Co-Culture of Plant Beneficial Microbes as Source of Bioactive Metabolites.

In microbial cultures the production of secondary metabolites is affected by experimental conditions, and the discovery of novel compounds is often prevented by the re-isolation of known metabolites. To limit this, it is possible to cultivate microorganisms by simulating naturally occurring interactions, where microbes co-exist in complex communities. In this work, co-culturing experiments of the biocontrol agent Trichoderma harzianum M10 and the endophyte Talaromyces pinophilus F36CF have been performed to elicit the expression of genes which are not transcribed in standard laboratory assays. Metabolomic analysis revealed that the co-culture induced the accumulation of siderophores for both fungi, while production of M10 harzianic and iso-harzianic acids was not affected by F36CF. Conversely, metabolites of the latter strain, 3-O-methylfunicone and herquline B, were less abundant when M10 was present. A novel compound, hereby named harziaphilic acid, was isolated from fungal co-cultures, and fully characterized. Moreover, harzianic and harziaphilic acids did not affect viability of colorectal cancer and healthy colonic epithelial cells, but selectively reduced cancer cell proliferation. Our results demonstrated that the co-cultivation of plant beneficial fungi may represent an effective strategy to modulate the production of bioactive metabolites and possibly identify novel compounds.

Secondary metabolites from the endophytic fungus Talaromyces pinophilus.

Endophytic fungi have a great influence on plant health and growth, and are an important source of bioactive natural compounds. Organic extracts obtained from the culture filtrate of an endophytic strain of Talaromyces pinophilus isolated from strawberry tree (Arbutus unedo) were studied. Metabolomic analysis revealed the presence of three bioactive metabolites, the siderophore ferrirubin, the platelet-aggregation inhibitor herquline B and the antibiotic 3-O-methylfunicone. The latter was the major metabolite produced by this strain and displayed toxic effects against the pea aphid Acyrthosiphon pisum (Homoptera Aphidiidae). This toxicity represents an additional indication that the widespread endophytic occurrence of T. pinophilus may be related to a possible role in defensive mutualism. Moreover, the toxic activity on aphids could promote further study on 3-O-methylfunicone, or its derivatives, as an alternative to synthetic chemicals in agriculture.

Chronic recording of hand prosthesis control signals via a regenerative peripheral nerve interface in a rhesus macaque.

OBJECTIVE: Loss of even part of the upper limb is a devastating injury. In order to fully restore natural function when lacking sufficient residual musculature, it is necessary to record directly from peripheral nerves. However, current approaches must make trade-offs between signal quality and longevity which limit their clinical potential. To address this issue, we have developed the regenerative peripheral nerve interface (RPNI) and tested its use in non-human primates. APPROACH: The RPNI consists of a small, autologous partial muscle graft reinnervated by a transected peripheral nerve branch. After reinnervation, the graft acts as a bioamplifier for descending motor commands in the nerve, enabling long-term recording of high signal-to-noise ratio (SNR), functionally-specific electromyographic (EMG) signals. We implanted nine RPNIs on separate branches of the median and radial nerves in two rhesus macaques who were trained to perform cued finger movements. MAIN RESULTS: No adverse events were noted in either monkey, and we recorded normal EMG with high SNR (>8) from the RPNIs for up to 20 months post-implantation. Using RPNI signals recorded during the behavioral task, we were able to classify each monkey's finger movements as flexion, extension, or rest with >96% accuracy. RPNI signals also enabled functional prosthetic control, allowing the monkeys to perform the same behavioral task equally well with either physical finger movements or RPNI-based movement classifications. SIGNIFICANCE: The RPNI signal strength, stability, and longevity demonstrated here represents a promising method for controlling advanced prosthetic limbs and fully restoring natural movement.

Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

Enhanced oncolytic potency of vesicular stomatitis virus through vector-mediated inhibition of NK and NKT cells.

Recombinant oncolytic viruses represent a promising alternative option for the treatment of malignant cancers. We have reported earlier the safety and efficacy of recombinant vesicular stomatitis virus (VSV) vectors in a rat model of hepatocellular carcinoma (HCC). However, the full potential of VSV therapy is limited by a sudden decline in intratumoral virus replication observed early after viral administration, a phenomenon that coincides with an accumulation of inflammatory cells within infected lesions. To overcome the antiviral function of these cells, we present a recombinant virus, rVSV-UL141, which expresses a protein from human cytomegalovirus known to downregulate the natural killer (NK) cell-activating ligand CD155. The modified vector resulted in an inhibition of NK cell recruitment in vitro, as well as decreased intratumoral accumulations of NK and NKT cells in vivo. Administration of rVSV-UL141 through hepatic artery infusion in immune-competent Buffalo rats harboring orthotopic, multi-focal HCC lesions resulted in a one-log elevation of intratumoral virus replication over a control rVSV vector, which translated to enhance tumor necrosis and substantial prolongation of survival. Moreover, these results were achieved in the absence of apparent toxicities. The present study suggests the applicability of this strategy for the development of effective and safe oncolytic agents to treat multi-focal HCC, and potentially a multitude of other cancers, in the future.

Efficient infection of tumor endothelial cells by a capsid-modified adenovirus.

Targeted antiangiogenic gene therapy is an attractive approach to treat metastatic cancer. However, the relative paucity of the receptors of the commonly used adenovirus serotype 5 in endothelial cells as compared with liver cells undermines the use of this vector for targeting the endothelial cells in tumors. To overcome this problem, we analyzed the ability of a hybrid Ad5/35 virus, where the serotype 5 fiber has been replaced with the fiber from serotype 35, to target tumor vasculature. Infection of human umbilical vein endothelial cells (HUVECs) with Ad5/35 at MOI 120 infected 100% of cells. In contrast, infection with Ad5 at the same MOI infected only 10% HUVECs. Ad5/35 was even more effective in transducing human aortic endothelial cells (HAECs), as infection with Ad5/35 at MOI 3.6 was sufficient to transduce 95% of cells. Gene expression analyses demonstrated that infection of HUVECs and HAECs with Ad5/35 resulted in between 1 and 3 orders of magnitude higher gene expression than infection with Ad5. Furthermore, various liver-derived cells were less infectable with Ad5/35 than Ad5, indicating a favorable toxicity profile for this virus. In a rat colon carcinoma tumor model, Ad5 was located mainly in the liver parenchyma after hepatic artery administration. In contrast, Ad5/35 was found only in the angiogenesis-rich border region of the tumor. Double immunostaining revealed that Ad5/35 colocalized with CD31 and Flk-1 positive endothelial cells. These results indicate that Ad5/35 may be useful in anticancer strategies targeting tumor endothelial cells.

The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants.

ABSTRACT Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations today available as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex, three-way cross-talk between the fungal antagonist, the plant, and microbial pathogens. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced resistance, proteins responsible for a gene-for-gene avirulent interaction between Trichoderma spp. and plants, mycoparasitism-related inducers, plant proteins specifically induced by Trichoderma, etc. We have transgenically demonstrated the ability of Trichoderma spp. to transfer heterologous proteins into plant during root colonization, and have used green fluorescent protein and other markers to study the interaction in vivo and in situ between Trichoderma spp. and the fungal pathogen or the plant.

The systemic administration of Ig-4-1BB ligand in combination with IL-12 gene transfer eradicates hepatic colon carcinoma.

We have previously shown that the local-membrane bound 4-1BB ligand and IL-12 gene transfer induced a significant antitumor response in a mouse colon carcinoma model. However, a high viral dose was required in order to achieve the best efficacy. In this study, we hypothesize that the systemic administration of soluble Ig-4-1BB ligand can give rise to better T-cell immune activation than local gene delivery. With potential clinical applications in mind, we further compare whether the natural 4-1BB ligand fused to mouse IgG2a (Ig-4-1BBL) would be as effective as the agonistic anti-4-1BB antibody. The dimeric form of Ig-4-1BBL was purified from HeLa cells transduced with a recombinant adenovirus (ADV/Ig-4-1BBL) expressing Ig-4-1BBL. Functional activity was confirmed by the ligand's ability to bind to activated splenic T cells or bone marrow (BM)-derived dendritic cells (DCs) that express 4-1BB receptor. The soluble Ig-4-1BBL efficiently costimulated CD3-activated T-cell proliferation in vitro. More importantly, it induced tumor-specific CTLs as effectively as the agonistic anti-4-1BB antibody. When combined with IL-12 gene transfer, systemic administration of the Ig-4-1BBL proved to be more potent than local gene delivery. In addition, the Ig-4-1BBL is as potent as the agonistic anti-4-1BB antibody for the treatment of hepatic MCA26 colon carcinoma, resulting in 50% complete tumor regression and long-term survival. In long-term surviving mice, both treatment modalities induced persistent tumor-specific CTL activity. In summary, these results suggest that the systemic delivery of Ig-4-1BBL can generate a better antitumor response than local gene delivery. Ig-4-1BBL had equivalent biological functions when compared to the agonistic anti-4-1BB antibody. Thus, soluble 4-1BBL dimmer can be developed as a promising agent for cancer therapy in humans.

Contribution of biomechanics, orthopaedics and rehabilitation: the past present and future.

Biomechanics is a field that has a very long history. From its beginnings in ancient Chinese and Greek literature, the field of orthopaedic biomechanics has grown in the areas of biomechanics of bone, articular cartilage, soft tissues, upper extremities, spine and so on. Bioengineers in collaboration with orthopaedic surgeons have applied biomechanical principles to study clinically relevant problems, improving patient treatment and outcome. In the past 30 years, my colleagues and I have focused our research on the biomechanics of musculoskeletal soft tissues, ligaments and tendons in particular. Therefore, in this review article, the function of the knee ligaments and the associated homeostatic responses secondary to immobilisation and exercise will be described. Research on healing of the medial collateral ligament (MCL) of the knee and possible future approaches in improving the healing of the knee ligaments will be presented. Finally, improvement of the understanding of ligament reconstruction, specifically of the anterior cruciate ligament (ACL), through the use of robotics technology will be included. Throughout the manuscript, specific scientific findings that have guided or changed the clinical management of injury to these soft tissues will be emphasised.

[Biomechanical tests for type II SLAP lesions of the shoulder joint before and after arthroscopic repair]. / Biomechanische Untersuchungen zur Typ-2-SLAP-Läsion am Schultergelenk vor und nach arthroskopischen Repair.

Superior labral anterior-to-posterior (SLAP) lesions can cause shoulder pain partly by causing glenohumeral instability. The purpose of this study was to examine the effect of a simulated type II SLAP lesion and subsequent repair on glenohumeral translation of the vented shoulder. In eight cadaver joints, a robotic/UFS testing system was used to measure joint translation by applying an anterior, posterior, or inferior load of 50 N to each shoulder. The "apprehension tests" for anterior and posterior instability were simulated by applying an anterior load of 50 N with an external rotation torque of 3 Nm or a posterior load of 50 N with an internal rotation torque of 3 Nm. Each loading condition was applied at 30 degrees and 60 degrees of glenohumeral abduction with a constant joint compressive load (44 N) to the intact, simulated SLAP lesion, and repaired shoulder. Repair of the type II SLAP was then performed by placing a Suretac through the labrum both anterior and posterior to the biceps anchor and testing was repeated. ANOVA was used to compare translation of the intact joint, the joint after the type II SLAP lesion had been simulated, and after repair. At 30 degrees of abduction, anterior translation of the intact vented shoulder joint from anterior loading was 18.7+/-8.5 mm and increased to 26.2+/-6.5 mm after simulation of the type II SLAP lesion ( p< or =0.05). The arthroscopic repair did not restore anterior translation (23.9+/-8.6 mm) to the same degree as the intact joint ( p> or =0.05). At 60 degrees of abduction, anterior translation of 16.6+/-9.6mm in the intact joint was not significantly increased at 19.4+/-10.1 after simulation of the type II SLAP lesion ( p=0.0527). AP loading also resulted in inferior translation. At 30 degrees of abduction it was 3.8+/-4.0 mm in the intact joint and increased to 8.5+/-5.4 mm after the type II SLAP lesion ( p< or =0.05. After repair the inferior translation decreased significantly to 6.7+/-5.3 mm ( p< or =0.05). Although inferior translations were less at 60 degrees of abduction, results were similar to those at 30 degrees after repair. There were no significant increases in translation after SI/AP combined external rotation torque or posterior-anterior combined internal rotation torque loading. In this study the repair of a type II SLAP lesion only partially restored translations to the same degree as an intact vented shoulder joint. Therefore, improved repair techniques or an anteroinferior capsulolabral procedure in addition to the type II SLAP lesion repair might be needed to restore normal joint function.

Telomerase-dependent oncolytic adenovirus for cancer treatment.

Conditionally replicative adenovirus (CRAD) is an attractive anticancer agent as it can selectively replicate in tumor cells. Expression of telomerase reverse transcriptase (TERT) is a unique tumor cell characteristic, being absent in normal postmitotic cells. Thus, we constructed a TERT promoter regulated CRAD for tumor-specific oncolysis by replacing the endogenous adenovirus E1A promoter with that of human TERT (Adv-TERTp-E1A). We showed that its replication was severely attenuated in TERT-negative cells, but that it replicated almost as efficiently as wild-type adenovirus in TERT-positive cells. Accordingly, Adv-TERTp-E1A conferred cytopathicity to TERT-positive, but not TERT-negative, cells. In vivo replication of Adv-TERTp-E1A after local administration into a xenograft model of human hepatocellular carcinoma in nude mice was demonstrated by an increase in adenovirus titers in tumor extracts by several orders of magnitude between 6 h and 3 days postvector injection. Furthermore, significant inhibition of tumor growth with substantial necrotic tumor areas staining positively for adenovirus was observed with Adv-TERTp-E1A, but not with a control replication-deficient adenovirus. There was also the absence of hepatotoxicity in tumor-bearing animals after intratumoral delivery of the CRAD. The results indicate that the TERT promoter-driven CRAD is capable of tumor-selective replication and oncolysis in vitro and in vivo, and can be utilized as an adjuvant treatment agent for cancer.

Transcriptional targeting of conditionally replicating adenovirus to dividing endothelial cells.

Conditionally replicating adenoviruses (CRADs) are a novel strategy in cancer treatment and clinical trials using CRADs targeted to tumor cells have been reported recently. We hypothesized that it would be possible to construct CRADs targeted to dividing endothelial cells, which are present in the tumor endothelium. We utilized the regulatory elements of Flk-1 and endoglin genes, which have been shown to be highly overexpressed in angiogenic endothelial cells, to construct two CRADs: Ad.Flk-1, which has adenoviral E1A gene under the control of the Flk-1 enhancer/promoter, and Ad.Flk-Endo, which harbors the same Flk-1 enhancer/promoter as Ad.Flk-1, plus it has the adenoviral E1B gene under control of the endoglin promoter. Viral titer measurements by plaque assay showed that in human umbilical vein endothelial cells (HUVECs), both CRADs replicated at levels comparable to that of wild-type adenovirus. In Flk-1 and endoglin negative Hep3B and A549 cells, however, the replication of Ad.Flk-1 and Ad.Flk-Endo was reduced by 30-fold and 600-fold, respectively. Cytotoxicity assays demonstrated that both CRADs killed HUVECs as effectively as wild-type adenovirus and their cytotoxicity in Hep3B and A549 cells was comparable to nonreplicating control adenovirus. Furthermore, there was a striking inhibition (83-91%) of capillary network formation in an in vitro angiogenesis assay when HUVECs were infected with Ad.Flk-1 or Ad.Flk-Endo as compared with the nonreplicating control virus. These results demonstrate that CRADs can be transcriptionally targeted to dividing endothelial cells with high specificity, and that the combined use of Flk-1 and endoglin regulatory elements has a synergistic effect on targeting specificity. This principle may be incorporated into novel therapeutic agents to develop anti-angiogenic treatment for cancer.

Tumor-specific transcriptional targeting of suicide gene therapy.

Transcriptional targeting of gene expression has been plagued by the weakness of tissue-specific promoters. Thus, to increase promoter strength while maintaining tissue specificity, we constructed a recombinant adenovirus containing a binary promoter system with a tumor-specific promoter (CEA; carcinoembryonic antigen) driving a transcription transactivator, which then activates a minimal promoter to express a suicide gene (HSV-tk; herpes simplex virus thymidine kinase). This ADV/binary-tk induced equal or greater cell killing in a CEA-specific manner in vitro compared with the CEA-independent killing of a vector with a constitutive viral promoter driving HSV-tk (ADV/RSV-tk). To monitor adenovirus-mediated HSV-tk gene expression in vivo, we employed noninvasive nuclear imaging using a radioiodinated nucleoside analog ([((1)31)I]-FIAU) serving as a substrate for HSV-tk. [((1)31)I]-FIAU-derived radioactivity accumulated after intratumoral injection of ADV/binary-tk only in the area of CEA-positive tumors with significantly less spread to the adjacent liver tissue than after administration of the universally expressed ADV/RSV-tk. Both viruses exhibited similar antitumor efficacy upon injection of liver metastases. Importantly, in vivo dose escalation studies demonstrated significantly reduced toxicity after intravenous administration of ADV/binary-tk versus ADV/RSV-tk. In summary, the increased therapeutic index of this novel, amplified CEA-driven suicide gene therapy vector is a proof of principle for the powerful enhancement of a weak tissue-specific promoter for effective tumor restricted gene expression.

The position of the tibia during graft fixation affects knee kinematics and graft forces for anterior cruciate ligament reconstruction.

Ten cadaveric knees (donor ages, 36 to 66 years) were tested at full extension, 15 degrees, 30 degrees, and 90 degrees of flexion under a 134-N anterior tibial load. In each knee, the kinematics as well as in situ force in the graft were compared when the graft was fixed with the tibia in four different positions: full knee extension while the surgeon applied a posterior tibial load (Position 1), 30 degrees of flexion with the tibia at the neutral position of the intact knee (Position 2), 30 degrees of flexion with a 67-N posterior tibial load (Position 3), and 30 degrees of flexion with a 134-N posterior tibial load (Position 4). For Positions 1 and 2, the anterior tibial translation and the in situ forces were up to 60% greater and 36% smaller, respectively, than that of the intact knee. For Position 3, knee kinematics and in situ forces were closest to those observed in the intact knee. For Position 4, anterior tibial translation was significantly decreased by up to 2 mm and the in situ force increased up to 31 N. These results suggest that the position of the tibia during graft fixation is an important consideration for the biomechanical performance of an anterior cruciate ligament-reconstructed knee.

Hepatic insulin production for type 1 diabetes.

Type 1 diabetes, along with its long-term complications, imposes a serious impact on public health. In spite of the development and application of various insulin formulations, exogenous insulin neither achieves the same degree of glycemic control as that provided by endogenous insulin, nor prevents the long-term complications associated with type 1 diabetes. As an alternative strategy, insulin gene transfer is being explored to restore endogenous insulin production in type 1 diabetes. Sustained hepatic insulin production has been shown to reverse ketonuria, prevent ketoacidosis, improve body weight gain and significantly ameliorate the adverse effects of insulin deficiency in diabetic animals. However, to achieve adequately regulated insulin production in response to changes in blood glucose concentrations remains a major hurdle. This article will review the most recent advances made to address this crucial limitation. In addition, based on the significance of maintaining basal plasma insulin for management of type 1 diabetes, we discuss the feasibility of developing basal hepatic insulin production as an auxiliary treatment to current insulin therapy for achieving tight glycemic control in type 1 diabetes.

Therapeutic elastase inhibition by alpha-1-antitrypsin gene transfer limits neointima formation in normal rabbits.

PURPOSE: Alpha-1-antitrypsin (AAT) is the major circulating elastase inhibitor. Deficiency of elastase inhibition leads to emphysema and vascular abnormalities including accelerated neointima. Because recent evidence suggests that tissue AAT levels determine inhibitory function, the authors hypothesize that local tissue-based expression of AAT limits elastase activity sufficiently to guide arterial response to injury. MATERIALS AND METHODS: Rabbit common femoral arteries were injured by mechanical overdilation and treated with buffer, viral control, or an adenovirus expressing AAT (Ad/AAT). After 3 and 28 days, intima-to-media (I/M) ratios were evaluated. Additionally, early changes in elastase inhibition potential (3 d), extracellular elastin and collagen content (3 d), and local macrophage and neutrophil infiltration (7 d) were determined. RESULTS: Ad/AAT significantly decreased neointima formation after mechanical dilation injury after 28 days: buffer controls exhibited mean I/M ratios of 0.76 +/- 0.06, whereas viral controls reached 0.77 +/- 0.09; in contrast, Ad/AAT reduced I/M ratios to 0.44 +/- 0.06. Both early elastin and collagen content were preserved in the Ad/AAT group relative to controls. The Ad/AAT group also reversed the local inflammation that characterized viral controls. CONCLUSIONS: This strategy demonstrates that local increases in elastase inhibition potential promote a neointima-resistant small-caliber artery, which may offer new promise in management of patients undergoing angioplasty.

A multidisciplinary study of the healing of an intraarticular anterior cruciate ligament graft in a goat model.

We evaluated knee function, tensile properties, and histologic appearance of a healing intraarticular bone-patellar tendon-bone autograft after anterior cruciate ligament reconstruction in a goat model. The patellar tendon graft was fixed such that both bone-to-bone (femoral tunnel) and bone-to-tendon (tibial tunnel) healing could be studied. The total anteroposterior translation significantly increased from 3 to 6 weeks, ranging from increases of 28.8% to 46.7%. In situ forces in the replacement graft decreased as much as 22.2% at 6 weeks. Conversely, tensile properties of the femur-anterior cruciate ligament graft-tibia complex did not change significantly from 3 to 6 weeks. However, the mode of failure changed from the graft pulling out of the tibial tunnel at 3 weeks to a mix of midsubstance failures (N = 2) and pullouts (N = 5) at 6 weeks. Histologic evaluations revealed progressive and complete incorporation of the bone block in the femoral tunnel, but only partial incorporation of the tendinous part of the graft in the tibial tunnel. The differences demonstrated at 3 and 6 weeks may be a result of the remodeling process of the midsubstance of the graft as the interfaces within the osseous tunnels mature.

Effect of capsular injury on acromioclavicular joint mechanics.

BACKGROUND: Traumatic disruption of the acromioclavicular joint capsule is associated with pain and instability after the injury and may lead to degenerative joint disease. The objective of this study was to quantify the effect of transection of the acromioclavicular joint capsule on the kinematics and the in situ forces in the coracoclavicular ligaments in response to external loading conditions. METHODS: Eleven fresh-frozen human cadaveric shoulders were tested with use of a robotic/universal force-moment sensor testing system. The shoulders were subjected to three loading conditions (an anterior, posterior, and superior load of 70 N) in their intact state and after transection of the acromioclavicular joint capsule. RESULTS: Transection of the capsule resulted in a significant (p < 0.05) increase in anterior translation (6.4 mm) and posterior translation (3.6 mm) but not in superior translation (1.6 mm). The effect of capsule transection on the forces in the coracoclavicular ligaments was also significant (p < 0.05) in response to anterior and posterior loading but not in response to superior loading. However, differences were found between the forces in the trapezoid and conoid ligaments. Under an anterior load, the mean in situ force (and standard deviation) in the trapezoid increased from 14 +/- 14 N to 25 +/- 19 N, while the mean force in the conoid increased from 15 +/- 14 N to 49 +/- 23 N, or 227%. In contrast, in response to a posterior load, the mean in situ force in the trapezoid increased from 23 +/- 15 N to 38 +/- 23 N, or 66% (p < 0.05), while the mean force in the conoid increased only 9%. CONCLUSIONS AND CLINICAL RELEVANCE: The large differences in the change of force in the conoid and trapezoid ligaments suggest that these ligaments should not be considered as one structure when surgical treatment is considered. Furthermore, transection of the capsule resulted in a shift of load to the coracoclavicular ligaments, which may render the intact coracoclavicular ligaments more likely to fail with anterior or posterior loading. The results of the present study also suggest that the intact coracoclavicular ligaments cannot compensate for the loss of capsular function during anterior-posterior loading as occurs in type-II acromioclavicular joint injuries.

Intratumoral adenovirus-mediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma: results of a phase I clinical trial.

Animal studies have shown that direct injection of an adenoviral vector (Adv.RSV-tk) expressing the herpes thymidine kinase gene into established tumors in the liver, followed by systemic ganciclovir administration, was effective in inducing tumor necrosis. Toxicities were minimal at therapeutically effective vector doses, although severe hepatic necroinflammation was seen at much higher supratherapeutic doses. We conducted a clinical phase I trial in patients with metastatic colorectal adenocarcinoma in the liver to assess the safety of intratumoral Adv.RSV-tk injection (escalating doses) followed by intravenous ganciclovir (fixed dose). The vector was injected into a metastatic tumor in the liver under local anesthesia by percutaneous needle placement with concurrent ultrasonographic monitoring to prevent injection or leakage into adjacent normal liver structures. We treated 16 patients in five dose level cohorts of Adv.RSV-tk, from 1.0x10(10) to 1.0x10(13) virus particles per patient. Hepatic toxicities were low, with transient grade 1 elevations in serum aminotransferase levels in 3 of 16 patients. Other toxicities were also transient: grade 2-3 fevers in 5 of 16 patients, grade 3 thrombocytopenia in 1 of 16 patients, and grade 2 leucopenia in 3 of 16 patients. These results indicate that Adv.RSV-tk can be safely administered by percutaneous intratumoral injection in patients with hepatic metastases at doses up to 1.0x10(13) virus particles per patient, and can provide the basis for future clinical trials involving intratumoral adenoviral vector injection.