in problematic fractures.

Noninvasive in vivo optical coherence tomography tracking of chronic otitis media in pediatric subjects after surgical intervention.

In an institutional review board-approved study, 25 pediatric subjects diagnosed with chronic or recurrent otitis media were observed over a period of six months with optical coherence tomography (OCT). Subjects were followed throughout their treatment at the initial patient evaluation and preoperative consultation, surgery (intraoperative imaging), and postoperative follow-up, followed by an additional six months of records-based observation. At each time point, the tympanic membrane (at the light reflex region) and directly adjacent middle-ear cavity were observed in vivo with a handheld OCT probe and portable system. Imaging results were compared with clinical outcomes to correlate the clearance of symptoms in relation to changes in the image-based features of infection. OCT images of most all participants showed the presence of additional infection-related biofilm structures during their initial consultation visit and similarly for subjects imaged intraoperatively before myringotomy. Subjects with successful treatment (no recurrence of infectious symptoms) had no additional structures visible in OCT images during the postoperative visit. OCT image findings suggest surgical intervention consisting of myringotomy and tympanostomy tube placement provides a means to clear the middle ear of infection-related components, including middle-ear fluid and biofilms. Furthermore, OCT was demonstrated as a rapid diagnostic tool to prospectively monitor patients in both outpatient and surgical settings.

A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas.

BACKGROUND: Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing. METHODS: The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used. RESULTS: Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability. CONCLUSIONS: A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (http://clinicaltrials.gov/ct2/show/NCT02220855).

TNFα and IL-1ß influence the differentiation and migration of murine MSCs independently of the NF-κB pathway.

INTRODUCTION: Mesenchymal stem cells (MSCs) have the ability to repair and regenerate tissue, home to sites of inflammation, and evade the host immune system. As such, they represent an attractive therapy for the treatment of autoimmune inflammatory diseases. However, results from in vivo murine studies in inflammatory arthritis have been conflicting, and this may be due to the genetic background of the MSCs used. It is known that the inflammatory milieu may influence properties of MSCs and that, in the case of human bone marrow-derived MSCs, this may be mediated by the nuclear factor-kappa-B (NF-κB) pathway. We sought to determine whether pro-inflammatory cytokines altered the differentiation and migration capacity of murine MSCs from different mouse strains and whether this was mediated by NF-κB. METHODS: The differentiation and migration of FVB and BALB/c MSCs were carried out in the presence of varying concentrations of tumor necrosis factor-alpha (TNFα) and interleukin (IL)-1ß, and the NF-κB pathway was inhibited in one of two ways: either by transduction of MSCs with an adenoviral vector expressing a super-repressor of NF-κB or by the addition of curcumin to culture media. RESULTS: Both BALB/c and FVB MSCs were sensitive to the effect of pro-inflammatory cytokines in vitro. TNFα and IL-1ß suppressed BALB/c osteogenesis and adipogenesis and FVB osteogenesis. The migration of both cell types toward media containing fetal bovine serum was augmented by pre-stimulation with either cytokine. In neither cell type were the cytokine effects reversed by abrogation of the NF-κB pathway. CONCLUSIONS: These data show that murine MSCs from different genetic backgrounds may be influenced by an inflammatory milieu in a manner that is not mediated by NF-κB, as is the case for human MSCs. This is not mediated by NF-κB. These findings are important and should influence how in vivo trials of murine MSCs are interpreted and the future development of pre-clinical studies in inflammatory diseases.

The effect of resistance exercise on all-cause mortality in cancer survivors.

OBJECTIVE: To examine the independent associations of leisure-time aerobic physical activity (PA) and resistance exercise (RE) on all-cause mortality in cancer survivors. PATIENTS AND METHODS: Patients included 2863 male and female cancer survivors, aged 18 to 81 years, who received a preventive medical examination between April 8, 1987, and December 27, 2002, while enrolled in the Aerobics Center Longitudinal Study in Dallas, Texas. Physical activity and RE were assessed by self-report at the baseline medical examination. Cox proportional hazards regression analysis was performed to determine the independent associations of PA and RE with all-cause mortality in participants who had a history of cancer. RESULTS: Physical activity in cancer survivors was not associated with a lower risk of all-cause mortality. In contrast, RE was associated with a 33% lower risk of all-cause mortality (95% CI, 0.45-0.99) after adjusting for potential confounders, including PA. CONCLUSION: Individuals who participated in RE during cancer survival had a lower risk for all-cause mortality. The present findings provide preliminary evidence for benefits of RE during cancer survival. Future randomized controlled trials examining RE and its effect on lean body mass, muscular strength, and all-cause mortality in cancer survivors are warranted.

Inflammatory cytokines induce a unique mineralizing phenotype in mesenchymal stem cells derived from human bone marrow.

Bone marrow contains mesenchymal stem cells (MSCs) that can differentiate along multiple mesenchymal lineages. In this capacity they are thought to be important in the intrinsic turnover and repair of connective tissues while also serving as a basis for tissue engineering and regenerative medicine. However, little is known of the biological responses of human MSCs to inflammatory conditions. When cultured with IL-1ß, marrow-derived MSCs from 8 of 10 human subjects deposited copious hydroxyapatite, in which authenticity was confirmed by Fourier transform infrared spectroscopy. Transmission electron microscopy revealed the production of fine needles of hydroxyapatite in conjunction with matrix vesicles. Alkaline phosphatase activity did not increase in response to inflammatory mediators, but PPi production fell, reflecting lower ectonucleotide pyrophosphatase activity in cells and matrix vesicles. Because PPi is the major physiological inhibitor of mineralization, its decline generated permissive conditions for hydroxyapatite formation. This is in contrast to MSCs treated with dexamethasone, where PPi levels did not fall and mineralization was fuelled by a large and rapid increase in alkaline phosphatase activity. Bone sialoprotein was the only osteoblast marker strongly induced by IL-1ß; thus these cells do not become osteoblasts despite depositing abundant mineral. RT-PCR did not detect transcripts indicative of alternative mesenchymal lineages, including chondrocytes, myoblasts, adipocytes, ligament, tendon, or vascular smooth muscle cells. IL-1ß phosphorylated multiple MAPKs and activated nuclear factor-κB (NF-κB). Certain inhibitors of MAPK and PI3K, but not NF-κB, prevented mineralization. The findings are of importance to soft tissue mineralization, tissue engineering, and regenerative medicine.

Recurrent post-tympanostomy tube otorrhea secondary to aerobic endospore-forming bacilli: a case report and brief literature review.

We report a unique case in which a 57-year-old man with an 8-year history of late recurrent post-tympanostomy tube otorrhea (PTTO) was found to harbor Bacillus subtilis, an aerobic endospore-forming bacillus that is typically resistant to chemical and physical agents because of its unique life cycle. Removal of the patient's tympanostomy tube resulted in complete resolution of his long-standing otorrhea. We also review the etiologies of and treatment strategies for early, late, chronic, and recurrent PTTO. We conclude that regardless of the etiology, a patient with persistent or recurrent PTTO should consider undergoing removal of the ventilation tube.

Improved facial nerve outcomes using an evolving treatment method for large acoustic neuromas.

OBJECTIVE: To describe a successful paradigm for the treatment of large acoustic neuromas (vestibular schwannomas). STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: The charts of 2,875 acoustic neuroma patients at Michigan Ear Institute were reviewed to identify 153 patients who underwent surgical resection for large acoustic neuromas (>=3 cm) between 2000 and 2009. INTERVENTION(S): Staged surgical resection or single stage surgery with or without adjuvant stereotactic radiosurgery. MAIN OUTCOME MEASURE(S): Postoperative facial nerve outcomes are reported using the House-Brackmann (HB) facial nerve grading scale and compared with historical controls from a literature review. Rates of adverse outcomes are also reported. RESULTS: Seventy-five patients underwent staged surgical resection of their tumors, whereas 78 patients underwent either single stage surgery or surgery with subsequent stereotactic radiosurgery. Eighty-one percent of patients in the staged surgical resection group had a postoperative HB Grade I or II facial nerve function compared with 75% in the single stage surgical group. Overall, 78% of patients in the current study had HB Grade I or II after treatment compared with a mean of 53% in the literature for similar sized tumors. Our methods including the decision to use staged surgery when necessary, dissection of tumor with stimulating dissector-directed intraoperative monitoring, and use of adjuvant stereotactic radiosurgery are described. CONCLUSION: Using the described paradigm, large acoustic neuromas can be successfully treated with either staged or single-stage surgical resection with or without adjuvant radiosurgery to obtain more favorable facial nerve outcomes than historically reported controls while minimizing morbidity for the patient.

EWS-FLI-1-targeted cytotoxic T-cell killing of multiple tumor types belonging to the Ewing sarcoma family of tumors.

PURPOSE: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types. DESIGN: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. RESULTS: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. CONCLUSIONS: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.

Genetic mismatch affects the immunosuppressive properties of mesenchymal stem cells in vitro and their ability to influence the course of collagen-induced arthritis.

INTRODUCTION: The immunological and homing properties of mesenchymal stem cells (MSCs) provide a potentially attractive treatment for arthritis. The objective of this study was to determine effects of genetic disparity on the immunosuppressive potential of MSCs in vitro and in vivo within collagen induced arthritis (CIA). METHODS: The ability of DBA/1, FVB and BALB/c MSC preparations to impact the cytokine release profile of CD3/CD28 stimulated DBA/1 T cells was assessed in vitro. The effect of systemically delivered MSCs on the progression of CIA and cytokine production was assessed in vivo. RESULTS: All MSC preparations suppressed the release of TNFα and augmented the secretion of IL-4 and IL-10 by stimulated DBA/1 T-cells. However, assessment of the ratio of IFNγ to IL-4 production indicated that the more genetically distant BALB/c MSCs had significantly less immunosuppressive capacity. Systemic delivery of BALB/c MSC resulted in an exacerbation of CIA disease score in vivo and a higher erosive disease burden. This was not seen after treatment with syngeneic or partially mismatched MSCs. An increase in serum levels of IL-1ß was observed up to 20 days post treatment with allogeneic MSCs. An initial elevation of IL-17 in these treatment groups persisted in those treated with fully mismatched BALB/c MSCs. Over the course of the study, there was a significant suppression of serum IL-17 levels in groups treated with syngeneic MSCs. CONCLUSIONS: These data demonstrate a significant difference in the immunosuppressive properties of syngeneic and allogeneic MSCs in vitro and in vivo, which needs to be appreciated when developing MSC based therapies for inflammatory arthritis.

Improvement of tendon repair using muscle grafts transduced with TGF-ß1 cDNA.

Tendon rupture is a common injury. Inadequate endogenous repair often leaves patients symptomatic, with tendons susceptible to re-rupture. Administration of certain growth factors improves tendon healing in animal models, but their delivery remains a challenge. Here we evaluated the delivery of TGF-ß1 to tendon defects by the implantation of genetically modified muscle grafts. Rat muscle biopsies were transduced with recombinant adenovirus encoding TGF-ß1 and grafted onto surgically transected Achilles tendons in recipient animals. Tissue regenerates were compared to those of controls by biomechanical testing as well as histochemical and immunohistochemical analyses. Healing was greatly accelerated when genetically modified grafts were implanted into tendon defects, with the resulting repair tissue gaining nearly normal histological appearance as early as 2 weeks postoperatively. This was associated with decreased deposition of type III collagen in favour of large fibre bundles indicative of type I collagen. These differences in tendon composition coincided with accelerated restoration of mechanical strength. Tendon thickness increased in gene-treated animals at weeks 1 and 2, but by week 8 became significantly lower than that of controls suggesting accelerated remodelling. Thus localised TGF-ß1 delivery via adenovirus-modified muscle grafts improved tendon healing in this rat model and holds promise for clinical application.

Evaluation of BMP-2 gene-activated muscle grafts for cranial defect repair.

Large, osseous, segmental defects heal poorly. Muscle has a propensity to form bone when exposed to an osteogenic stimulus such as that provided by transfer and expression of cDNA encoding bone morphogenetic protein-2 (BMP-2). The present study evaluated the ability of genetically modified, autologous muscle to heal large cranial defects in rats. Autologous grafts (8 mm × 2 mm) were punched from the biceps femoris muscle and transduced intraoperatively with recombinant adenovirus vector containing human BMP-2 or green fluorescent protein cDNA. While the muscle biopsies were incubating with the vector, a central parietal 8 mm defect was surgically created in the calvarium of the same animal. The gene-activated muscle graft was then implanted into the cranial defect. After 8 weeks, crania were examined radiographically, histologically, and by micro-computed tomography and dual energy X-ray absorptiometry. Although none of the defects were completely healed in this time, muscle grafts expressing BMP-2 deposited more than twice as much new bone as controls. Histology confirmed the anatomical integrity of the newly formed bone, which was comparable in thickness and mineral density to the original cranial bone. This study confirms the in vivo osteogenic properties of genetically modified muscle and suggests novel strategies for healing bone.

Analysis of intersegmental trough and proximal latency of smooth muscle contraction using high-resolution esophageal manometry.

BACKGROUND AND AIMS: Intersegmental troughs (ISTs) between striated and smooth muscle contraction segments on high-resolution manometry (HRM) have been linked to hypomotility disorders. We investigated the relationship between ISTs, latency of initiation of smooth muscle contraction, and motor patterns in symptomatic patients and normal controls. METHODS: HRM Clouse plots were analyzed in 199 participants (47.2±1.2 y, 112F/87M), categorized into 110 participants with gastroesophageal reflux disease (GERD), 74 symptomatic participants without GERD, and 15 healthy controls. IST length was measured in centimeters and percentage esophageal length, designated extended when ≥20% esophageal length on >30% swallows. Proximal latency was measured as the time interval between onset of skeletal and smooth muscle contraction segments, and designated prolonged when ≥4s in ≥50% of swallows. RESULTS: ISTs of any length were noted in 74.6% swallows and in 92.5% of participants, with a similar frequency across the 3 groups. ISTs and proximal latency were both longer in the GERD group, especially when Barrett esophagus was present, compared with non-GERD patients or controls (P≤0.03 across groups); extended IST and prolonged proximal latency followed similar trends. On multivariate logistic regression, extended IST predicted GERD [odds ratio (OR), 2.30; 95% confidence intervals (CI) 1.18-4.47], as did lower esophageal sphincter pressure <5 mm Hg (OR, 3.79-3.96; 95% CI 1.77-8.49), after controlling for age and sex; prolonged proximal latency predicted both GERD (OR, 2.03; 95% CI 1.01-4.12) and Barrett esophagus (OR 1.91, 95% CI 1.24-2.94). CONCLUSIONS: Measurement of IST and proximal latency add value to HRM analysis, and may be markers of esophageal hypomotility.