Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Potential predictive value of axial T2 mapping at 3 Tesla MRI in patients with untreated patellar cartilage defects over a mean follow-up of four years.

OBJECTIVE: The objective was to demonstrate the potential of axial T2 mapping for quantification of untreated early-stage patellar cartilage lesions over time and to assess its capability as a potential predictive marker for future progression. STUDY DESIGN & METHODS: Thirty patients (mean age, 36.7 ± 11.1 years; 16 males), with early-stage patellar cartilage defects (≤ICRS grade 2) at baseline and no treatment during follow up (4.0 ± 1.6 years) were enrolled. Morphological cartilage changes over time were subdivided into a Progression, Non-Progression Group and Regression Group. Quantitative analysis of cartilage defects and healthy reference was performed by means of global and zonal T2 mapping (deep and superficial cartilage T2 values) at both time points. Statistical evaluation included analysis of variance (ANOVA), paired t Test's and ROC analysis. RESULTS: The Progression Group (N = 11) had significantly higher global T2 values at baseline (57.4 ± 7.8 ms) than patients without (N = 17) (40.6 ± 6.9 ms) (P < 0.01). Furthermore the Non-Progression Group showed only a minor increase in global T2 relaxation times to 43.1 ± 7.9 ms (P = 0.07) at follow up, whereas in the progression group global (68,7 ± 19 ms: P = 0.02) and superficial T2 values (65,8 ± 8.2-79.8 ± 24.4 ms; P = 0.03) increased significantly. T2 values for healthy reference cartilage remained stable. In 2 patients an improvement in ICRS grading was observed (Regression Group) with decreasing T2 values. The ROC analysis showed an area under the curve of 0.92 (95%CI 0.82-1.0). At a cut-off value of 47.15 ms, we found a sensitivity of 92% (false-positive rate of 18%) for future progression of cartilage defects. CONCLUSIONS: This study provides evidence regarding the possible potential of axial T2 mapping as a tool for quantification and prediction of patellar cartilage defect progression in untreated defects.

An ex vivo RNA trans-splicing strategy to correct human generalized severe epidermolysis bullosa simplex.

BACKGROUND: Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans-splicing-based therapeutic approach to correct the phenotype. OBJECTIVES: To correct a mutation within exon 1 of the KRT14 gene, using a 5'-trans-splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. METHODS: A therapeutic RNA trans-splicing molecule containing wild-type exons 1-7 was stably transduced into an EBS patient-derived keratinocyte line. Trans-splicing was confirmed via reverse-transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post-transplantation for regular epidermal stratification, trans-splicing-induced green fluorescent protein expression and blistering. RESULTS: Transplanted skin equivalents generated from trans-splicing-corrected patient keratinocytes showed a stable and blister-free epidermis. KRT14 correction disrupted EBS-gen sev-associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism. CONCLUSIONS: Our data demonstrate that trans-splicing-mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS-gen sev phenotype and stabilization of the epidermis in a xenograft mouse model.

Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations.

RNA trans-splicing has become a versatile tool in the gene therapy of monogenetic diseases. This technique is especially valuable for the correction of mutations in large genes such as COL7A1, which underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa. Over 800 mutations spanning the entire length of the COL7A1 gene have been associated with defects in type VII collagen, leading to excessive fragility of epithelial tissues, the hallmark of dystrophic epidermolysis bullosa (DEB). In the present study, we designed an RNA trans-splicing molecule (RTM) that is capable of repairing any given mutation within a 4200 nucleotide region spanning the 3' half of COL7A1. The selected RTM, RTM28, was able to induce accurate trans-splicing into endogenous COL7A1 pre-mRNA transcripts in a type VII collagen-deficient DEB patient-derived cell line. Correct trans-splicing was detected at the RNA level by semiquantitative RT-PCR and correction of full-length type VII collagen was confirmed at the protein level by immunofluorescence and western blot analyses. Our results demonstrate that RTM28, which covers >60% of all mutations reported in DEB and is thus the longest RTM described so far for the repair of COL7A1, represents a promising candidate for therapeutic applications.

[The German centre for lung research - translational research for the prevention, diagnosis and treatment of respiratory diseases]. / Das Deutsche Zentrum für Lungenforschung - Translationale Forschung für Prävention, Diagnose und Therapie von Atemwegserkrankungen.

Respiratory diseases are one of the most important causes of mortality with tremendous costs for health care systems, not only in Germany, but worldwide. Up to now treatment options for most of these chronic diseases are limited. The German Ministry for Research and Education (BMBF) - following the example of the US National Institute of Health have supported the foundation of a German Centre for Lung Research (DZL) to speed up the development of preventive, diagnostic and therapeutic measures. Not only universities, but also non-university based research institutes are part of the DZL. To allow the translation from basic research experience into clinical practice to improve patient care, basic research orientated approaches will be combined with disease and patient focused approaches. The DZL is one of six German Centres for Health Care Research (neurological diseases, diabetes, cardiovascular diseases, infectious diseases, cancer, and lung diseases) for the optimisation of translational processes to overcome the burden of major diseases.

A tissue engineering approach to meniscus regeneration in a sheep model.

OBJECTIVE: Regeneration of the meniscal tissue occurs to a limited extent, and the loss of meniscal tissue leads to osteoarthritis. A new biomaterial consisting of hyaluronic acid and polycaprolactone was used as a meniscus substitute in sheep to evaluate the properties of the implant material with regard to size, biomechanical stability, tissue ingrowth, and integration. METHODS: Eight sheep (right stifle joints) were treated with three total and three partial meniscus replacements while two meniscectomies served as empty controls. The animals were euthanized after 6 weeks. The specimens were assessed by gross inspection and histology, and compared with the nonoperated left joints. RESULTS: The surgical technique was found to be feasible. The implants remained in position, did not tear, and showed excellent tissue ingrowth to the capsule. Tissue integration was also observed between the original meniscus and the implant. However, graft compression and extrusion occurred. The histological investigation revealed tissue formation, cellular infiltration and vascularization. Cartilage degeneration was more severe in the operated joints. CONCLUSION: The present study shows promising results concerning the qualities of this biomaterial with regard to implantation technique, stability and tissue ingrowth.

Initially elevated osteoprotegerin serum levels may predict a perioperative myocardial lesion in patients undergoing coronary artery bypass grafting.

OBJECTIVE: We investigated whether osteoprotegerin (OPG), an important regulator in the genesis of arteriosclerosis and bone formation, is able to identify patients at risk for perioperative myocardial infarction measured as cardiac troponin I (cTNI) and signs of myocardial ischemia in the electrocardiogram after coronary artery bypass grafting (CABG). DESIGN: Observational study. SETTING: Post-surgical intensive care unit of a tertiary care center. PATIENTS: Ninety-seven patients undergoing elective CABG. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: OPG and cTNI were measured before and 24 hrs after CABG. Additionally, cTNI was measured after 12 hrs. Electrocardiography was done before and immediately after CABG. OPG before CABG (OPGpre) measurements correlated with cTNI measurements after 12 hrs (cTNI12) (r = 0.56; p < .0001) and with cTNI measurements after 24 hrs (cTNI24) (r = 0.77; p < .0001). OPGpre measurements correlated with electrocardiographic findings after surgery (r = 0.65; p < .0001). There was a positive correlation between OPGpre value and the number of bypasses (r = 0.95; p < .0001). A strong correlation was found between OPGpre and homocysteine (r = 0.96; p < .0001). The median OPG presurgical level for the four patients with cardiac complications was found to be notably elevated (28.1 [26.6/31.0] pmol/L) in comparison with that for patients without complications (10.2 [3.7/16.9] pmol/L). CONCLUSIONS: OPG appears to be a useful marker for estimating risk for perioperative myocardial infarction in patients undergoing CABG, as demonstrated by signs of ischemia on electrocardiography.

Ultrasonographic findings in cows with dilatation, torsion and retroflexion of the caecum.

Thirty cows with caecal dilatation underwent clinical and ultrasonographic examinations, followed by a right flank laparotomy and surgical correction. The intraoperative findings were compared with the results of the ultrasonographic examination. The appearance, position, dimensions, diameter and nature of the contents of the caecum and proximal and spiral ansa of the colon were determined with a 3.5 MHz linear transducer. The wall of the proximal ansa of the colon and of the dilated caecum closest to the abdominal wall was visible in all the cows and appeared as an echogenic semicircular line immediately adjacent to the peritoneum. The contents of the caecum and of the proximal and spiral ansa of the colon were not visible in 21, 25 and 25 cows, respectively, owing to gas. In the remaining cows, the contents were hypoechogenic to echogenic in appearance. In all of the cows, the dilated caecum was imaged from the right abdominal wall at the level of the tuber coxae. The caecum was imaged from the 12th, 11th and 10th intercostal spaces in 11, five and three cows, respectively. The caecum and proximal ansa of the colon were situated immediately adjacent to the right abdominal wall in 28 cows, but in the other two cows parts of these structures were pushed away from the abdominal wall by the liver or gall bladder. The diameter of the caecum, measured at various sites varied from 7.0 to 25.0 cm. Caecal dilatation was diagnosed on the basis of the results of rectal examinations in 28 of the cows, but in all 30 cows on the basis of the results of the ultrasonographic examinations. Dilatation and caudal displacement of the caecum were diagnosed in 18 cows; dilatation and cranioventral retroflexion of the caecum were diagnosed in six cows, and dilatation and craniodorsal retroflexion of the caecum were diagnosed in two cows. In the four other cows, the direction of the retroflexed caecum could not be determined. The diagnosis of caecal dilatation based on the ultrasonographic findings was confirmed in all the cows during exploratory laparotomy. The results of ultrasonography and exploratory surgery with regard to the position of the dilated and sometimes retroflexed or twisted caecum were in complete agreement in 18 cases, in partial agreement in eight cases, but in four cases did not agree.

Effect of therapeutic dietary biotin on the healing of uncomplicated sole ulcers in dairy cattle--a double blinded controlled study.

The objective of this study was to investigate the influence of orally administered biotin on the healing of uncomplicated sole ulcers in dairy cattle. In a double-blind controlled study, 24 dairy cows with a mild, uncomplicated sole ulcer on a lateral hind claw were given either 40 mg biotin per day or a placebo feed over a period of 50 days. An orthopaedic shoe was fitted to the medial claw of the affected foot. The healing process was assessed clinically and by histological examination of horn samples. In the biotin-treated animals, the newly formed epidermis covering the sole ulcers was found to be of significantly better histological quality after 50 days than at the start of the study. The significant improvement in histological horn quality found in the biotin-treated animals suggests that biotin exerts a positive influence on the healing of sole ulcers, however the study period of 50 days appears to have been too short to permit macroscopic detection of the improvement in horn quality.

[Implantation of synthetic mesh for the closing of abdominal wall ruptures in the ventral flank of cows: a retrospective study of 16 cases]. / Implantation von synthetischen Netzen zum Verschluss von Bauchwandrupturen in der ventralen Flanke bei der Kuh: Eine retrospektive Studie über 16 Fälle.

In this paper the technique and long-term results for abdominal wall ruptures in the ventral flank are described in 16 cows that underwent surgery between January 1990 and October 1999. Most injuries were caused by a horn of another cow. In three cases the rupture was repaired longer than 6 weeks after traumatic injury. The other defects were treated surgically 6.4 (0-25) days after they occurred. The muscle defects were closed layer by layer with simple continuous sutures (polyglactin 910, 6 metric) under general anesthesia in lateral recumbency and the sutured defect was reinforced with a synthetic mesh (polyester or polyglactin 910) fixed to the outside of the external oblique abdominal muscle. The most frequent postoperative complication was subcutaneous seroma. It was treated successfully by incision and drainage. All patients were released 11.6 +/- 5.5 (6-23) days after surgery. A telephone survey 53 +/- 26 (7-106) months after surgery revealed that the patients had an average survival time of 30 (2-104) months, had born one to seven calves without any complications and that only one cow had had an unsatisfactory milk yield. One cow had to be slaughtered 2 months after surgery because of a relapse. Synthetic mesh was used successfully to close the defect in 15 animals. The functional as well as the cosmetic result of the described operation was good to excellent.

Strangulation of the duodenum by the uterus during late pregnancy in two cows.

Two Swiss Braunvieh cows in late pregnancy underwent surgery because of a rare form of ileus due to strangulation of the duodenum at its caudal flexure by the gravid uterus. The whole uterus had passed through a gap between the mesoduodenum and duodenum and with increasing weight had led to strangulation of the duodenum. This was possible since the mesoduodenum and both walls of the greater omentum adjacent to its caudal edge were not connected with the duodenum, probably due to a congenital inhibitory malformation. A transsection and an end-to-end anastomosis of the duodenum were necessary in both cases since it was impossible to retract the gravid uterus through the defect. Postoperative recovering was uneventful in both cows, which were discharged after seven and five days respectively and calved normally about two months later.

Imaging findings in a cow with a retropharyngeally displaced magnet.

This report describes the ultrasonographic and radiographic findings and the treatment of a Swiss Brown cow with a retropharyngeal abscess caused by improper administration of a magnet.

Hyaluronidase additional to standard chemotherapy improves outcome for children with malignant brain tumors.

Ex vivo experiments with vital brain tumor samples show that hyaluronidase enhances the permeation of carboplatin into tumor tissue with a matrix rich in hyaluronic acid. We achieved long-lasting second remissions for children with relapsed malignant brain tumors treated with carboplatin, etoposide and this enzyme. Thereafter, we initiated a pilot study where we added hyaluronidase to the first line standard therapy to prevent the deadly relapses right from the beginning. All 19 patients with malignant brain tumors admitted to our pediatric neurooncological center from 1992 to 1994 were included in the study. Kaplan-Meier estimation of event-free survival and overall survival after 3 years follow-up indicates a significantly better outcome for the hyaluronidase-treated group. The children receiving supportive hyaluronidase suffered significantly less relapses (P = 0.034) and had a significantly better chance for survival (P = 0.045) compared to the historical control of 21 children treated with the same standard regimen but without supportive hyaluronidase (product limit analysis and the log-rank test, P < 0.05). Children aged >3 years receiving hyaluronidase together with primary treatment seemed to gain the most benefit.

Immunophenotypic characterization of myelomonocytic cells in patients with myelodysplastic syndrome.

Infections, an important determining factor in the clinical course of myelodysplastic syndromes (MDS), result in activation of myelomonocytic cells. In this study we demonstrate activation-associated immunophenotypic changes of cell surface antigens on monocytes and granulocytes observed in two groups of MDS patients, one with low and another one with high clinical risk, and compared them to healthy individuals. Significantly changed expression of the complement receptors 1 (CD35) and 3 (CD11b), the Fc gamma receptor I (CD64), the leucocyte-homing receptor (CD44) and the activation associated membrane proteins CD67 and M5 were found on monocytes and/or granulocytes of MDS patients. In low-risk MDS patients we observed activation-associated phenotypic changes only in monocytes, whereas in high-risk MDS patients, both monocytes and granulocytes showed such changes. Additionally, we performed respiratory burst experiments and observed an impaired response of monocytes and granulocytes derived from MDS patients. Despite the fact that all patients were free of infection by clinical criteria, cell surface phenotyping as well as the reduced respiratory burst capacity of myelomonocytic cells suggests in vivo preactivation of these cells.

Prognostic significance of myeloid-associated antigen expression on blast cells in children with acute lymphoblastic leukemia. The Austrian Pediatric Oncology Group.

The prognostic significance of expression of myeloid-associated antigens in childhood acute lymphoblastic leukemia (myA+ALL) was evaluated. From 1984 to 1990, 251 children with immunologically verified ALL were treated in two prospective consecutive Austrian studies. Complete immunophenotyping was performed in 206 cases (82%). Out of these 175 cases were classified as B-cell precursor ALL, 31 cases as T-ALL. Expression of myeloid-associated antigens was demonstrated in 23 cases (13.1%) of childhood B-cell precursor ALL, particularly in immature (CD10 negative) forms (P < .0001), and in 1 case (3.2%) of T-ALL. CDw65 was expressed most frequently (12 cases), followed by CD13 and CD15 (5 cases each), CD33 (4 cases), and blood-group H (3 cases). Compared to myA- ALL prognosis of children with myA+ B-cell precursor ALL was poor, despite intensive multiagent chemotherapy according to BFM protocols. Remission rates were not impaired, but pEFS was 74.6% for myA- ALL, and only 37.8% for myA+ ALL (P = .0001). As demonstrated by multivariate analysis the expression of myeloid-associated antigens was the most important prognostic variable for EFS in B-cell precursor ALL, whether or not CD10 was expressed.

Prognostic impact of karyotype and immunologic phenotype in 125 adult patients with de novo AML.

One hundred-twenty-five adult patients with de novo acute myeloid leukemia (AML) were treated according to a standard 7 + 3 induction regimen. Karyotype and immunological phenotype of blasts examined prior to treatment were correlated with each other, with response to treatment and duration of survival. The following monoclonal antibodies (mAbs) were used for immunological phenotyping: VIM-D5 (CD15), MY7 (CD13), MY9 (CD33), VIM-2 (CDw65), VIM-13 (CD14), 63D3 (CD14), VID-1 (anti HLA-DR), WT1 (CD7), CLB-Ery3 (antiblood group H antigen), C17-27 (CD61), and an antiserum against TdT. Despite a considerable overlap between the individual groups, patients with specific aberrations as defined by the MIC classification (n = 39) showed distinct, characteristic, myeloid or myelomonocytic immunophenotypes. In M2/t(8;21) there was a significant association with negativity to CD13, in M3/t(15;17) with negativity to CD15 and HLA-DR, whereas in M4/inv(16) expression of blood group H antigen was unexpectedly found. The response to therapy, as well as rate of complete remission as duration of survival, was better in patients with M2/t(8;21), M3/t(15;17), and M4Eo/inv(16) as compared to all other patients and significantly worse in patients with M5a/t/del(11)(q23). In 35 patients with normal karyotype and 16 patients with cytogenetic anomalies not presently associated with FAB subtypes the expected correlations of rather immature myeloid immunologic phenotypes with M1 and M2 morphology and CD14 expression in monoblastic leukemias was found. Remission rate and survival were significantly worse in 19 patients with complex nonrandom aberrations, where blast cell expression of blood group H antigen and of TdT were significantly increased.

Paraparesis secondary to a spinal mass as the presenting feature of erythroleukaemia in a 10-month-old child.

Severe neurological impairment as the first symptom of acute leukaemia is a rather uncommon finding. We report the case of a 10-month-old infant who presented with acute paralysis of the lower extremities due to cord compression by an epidural tumour composed of malignant erythrocyte precursor cells. Diagnosis of erythroleukaemia (EL) was made by needle biopsy of the spinal epidural mass and confirmed by bone marrow aspiration. Antileukaemic treatment in combination with radiotherapy to the epidural tumour led to haematological remission and neurological recovery with disappearance of the mass lesion as demonstrated by MRI. However, haematological relapse occurred with death of the patient 7 months after diagnosis. This is the first reported case of EL presenting with paraparesis due to an epidural tumour. The clinical symptoms, results of cytogenetic and immunological studies and the clinical course are described.