Tendon healing induced by chemically modified mRNAs.

Tendon disorders are frequent both in human and veterinary medicine with high re-injury rates and unsatisfactory therapeutic treatments. Application of naked, chemically-modified mRNA (cmRNA), encoding for therapeutic proteins, is an innovative approach to address tendon healing. In the current study, we demonstrated that injection of naked cmRNA, diluted in a glucose-containing solution, into tendons resulted in high protein expression in healthy and experimentally-injured tendons. Injection of bone morphogenetic protein 7 (BMP-7)-encoding cmRNA resulted in a significantly higher expression of BMP-7 protein and reduced formation of collagen type III, compared to vehicle control. Moreover, in a large animal model, reporter protein expression was detectable not only in healthy, but also in experimentally-injured, severely inflamed tendons. Summarising, these results demonstrated the potential of cmRNAs encoding for therapeutic proteins as a new class of drugs for the treatment of tendon disorders.

Red ear syndrome and auricular erythromelalgia: the same condition?

Several cases of relapsing attacks during which the ear becomes red and patients experience a burning sensation have been reported in the literature. This combination of symptoms has been described as 'red ear syndrome' (RES). We report on a 7-year-old boy who had episodes of reddening, swelling and a burning sensation in one ear with local hyperthermia persisting for 3 years. The differential diagnosis included RES and erythromelalgia, as isolated auricular variants of erythromelalgia have been described and the symptoms are difficult to distinguish from RES. In this report, we discuss the similarities and differences between RES and erythromelalgia.

The effect of selective inhibition of inducible nitric oxide synthase on cytochrome P450 after liver transplantation in a rat model.

BACKGROUND: Activity levels of cytochrome P450 (CYP) provide markers for liver function and graft rejection episodes after orthotopic liver transplantation (OLT). Some in vitro studies have shown decreased CYP activation of inducible nitric oxide synthase (iNOS) in rejecting liver grafts. The aim of this study was to evaluate CYP isoenzyme activity changes in vivo and to examine histopathologic aspects during inhibition of iNOS after treatment with aminoguanidine (AG) using OLT in the rat. MATERIALS AND METHODS: Thirty DA-(RT1av1) rats that served as donors and LEWIS-(RT(1)) rats as recipients were divided into three groups: group I (controls, syngeneic rats; n = 6), group II (allogeneic rats without immunosupression; n = 11), and group III (allogeneic rats with AG treatment; n = 13). On postoperative days 5, 8, and 10 we performed laboratory investigations and liver biopsies for histopathologic investigations. RESULTS: On postoperative day 5, activities of CYP-1A1 and -3A4 were significantly lower (P = .022) in group III and the activity of CYP-1A2 higher (P < .05) compared with group II. At postoperative days 8 and 10, the activities of all CYP isoenzymes were significant higher in AG-treated rats (group III) in contrast with group II after allogeneic OLT without immunosuppression. Histopathologic findings revealed less distinct rejection signs in group III specimens after AG treatment compared with group II. CONCLUSION: Summarizing our results, we concluded that AG treatment led to increased CYP activity and less distinction of graft rejection after OLT in rats.

Intra-tumoral gene delivery of feIL-2, feIFN-gamma and feGM-CSF using magnetofection as a neoadjuvant treatment option for feline fibrosarcomas: a phase-I study.

Despite aggressive pre- or postoperative treatment, feline fibrosarcomas have a high relapse rate. In this study, a new treatment option based on immune stimulation by intra-tumoral delivery of three feline cytokine genes was performed. The objective of this phase-I dose-escalation study was to determine a safe dose for further evaluation in a subsequent phase-II trial. Twenty-five client-owned cats with clinical diagnosis of fibrosarcoma - primary tumours as well as recurrences - entered the study. Four increasing doses of plasmids coding for feIL-2, feIFN-gamma or feGM-CSF, respectively, were previously defined. In groups I, II, III and IV these doses were 15, 50, 150 and 450 microg per plasmid and a corresponding amount of magnetic nanoparticles. Two preoperative intra-tumoral injections of the magnetic DNA solution were followed by magnetofection. A group of four control cats received only surgical treatment. Side effects were registered and graded according to the VCOG-CTCAE scale and correlated to treatment. Statistical analyses included one-way anova, post hoc and Kruskal-Wallis tests. ELISA tests detecting plasma feIFN-gamma and plasma feGM-CSF were performed. One cat out of group IV (450 microg per plasmid) showed adverse events probably related to gene delivery. As these side effects were self-limiting and occurred only in one of eight cats in group IV, this dose was determined to be well tolerable. Altogether six cats developed local recurrences during a 1-year observation period. Four of these cats had been treated with dose IV. Regarding these observations, a subsequent phase-II trial including a representative amount of cats should be tested for the efficacy of dose IV as well as dose III.

Allogeneic retrovirally transduced, IL-2- and IFN-gamma-secreting cancer cell vaccine in patients with hormone refractory prostate cancer--a phase I clinical trial.

BACKGROUND: The purpose of this vaccine study was to determine the safety and feasibility of vaccination with an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and to evaluate the efficacy of inducing tumor-specific immune responses in HLA-A2-matched patients with hormone refractory prostate cancer (HRPC). METHODS: In a dose-escalating phase I study, HLA-A2-matched HRPC patients received four vaccinations of irradiated allogeneic LNCaP cells retrovirally transduced to secrete IL-2 and IFN-gamma at study day 1, 15, 29 and 92 and subsequently every 91 days unless tumor progression was evident. RESULTS: Three patients receiving the first dose level (7.5 million cells) showed no evidence of dose-limiting toxicity or vaccine-related adverse events including autoimmunity. One of three patients receiving the second dose level (15 million cells) developed a transient self-limiting grade 3 local injection site reaction (ulceration) after the eighth vaccination. Vaccine-induced immune responses against a broad array of prostate tumor associated antigens were detected in all six patients. Two of the three patients receiving the higher dose showed a decline in serum prostate-specific antigen (PSA) values of more than 50%, with one patient remaining on protocol for 3 years. CONCLUSIONS: Immunisation with the allogeneic LNCaP/IL-2/IFN-gamma vaccine is safe and feasible without any dose-limiting toxicity or autoimmunity. A 50% PSA decline was achieved in two of the six patients. This encouraging data provides the scientific rationale for further investigation of the vaccine in a phase II trial.

Prediction of flap necrosis with laser induced indocyanine green fluorescence in a rat model.

Prediction of necrosis has a clinical relevance in all fields of plastic surgery. The new application of indocyanine green (ICG) fluoroscopy in plastic surgery allows an objective quantification of skin perfusion and a high topographical resolution. The aim of the present study is to determine threshold values for flap perfusion under well-defined experimental conditions. Twenty random pattern flaps with a length to width ratio of 4:1 (8 x 2 cm(2)) were dissected on the anterior abdominal wall of 20 male Sprague-Dawley rats. ICG fluoroscopy was performed at the end of the operation. The animals were sacrificed at the seventh postoperative day with a reliable necrosis of the distal part of the flaps. Postoperative ICG fluoroscopy then was analysed both in regions that will survive and undergo necrosis. At day 7 a mean area of 5.5 cm(2) (57% of the total flap area) survived and a mean of 3.8 cm(2) (43%) became necrotic. The surviving part of the flap had a mean perfusion index of 62% compared to reference skin. The distal parts of the flap that necrotised showed an average perfusion index of only 19% postoperatively. Differences were statistically highly significant (p<0.001). Indocyanine green fluoroscopy is a useful tool to evaluate perfusion topographically and predict necrosis. From a statistical point of view a perfusion index of less than 25% of the reference skin can be considered as a sign of developing flap necrosis.

Nerve replacement strategies for cavernous nerves.

OBJECTIVE: This article reviews novel restorative therapies for cavernous nerves that may be used to replace resected cavernous nerves at the time of pelvic surgery. METHODS: A literature-based presentation (Medline search) on current nerve replacement strategies was conducted with emphasis on neurobiological factors contributing to the restoration of erectile function after cavernous nerve injuries. RESULTS: A promising alternative to autologous nerve grafts for extending the length of successful nerve regeneration are artificial nerve guides. The addition of neurotrophic factors, extracellular matrix components and Schwann cells has been shown to promote cavernous nerve regeneration. Neurotrophic factors can be incorporated in the scaffold or can be supplied by cells seeded into the stroma. The regenerative capacity of these cells can be further enhanced by genetic modification with neurotrophic factor encoding genes. CONCLUSIONS: Artificial nerve guides, especially biodegradable ones containing growth-promoting factors or cells, are a promising option for the repair of cavernous nerve lesions.

[Tissue engineering of erectile nerves]. / Tissue Engineering erektiler Nerven.

Dissection of the cavernous nerves eliminates spontaneous erections and may lead to irreversible erectile dysfunction due to degeneration of cavernous tissue. Novel procedures to reconstruct penile innervation include cavernous nerve interposition grafting and neurotrophic treatments to revitalize penile neural input, evaluated thus far in various preclinical models of cavernous nerve injury. Schwann cells crucially contribute to successful axonal regeneration by mechanical and paracrine mechanisms in the injured nerve, and Schwann cells seeded into guidance channels have been successfully employed to support regeneration in animal models of cavernous nerve injury. Gene therapy, tissue engineering, and reconstructive techniques have been combined to deliver neurotrophic factors and recover erectile function.

Inducible nonviral gene expression in the treatment of osteochondral defects.

OBJECTIVE: The repair of osteochondral defects with chondrocytes genetically modified to express desired growth factors promises great potential in orthopaedic therapy. Controlled expression of the transgenes is required in many instances. The objective of the present study was to demonstrate the inducibility of tetracycline-responsive transgene expression in osteochondral defects in the knee joint filled with genetically modified chondrocyte implants. METHODS: An expression plasmid containing the lacZ gene under the control of the minimal CMV promoter fused to the Tet-responsible element (TRE) as well as the reverse transactivator (rtTA2s-M2) was constructed and used to transfect isolated articular chondrocytes from New Zealand white rabbits. rtTA2s-M2 binds to the TRE in the presence of tetracycline and leads to the transcription of the transgene. Different concentrations of DNA and various DNA:lipid ratios were tested to determine best transfection conditions. Transfection efficiency and inducibility were analysed by histochemical analysis and flow-cytometry. To evaluate the system in vivo, collagen-sponges were seeded with transfected autologous chondrocytes and implanted in osteochondral defects in the knees of NZW-rabbits. Gene expression was induced by doxycycline and 3 weeks later, LacZ-expression in isolated knee joints was evaluated in histological sections by X-gal staining. RESULTS: In vitro 13.5% (+/-1.32) of induced primary chondrocytes were LacZ-positive, while non-induced controls showed a background-staining in 0.6% (+/-0.2). In vivo, upon doxycycline treatment, induction of lacZ-gene-expression could be demonstrated in chondrocytes in 3-week-old, well-integrated implants. CONCLUSION: For the first time, tetracycline-inducible gene expression is demonstrated to work in the treatment of osteochondral defects. This demonstrates the feasibility for a gene therapy-assisted approach using controlled expression of therapeutic growth factors from transplanted genetically modified chondrocytes.

Uptake of radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil in cardiac cells after adenoviral transfer of the herpesvirus thymidine kinase gene: the cellular basis for cardiac gene imaging.

BACKGROUND: Gene therapy is a promising approach for the treatment of cardiac diseases. Coexpression of therapeutic genes with a suitable marker gene would allow for the noninvasive imaging of successful gene transfer and expression via radiolabeled marker substrates. In the present study, such an approach was first applied to cardiac tissue. METHODS AND RESULTS: The combination of the herpesvirus thymidine kinase reporter gene (HSV1-tk) and radiolabeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyluracil (FIAU) was evaluated. H9c2 rat cardiomyoblasts were infected in vitro with a replication-defective HSV1-tk-containing adenovirus and a negative control virus. The intracellular uptake of [(14)C]FIAU increased with increasing multiplicity of infection and with time after infection. Uptake in negative controls remained <15% of positive controls. Additionally, vectors were applied intramyocardially in Wistar rats. The marker substrate [(125)I]FIAU was injected intravenously 3 days later, and animals were killed after 24 hours. Autoradiographically, regional transgene expression was clearly identified in animals receiving the adenovirus containing HSV1-tk (3. 4+/-2.2-fold increase of radioactivity at vector administration site compared with remote myocardium), whereas nonspecific uptake in negative controls was low (<10% of positive controls). CONCLUSIONS: Using an adenoviral vector, HSV1-tk can be successfully expressed in cardiac cells in vitro and in vivo, yielding high uptake of radiolabeled FIAU. The results suggest that imaging transgene expression in the heart is feasible and may be used to monitor gene therapy noninvasively.

Enhanced cardiac contractility after gene transfer of V2 vasopressin receptors In vivo by ultrasound-guided injection or transcoronary delivery.

BACKGROUND: Systemic levels of arginine vasopressin (AVP) are increased in congestive heart failure, resulting in vasoconstriction and reduced cardiac contractility via V(1) vasopressin receptors. V(2) vasopressin receptors (V2Rs), which promote activation of adenylyl cyclase, are physiologically expressed only in the kidney and are absent in the myocardium. Heterologous expression of V2Rs in the myocardium could result in a positive inotropic effect by using the endogenous high concentrations of AVP in heart failure. METHODS AND RESULTS: We tested gene transfer with a recombinant adenovirus for the human V2R (Ad-V2R) to stimulate contractility of rat or rabbit myocardium in vivo. Ultrasound-guided direct injection or transcoronary delivery of adenovirus in vivo resulted in recombinant receptor expression in the myocardial target area, leading to a substantial increase in [(3)H]AVP binding. In 50% of the cardiomyocytes isolated from the directly injected area, single-cell shortening measurements detected a significant increase in contraction amplitude after exposure to AVP or the V2R-specific desmopressin (DDAVP). Echocardiography of the target myocardial area documented a marked increase in local fractional shortening after systemic administration of DDAVP in V2R-expressing animals but not in control virus-treated hearts. Simultaneous measurement of global contractility (dP/dt(max)) confirmed a positive inotropic effect of DDAVP on left ventricular function in the Ad-V2R-injected animals. CONCLUSIONS: Adenoviral gene transfer of the V2R into the myocardium increases cardiac contractility in vivo. Heterologous expression of cAMP-forming receptors in the myocardium could lead to novel strategies in the therapy of congestive heart failure by bypassing the desensitized beta-adrenergic receptor-signaling cascade.

Pharmacokinetics and pharmacodynamics of vecuronium in rats with systemic inflammatory response syndrome: treatment with NG-monomethyl-L-arginine.

BACKGROUND: Insufficient detoxification caused by nitric oxide-related inhibition of cytochrome P450 may be important for metabolism of numerous drugs, including vecuronium. The present study investigated the pharmacodynamics and pharmacokinetics of vecuronium in rats with inflammatory liver dysfunction. METHODS: Male Sprague-Dawley rats (n = 56) were randomly allocated into two groups: In the sepsis group, liver inflammation was established by injection of 56 mg/kg heat-killed Corynebacterium parvum; control rats received the solvent. At day 4, groups were subdivided according to treatment with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (250 mg/kg) or placebo. The aminopyrine breath test was performed to assess cytochrome P450 activity. Rats were anesthetized with propofol and mechanically ventilated. Duration of action of vecuronium (1.2 mg/kg) was measured by evoked mechanomyography (stimulation of the sciatic nerve, contraction of the gastrocnemius muscle). In seven rats of each subgroup a 50% neuromuscular blockade was established by a continuous vecuronium infusion. Vecuronium plasma levels were measured and plasma clearance of vecuronium was calculated. Nitric oxide synthesis was assessed by measuring nitrite/nitrate serum levels. RESULTS: In sepsis/placebo rats, vecuronium-induced neuromuscular blockade was prolonged (144% of contro/placebo), vecuronium plasma levels at 50% neuromuscular blockade were increased (122% of control/placebo), and plasma clearance was decreased (68% of control/placebo). N(G)-monomethyl-L-arginine therapy in rats with sepsis improved cytochrome P450 activity and plasma clearance of vecuronium, shortened duration of action of vecuronium, but did not alter the elevated vecuronium plasma levels. CONCLUSIONS: A systemic inflammatory response syndrome with liver dysfunction results in decreased sensitivity to and a decreased elimination of vecuronium. Modulation of nitric oxide synthesis may be a strategy that can be used in the future to improve xenobiotic metabolism in sepsis.

Hand parameter differences between psychiatric patients and normal controls: a preliminary evaluation.

The basic assumption of this study was that it is possible to identify unusual hand features in the hands of unusual populations, and that a combination of certain unusual hand features can be associated with mental illness. One hundred and seventeen people were studied in two groups: 63 mental patients (40 males, 23 females) hospitalized in the Abarbanel Hospital, Israel, and a control group of 54 people (27 males, 27 females). Twelve hand parameters were studied, and 11 of them were found to be significantly different. Out of them, 7 show the significant result of p < 0.001. The results of this study suggest that the features of the hand may be considered as a part of the phenomenon of minor physical anomalies which are apparently neurodevelopmental markers. It suggests also the desirability of further research in this area.

Spectroscopy of Hydrothermal Reactions. 8. Kinetics of Aqueous NH(4)XCN (X = O, S) and OCS by Flow Reactor-Infrared Spectroscopy.

The kinetics and pathway of hydrothermolysis of 1 m NH(4)SCN to CO(2), NH(3), and H(2)S were determined at 543-573 K and 275 bar by the use of FTIR spectroscopy and a Pt/Ir flow reactor with diamond windows or a 316 stainless steel flow reactor with sapphire windows. The rates of SCN(-) loss and CO(2) formation were the same. The reaction is (pseudo) second-order with E(a) = 113 +/- 11 kg/mol and ln(A, kg/(mol.s)) = 21 +/- 2. DeltaS() = -84 J/(mol.K), which suggests a bimolecular, rate-determining, initial decomposition step for NH(4)SCN. A reaction scheme is proposed in which OCS and a monothiocarbamate species are undetected intermediates. The absence of OCS is explained by the rapid hydrothermolysis rate of OCS to CO(2) and H(2)S which was determined by IR spectroscopy at 393-423 K under 275 bar to be E(a) = 44 +/- 5 kJ/mol and ln(A/s) = 13 +/- 1 for OCS. The resulting rate is about 10(3) times faster than the hydrothermolysis rate of NH(4)SCN at 543 K. The results are compared to the equivalent reaction for NH(4)OCN. NH(4)OCN reacts about 3 x 10(3) times faster than NH(4)SCN at 543 K. The trend in the rates is consistent with the charge distribution and the trend in the bond distances, which resulted from ab initio quantum mechanical calculations at the HF/N311G//HF/N31G level in the OCN(-) and SCN(-) ions and the proposed carbamate and monothiocarbamate intermediates.

Inhibition of nitric oxide synthesis improves detoxication in inflammatory liver dysfunction in vivo.

Inflammatory stimulation of the liver induces nitric oxide (NO) biosynthesis and suppression of detoxication. In this study the effect of NO biosynthesis on cytochrome P-450 (CYP) enzyme activity was investigated by comparing in vivo and in vitro assays. To establish liver inflammation, CD rats were injected with Corynebacterium parvum (C. parvum) suspension. After 5 days NO biosynthesis was highly induced as indicated by increased NO2- plus NO3- serum concentrations. At the same time the aminopyrine breath test (ABT), measuring CYP activity in vivo, was reduced to 42% and the in vitro assay of aminopyrine turnover was suppressed to 12% of NaCl- injected controls. When C. parvum-injected animals were treated with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), CYP activities significantly improved with an ABT of 76% and an in vitro aminopyrine turnover of 47% of controls. Neither C. parvum injections nor L-NMMA treatment resulted in a significant change of CYP protein concentrations. These data indicate that suppression of xenobiotic metabolism can be attenuated by inhibition of NO biosynthesis during an ongoing process of inflammation.

[Isoflurane anesthesia in rabbits in a closed anesthetic system]. / Zur Isoflurannarkose beim Kaninchen im geschlossenen Narkosesystem.

Using the Stephens anaesthetic apparatus-which is a closed system with an in-circuit, nonprecision vaporizer-and isoflurane as anaesthetic gas, 18 rabbits were anaesthetized and showed sufficient hypnosis, analgesia, and muscle relaxation during bone surgery. Induction of anaesthesia was achieved with intravenous propofol and all rabbits were intubated afterwards. During the following isoflurane inhalation anaesthesia the mean arterial blood pressure decreased considerably (compared to control measures before induction), the heart rate remained the same or showed a slight increase, and the respiratory rate decreased. The arterial pO2 decreased corresponding to the respiratory depression after propofol induction and increased again during spontaneous ventilation with 100% oxygen. The changes in arterial pCO2 and pH were representative for a rise in the CO2-stimulation threshold. A moderate metabolic acidosis could be seen due to preanaesthetic excitement of the animals. Recovery time was short (between one and 11 minutes) and no signs of excitation could be detected. The consumed volume of isoflurane was 0.80 ml/kg BW/h.

[Carbon dioxide stunning for the slaughter of turkeys]. / CO2-Betäubung zur Schlachtung von Puten.

In an experimental study on stunning of 460 turkeys in a CO2 enriched atmosphere (60-70% CO2 in air), as a more animal protecting and more economic alternative to conventional electrical stunning, the following results are obtained: all animals show respiratory arrest within 100 seconds. After being dipped in a CO2 enriched atmosphere for a few seconds the turkeys show head shaking and forced respiration which has to be considered as a strain the animals are conscious of. The meat hygienic results after CO2 stunning are much better than after electrical stunning.

[Clinical investigations of an i.m. combination anesthesia with fentanylclimazolam/xylazine and postoperative i.v. antagonism with naloxone/sarmazenil/yohimbine in guinea pigs]. / Klinische Untersuchungen zur i.m. Kombinationsanästhesie mit Fentanyl/Climazolam/Xylazin und postoperativer i.v. Antagonisierung mit Naloxon/Sarmazenil/Yohimbin beim Meerschweinchen.

Aim of the study was the clinical use of a completely antagonisable anaesthesia in guinea pigs. The dosage based on experimental studies. Fentanyl, climazolam and xylazine (0.05/2.0/2.0 mg/kg) were injected i.m. for combination anaesthesia and naloxone, sarmazenil and yohimbine (0.03/0.3/2.0 mg/kg) i.v. were used for antagonisation. 18 guinea pigs (17 female, one male) with an average body weight of 1177 g aged between eight months and five years were anaesthetised for various surgical procedures. The following clinical parameters were measured: body temperature, muscle relaxation, analgesia, hyperacusia, righting-, interdigital- and palpebral reflex. After antagonisation the return of righting reflex, the appearance of muscle trembling, the first walking without ataxia and the first food uptake were observed. The investigations showed that this kind of anaesthesia is suitable for surgical procedures in the guinea pig and is very safe because of the complete antagonisation of the depressing respiratory and circulatory effects of anaesthesia.