Rothmund-Thomson Syndrome-Like RECQL4 Truncating Mutations Cause a Haploinsufficient Low-Bone-Mass Phenotype in Mice.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of RECQL4. As existing murine models employ Recql4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating Recql4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4 and resulted in homozygous embryonic lethality and a haploinsufficient low-bone mass phenotype. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine Recql4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.

Effects of chemotherapy agents used to treat pediatric acute lymphoblastic leukemia patients on bone parameters and longitudinal growth of juvenile mice.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Therapies for pediatric ALL have improved such that more than 80% of patients survive to 5 years post-therapy, and most survive to adulthood. These ALL patients experience long-term side effects that permanently affect their quality of life, with bone loss and reduced longitudinal growth being the most common skeletal complications. To determine the effects of the chemotherapeutic agents used in ALL induction therapy on bone density and longitudinal growth in mice, we treated juvenile mice with doxorubicin, dexamethasone, vincristine, l-asparaginase, or combination therapy. At adulthood, mice were culled and bones collected and scanned by micro-computed tomography (micro-CT). Mice that received doxorubicin and combination therapy exhibited reduced longitudinal growth and significant reductions in trabecular bone volume, trabecular thickness, and trabecular number, with increased trabecular separation. Mean cortical thickness, cortical area, marrow area, endocortical perimeter, and polar moment of inertia were significantly reduced by doxorubicin and combination therapy. Vincristine treatment significantly decreased trabecular bone volume, trabecular number, and increased trabecular separation but had no effects on cortical bone. Dexamethasone treatment increased trabecular bone separation, cortical marrow area, and cortical bone periosteal perimeter. Mice treated with l-asparaginase did not have any bone phenotypes. In conclusion, these data indicate that the majority of the chemotherapy agents used in induction therapy for pediatric ALL have long-term effects on bone in mice. A single dose of doxorubicin in juvenile mice was sufficient to cause the majority of the bone phenotypes, with combination therapy intensifying these effects.

Osteosarcoma in the Post Genome Era: Preclinical Models and Approaches to Identify Tractable Therapeutic Targets.

PURPOSE OF REVIEW: Osteosarcoma (OS) is the most common cancer of bone, yet is classified as a rare cancer. Treatment and outcomes for OS have not substantively changed in several decades. While the decoding of the OS genome greatly advanced the understanding of the mutational landscape of OS, immediately actionable therapeutic targets were not apparent. Here we describe recent preclinical models that can be leveraged to identify, test, and prioritize therapeutic candidates. RECENT FINDINGS: The generation of multiple high fidelity murine models of OS, the spontaneous disease that arises in pet dogs, and the establishment of a diverse collection of patient-derived OS xenografts provide a robust preclinical platform for OS. These models enable evidence to be accumulated across multiple stages of preclinical evaluation. Chemical and genetic screening has identified therapeutic targets, often demonstrating cross species activity. Clinical trials in both PDX models and in canine OS have effectively tested new therapies for prioritization. Improving clinical outcomes in OS has proven elusive. The integrated target discovery and testing possible through a cross species platform provides validation of a putative target and may enable the rigorous evaluation of new therapies in models where endpoints can be rapidly assessed.

ATP-dependent helicase activity is dispensable for the physiological functions of Recql4.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, juvenile cataracts, sparse or absent hair, and predisposition to specific malignancies such as osteosarcoma and hematological neoplasms. RTS is caused by germ-line mutations in RECQL4, a RecQ helicase family member. In vitro studies have identified functions for the ATP-dependent helicase of RECQL4. However, its specific role in vivo remains unclear. To determine the physiological requirement and the biological functions of Recql4 helicase activity, we generated mice with an ATP-binding-deficient knock-in mutation (Recql4K525A). Recql4K525A/K525A mice were strikingly normal in terms of embryonic development, body weight, hematopoiesis, B and T cell development, and physiological DNA damage repair. However, mice bearing two distinct truncating mutations Recql4G522Efs and Recql4R347*, that abolished not only the helicase but also the C-terminal domain, developed a profound bone marrow failure and decrease in survival similar to a Recql4 null allele. These results demonstrate that the ATP-dependent helicase activity of Recql4 is not essential for its physiological functions and that other domains might contribute to this phenotype. Future studies need to be performed to elucidate the complex interactions of RECQL4 domains and its contribution to the development of RTS.

Efficacy of N,N'bis-(2-mercaptoethyl) isophthalamide on mercury intoxication: a randomized controlled trial.

BACKGROUND: Chronic mercury intoxication is a severe health issue and occurs especially in gold mining communities. Common chelators used for improving mercury elimination are not everywhere available and challenged by poor cell wall penetration. This study is part of a feasibility trial and the aim was to gather first information about the efficacy of the newly developed chelator N,N'bis-(2-mercaptoethyl) isophthalamide (NBMI) on chronic mercury intoxication. METHODS: In this three-armed, placebo-controlled randomized trial, 36 miners with mercury urine levels exceeding 15 µg/l were administered 100 mg NBMI, 300 mg NBMI or placebo for 14 days. Levels of mercury in urine [µg/l and µg/g creatinine] and plasma l were analyzed. Therapeutic effect was assessed using the medical intoxication score (MIS) and its single health outcomes (e.g. excessive salivation, sleeping problems), fatigue scores, a neuromotoric test battery (CATSYS) and a neurological outcome (Finger to nose test). RESULTS: Physical fatigue was significantly decreased in the 300 mg NBMI group compared to the control. Mercury concentration in urine following 300 mg NBMI treatment was significantly lowered compared to control, however, this effect was less distinct with adjustment for creatinine. CONCLUSION: NBMI showed an effect on physical fatigue and there were indications to positive effects on other symptoms as well. More comprehensive studies are mandatory to verify the effects of NBMI as a novel tool for treating mercury intoxications. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02486289 . Date of registration: June 24, 2015.

Murine models of osteosarcoma: A piece of the translational puzzle.

Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome.

Spanish translation, cross-cultural adaptation, and validation of the Questionnaire for Diabetes-Related Foot Disease (Q-DFD).

PURPOSE: To translate, cross-culturally adapt, and validate the Questionnaire for Diabetes-Related Foot Disease (Q-DFD), originally created and validated in Australia, for its use in Spanish-speaking patients with diabetes mellitus. PATIENTS AND METHODS: The translation and cross-cultural adaptation were based on international guidelines. The Spanish version of the survey was applied to a community-based (sample A) and a hospital clinic-based sample (samples B and C). Samples A and B were used to determine criterion and construct validity comparing the survey findings with clinical evaluation and medical records, respectively; while sample C was used to determine intra- and inter-rater reliability. RESULTS: After completing the rigorous translation process, only four items were considered problematic and required a new translation. In total, 127 patients were included in the validation study: 76 to determine criterion and construct validity and 41 to establish intra- and inter-rater reliability. For an overall diagnosis of diabetes-related foot disease, a substantial level of agreement was obtained when we compared the Q-DFD with the clinical assessment (kappa 0.77, sensitivity 80.4%, specificity 91.5%, positive likelihood ratio [LR+] 9.46, negative likelihood ratio [LR-] 0.21); while an almost perfect level of agreement was obtained when it was compared with medical records (kappa 0.88, sensitivity 87%, specificity 97%, LR+ 29.0, LR- 0.13). Survey reliability showed substantial levels of agreement, with kappa scores of 0.63 and 0.73 for intra- and inter-rater reliability, respectively. CONCLUSION: The translated and cross-culturally adapted Q-DFD showed good psychometric properties (validity, reproducibility, and reliability) that allow its use in Spanish-speaking diabetic populations.

Spontaneous regression of splenic artery pseudoaneurysm: a rare complication of acute pancreatitis.

Spontaneous pseudoaneurysm regression is a rare event. In particular, the spontaneous regression of a splenic artery pseudoaneurysm has, to our knowledge, been previously documented in only two case reports. Furthermore, the pathophysiological mechanism of this event remains unclear. However, it is fully known that this vascular complication is potentially life-threatening and presents a high mortality rate if untreated. We report the case of a 49-year-old man affected by acute pancreatitis. Computed tomography was performed, and showed a pseudoaneurysm of the splenic artery. This patient underwent endoscopic retrograde cholangiopancreatography to treat the pancreatitis, while the vascular complication was managed with a careful and conservative treatment. On day 6 of hospitalization, a second computed tomography scan was performed and revealed complete regression of the pseudoaneurysm. This case describes the diagnosis and management of splenic artery pseudoaneurysm following acute pancreatitis and its spontaneous regression.

Pathogenesis and therapeutics of interstitial lung disease in systemic sclerosis.

Interstitial lung disease is a common manifestation in systemic sclerosis and is considered as one of the two main causes of death among these patients. Although the pathogenesis of interstitial lung disease related to systemic sclerosis (SSc-ILD) is very complex and not yet fully understood, diverse mechanisms such as vascular injury, altered immunological response and inflammatory activation have been proposed. Vascular injury is considered as the earliest event in the pathogenesis of this disease and has been associated with an excessive formation of alveolar capillaries, circulating endothelial cells, and increased expression of endothelin-1. Different cells like myofibroblasts, fibroblasts, endothelial cells and T lymphocytes are involved in the inflammatory activation. Meanwhile, lymphocyte activation, release of several cytokines and autoantibody production play an important role in the immunological response. To date, the treatment of SSc-ILD is not totally defined, as studies have shown mixed results. Given the high progression of the disease, it is difficult to enroll patients for clinical trials. Therefore, there is lack of evidence to guide therapeutic approaches. Throughout this paper, we present evidence supporting that the combination of glucocorticoids and cyclophosphamide is considered the best regimen for patients with SSc-ILD. In addition, we present data regarding the use of azathioprine, mycophenolate, anti-fibrosing agents, bosentan, rituximab, and imatinib mesylate as alternative therapies. Finally, for patients who are unresponsive to pharmacologic interventions, we present data regarding the efficacy of highdose immunosuppression with autologous transplantation of hematopoietic stem cells, and lung transplantation.