Discovery of a periosteal stem cell mediating intramembranous bone formation.

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear1-5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

DeTiN: overcoming tumor-in-normal contamination.

Comparison of sequencing data from a tumor sample with data from a matched germline control is a key step for accurate detection of somatic mutations. Detection sensitivity for somatic variants is greatly reduced when the matched normal sample is contaminated with tumor cells. To overcome this limitation, we developed deTiN, a method that estimates the tumor-in-normal (TiN) contamination level and, in cases affected by contamination, improves sensitivity by reclassifying initially discarded variants as somatic.

Mutational heterogeneity in cancer and the search for new cancer-associated genes.

Major international projects are underway that are aimed at creating a comprehensive catalogue of all the genes responsible for the initiation and progression of cancer. These studies involve the sequencing of matched tumour-normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false-positive findings that overshadow true driver events. We show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumour-normal pairs and discover extraordinary variation in mutation frequency and spectrum within cancer types, which sheds light on mutational processes and disease aetiology, and in mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and enable the identification of genes truly associated with cancer.

Immune reconstitution is preserved in hematopoietic stem cell transplantation coadministered with regulatory T cells for GVHD prevention.

Recipient-specific regulatory T cells (rsTreg) can prevent graft-versus-host disease (GVHD) by inhibiting donor T-cell expansion after hematopoietic stem cell transplantation (HSCT) in mice. Importantly, in adult humans, because of thymus involution, immune reconstitution during the first months after HSCT relies on the peripheral expansion of donor T cells initially present in the graft. Therefore, we developed a mouse model of HSCT that excludes thymic output to study the effect of rsTreg on immune reconstitution derived from postthymic mature T cells present within the graft. We showed that GVHD prevention with rsTreg was associated with improvement of the limited immune reconstitution compared with GVHD mice in terms of cell numbers, activation phenotype, and cytokine production. We further demonstrated a preserved in vivo immune function using vaccinia infection and third-party skin-graft rejection models, suggesting that rsTreg immunosuppression was relatively specific of GVHD. Finally, we showed that rsTreg extensively proliferated during the first 2 weeks and then declined. In turn, donor Treg proliferated from day 15 on. Taken together, these results suggest that rsTreg GVHD prevention is associated with improved early immune reconstitution in a model that more closely approximates the biology of allogeneic HSCT in human adults.

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy.

BACKGROUND: Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4(+)CD25(+)Foxp3(+) natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells. DESIGN AND METHODS: We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease. RESULTS: In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells. CONCLUSIONS: Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.

Regulatory T cell content in the bone marrow graft does not predict the occurrence of acute GVHD.

The subpopulation of regulatory T cells (Treg) was shown to play a key role in alloreactive responses. In allogeneic hematopoietic stem cell transplantation, several groups tested whether Treg content in transplants correlates with graft-versus-host disease (GVHD) with controversial results. In a retrospective study of 49 consecutive HLA-matched sibling transplantations, we studied the relationship between Treg content in bone marrow transplants and acute GVHD (aGVHD) occurrence. We observed a large variability in Treg in bone marrow grafts. However, contrary to previous observations in peripheral blood stem cells transplantation, we report that the Treg content of allogeneic bone marrow transplantation did not predict the occurrence of aGVHD.

Correlation of clinical and virologic responses to antiviral treatment and regulatory T cell evolution in patients with hepatitis C virus-induced mixed cryoglobulinemia vasculitis.

OBJECTIVE: Mixed cryoglobulinemia (MC) vasculitis is an autoimmune disorder associated with chronic hepatitis C virus (HCV) infection. We previously reported that MC vasculitis is associated with a quantitative defect of peripheral blood regulatory T cells. The aim of this study was to prospectively evaluate the evolution of this defect during the course of antiviral treatment. METHODS: Treg cell frequencies and numbers were analyzed in 131 patients with chronic HCV infection (including 66 with MC vasculitis) and 20 healthy volunteer donors. Measurements were taken before, during, and after treatment with PEGylated interferon alfa-2b plus ribavirin. RESULTS: At baseline, patients with MC vasculitis had a significantly lower frequency and number of Treg cells than did patients without MC vasculitis. Complete remission of MC vasculitis following antiviral treatment was associated with a significant increase in Treg cell levels compared with baseline. In contrast, Treg cell levels in nonresponders or partial responders, which did not differ from those in complete responders at baseline, remained unchanged over the course of the study. CONCLUSION: The strong positive correlation between clinical responses and Treg cell levels provides further support for the central role of Treg cells in the pathogenesis of HCV-induced MC vasculitis and emphasizes the dual role of Treg cells in chronic HCV infection: while Treg cells may hinder viral elimination, they also limit autoimmune injury.

Role of air-medical evacuation in mass-casualty incidents--a train collision experience.

BACKGROUND: On 21 June 2005, a passenger train collided with a truck near Revadim, Israel. The collision resulted in a multiple-scene mass-casualty incident in an area characterized by difficult access and a relatively long distance from trauma centers. A major disaster response was initiated by civilian and military medical forces including the Israeli Air Force (IAF) Search and Rescue teams. The air-medical evacuation from the accident site to the trauma centers, the activities of the airborne medical teams, and the lessons learned from this event are described in this report. METHODS: A retrospective analysis of data gathered from relevant elements that participated in management, treatment, and evacuation from the accident site was conducted. RESULTS: The accident resulted in 289 injured passengers and seven of the injured were killed. Six helicopters (performing nine sorties) participated. Helicopters evacuated trauma victims and aided in transporting air-medical teams to the site of the collision. Overall, 35 trauma victims (10 urgent) were evacuated by air to trauma centers. The length of time between the first helicopter landing and completion of the air evacuation was 83 minutes. The air-medical evacuation operation was controlled by the commander of the IAF Search and Rescue. Different crew compositions were set in real time. CONCLUSIONS: Air-medical evacuation during this unique event enabled prompt transportation of casualties from the scene to trauma centers and provided reasonable distribution of patients between various centers in the region. This operation highlighted the necessity for flexibility in medical decision-making and the need for nonconventional solutions regarding crew compositions during management of an airborne evacuation in similar settings. Air-medical evacuation should be considered as a part of responses to mass-casualty incidents, especially when the site is remote or characterized by accessibility difficulties.

EpiMethylTag: simultaneous detection of ATAC-seq or ChIP-seq signals with DNA methylation.

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a fast, low-input, low sequencing depth method, EpiMethylTag, that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA. Here we demonstrate that EpiMethylTag can be used to study the functional interplay between chromatin accessibility and TF binding (CTCF and KLF4) at methylated sites.

The prevalence of gastrointestinal diseases in Israeli adolescents and its association with body mass index, gender, and Jewish ethnicity.

OBJECTIVE: The objectives of this study were to describe gastrointestinal (GI) disease prevalence in Israeli adolescents, and possible associations between prevalence and body mass index (BMI), sex and Jewish ethnicity. DESIGN: A retrospective analysis of screening for GI disease performed by the Israeli Defense Forces recruiting office between 1998 and 2003. SETTING: Screening was performed at the recruiting office, and included detailed history and physical examination performed by a general practitioner. Further testing was performed as needed and the final diagnosis was established by a gastroenterologist at the recruiting office. PATIENTS: Seventeen-year-old Israeli nationals. RESULTS: During the study period, 466,855 (58.5% male) adolescents were screened for GI disease. Peptic ulcer disease, irritable bowel syndrome, and nonulcer dyspepsia were the most prevalent disorders affecting 466/10, 460/10, and 296/10, respectively. There was an increase in the prevalence of lactose intolerance during the study period and also an increase in the prevalence of peptic ulcer disease in females. The prevalence of inflammatory bowel disease has also increased from 100/10 to 149/10, although this trend failed to reach statistical significance (P=0.097). Higher BMI was associated with statistically significant higher prevalence rates of gastroesophageal reflux disease (P<0.05). A stronger association in females was found in gallbladder disease (P<0.001). Lower BMI was associated with higher prevalence rates of irritable bowel syndrome (P<0.001), and higher rates of inflammatory bowel disease and lactose intolerance in males (P<0.01 and <0.001, respectively). CONCLUSIONS: GI diseases are not uncommon among adolescents, and for some disorders prevalence is rising. The association between BMI and prevalence has been further clarified.

Physical examination and ECG screening in relation to echocardiography findings in young healthy adults.

BACKGROUND AND AIMS: Cardiovascular screening in young adults is an important tool in many occupational settings. Our aim was to test whether screening physical examination and ECG influence the rate of abnormal echocardiogarphic findings in young healthy subjects. METHODS: Consecutive echocardiography results of 18- to 20-year-old flight candidates were analyzed retrospectively. Echocardiographies were performed as part of a screening protocol, which includes ECG, physical examination and referral for echocardiography for any positive finding. A second stage includes universal echocardiography for all candidates. RESULTS: 1,066 subjects were evaluated; 489 subjects underwent echocardiography following referral because of abnormal auscultatory or ECG findings. Findings (mostly mild valvular insufficiencies) were demonstrated in 12.7%, with only 0.6% of subjects disqualified. In subjects who underwent universal echocardiography (n = 577), findings (mostly mild valvular insufficiencies) were detected in 18%, with only 0.5% of subjects disqualified. CONCLUSIONS: The rate of significant echocardiography findings is extremely low in this young and healthy population. The presence of abnormal findings on either physical examination or ECG screening was not demonstrated to alter the rate of abnormal echocardiographic findings. We suggest that the low yield of screening should be weighed against the cost of an unidentified congenital cardiac lesion in the specific setting.

Detection of occult lung impairment in welders by induced sputum particles and breath oxidation.

BACKGROUND: We evaluated particulate matter in combined induced sputum (IS) and oxidation in exhaled breath condensate (EBC) to test whether underlying inflammatory changes are present in asymptomatic welders. METHODS: Thirty welders from the Israel Defense Forces exposed to aluminum/iron (Group 1) or to cadmium/chromium/iron/lead/nickel (Group 2, N = 16) and 27 non-exposed administrators were studied. IS was recovered, particle size distribution, hydrogen peroxide and pH were measured, and exhaled breath condensate was collected. RESULTS: Group 2 had a higher % neutrophils than all other participants (P = 0.0001) and a higher % particles >2 microm in diameter (P = 0.0017). Percent particles and years of exposure highly correlated (P = 0.051). All welders EBC samples had higher concentrations of hydrogen peroxide than controls (P = 0.0001). pH was lower only for Group 2 (P = 0.0001). CONCLUSIONS: Combined IS and EBC measurements detect underlying inflammation in airways of asymptomatic welders. It emerged that airway inflammation is present in asymptomatic welders, and that the particle burden, inflammatory cells, and level of oxidative stress are a function of the type and the duration of welding. Am. J. Ind. Med. 51:503-511, 2008.

The pathophysiology of HCV induced B-cell clonal disorders.

Hepatitis C virus (HCV) has been shown in epidemiologic studies to be associated with immune system disorders. Primarily disorders that stem from B-cell regulatory control disturbance, such as mixed cryoglobulinemia (MC) and non-Hodgkin's lymphoma (NHL). The causative role of HCV in these disorders is supported by the response to anti-viral treatment. The understanding of the pathophysiological process leading from HCV infection to B-cell clonal expansion has improved significantly. Data supports an antigen-driven indirect stimulation of clonal expansion model, leading from oligoclonal to monoclonal expansion and in some instances to frank malignancy. HCV-E2 antigen has been suggested as a candidate antigen as well as NS3. Binding of the B-cell receptor by viral antigens coupled with direct binding of CD-81 by HCV-E2 has been shown to provide a strong proliferative signal. Additional regulatory elements are also affected in HCV-related B-cell clonal expansion, including the Fas and BLyS signaling mechanisms. Finally, genetic events such as bcl-2 rearrangement may also be involved in clonal expansion. In this review, evidence linking HCV with MC and NHL, as well as known events in the pathophysiological process are described.

Doctors--A species on the verge of extinction? A visit to the 22nd century clinic.

Medicine is undergoing profound change, but the basic format of the medical encounter has remained unchanged. Nevertheless, medicine in the 22nd century may be fully computerized, and a possible model is shortly depicted in this paper. Computer applications are constantly increasing their share in medical diagnosis, and may ultimately replace physicians. Treatment decisions have been submitted to standardized treatment guidelines, which may be applied more efficiently by computer applications. Although hundreds of studies have evaluated computerized tools in diagnosis and treatment, the possibility that computer applications may replace human physicians in the future is rarely raised. The effects of this process on doctors and medicine may be tremendous and will probably be felt even in early stages, and therefore, this process should be a subject of open discussion.

Patients' views on optimal visit length in primary care.

Visit length (VL) has been decreasing over the last decades. Patients and physicians alike hold that this may have adverse affects on quality of care and patient-doctor rapport. Our aim was to study the optimal VL as viewed by the patients as well as possible related factors such as demographicparameters and patient satisfaction. By using surveys, we determined that patient satisfaction was highly correlated with VL and satisfaction from VL. Optimal VL as viewed by the patients was 15.4 minutes on average with considerable variation. Longer expected VL was associated with longer visits. Patient satisfaction from the visit was significantly decreased when expressed optimal VL exceeded the estimated duration of the visit. The surveys demonstrated that in young adults, satisfaction is highly correlated with VL. When asked, patients expect attainable VLs, which are on average only somewhat longer then the scheduled duration. We believe that patients should take part in decisions regarding visit scheduling. We suggest that individualization may contribute to the cost-effectiveness of physicians' time.

Chromosomal Instability as a Driver of Tumor Heterogeneity and Evolution.

Large-scale, massively parallel sequencing of human cancer samples has revealed tremendous genetic heterogeneity within individual tumors. Indeed, tumors are composed of an admixture of diverse subpopulations-subclones-that vary in space and time. Here, we discuss a principal driver of clonal diversification in cancer known as chromosomal instability (CIN), which complements other modes of genetic diversification creating the multilayered genomic instability often seen in human cancer. Cancer cells have evolved to fine-tune chromosome missegregation rates to balance the acquisition of heterogeneity while preserving favorable genotypes, a dependence that can be exploited for a therapeutic benefit. We discuss how whole-genome doubling events accelerate clonal evolution in a subset of tumors by providing a viable path toward favorable near-triploid karyotypes and present evidence for CIN-induced clonal speciation that can overcome the dependence on truncal initiating events.

Long-term survivors of childhood malignancies--aeromedical dilemmas and implications.

Survival rates from childhood cancer have dramatically improved over the past three decades; average overall 5-yr survival rates are now > 75%. However, this has been achieved by treatments associated with significant morbidity that may present many years later. This review seeks to delineate the basic information necessary to evaluate flight-training candidates with a history of childhood cancer. We performed a literature review using the Medline database with appropriate search terms related to delayed morbidity and mortality associated with childhood cancer; we did not attempt to evaluate the risk of recurrent cancer. The neurological, cardiovascular, and pulmonary systems were identified areas of aeromedical concern. Central nervous disease and treatment-related effects may increase the risk of seizures or other neurocognitive sequelae. The cardiac toxicity of chemotherapeutic agents such as anthracyclines and radiation may cause late-occurring arrhythmia, cardiac failure, and sudden death, while available screening modalities are of limited value. Pulmonary disease and related treatment effects may cause a 9-fold increase of late-occurring pulmonary fibrosis and lung cancer, with increasing prevalence as long as 25 yr following the cancer diagnosis. Additionally, second malignancies may occur in up to 12.5% of cancer survivors at 25 yr after cancer diagnosis, affecting medical clearance for flight training. In summary, this review discusses the relevant aeromedical issues, including disabilities with specific relevance to the flying environment, risk estimation of late-occurring treatment complications, and possible interactions with occupational exposures in aircrew.

Cervical and lumbar MRI findings in aviators as a function of aircraft type.

BACKGROUND & AIMS: Neck pain and lower back pain (LBP) are frequently reported by military helicopter pilots (HP) and fighter pilots. A small number of studies have used imaging methods to evaluate spinal cervical degenerative findings in pilots exposed to high +Gz, with results indicating an increase in cervical disk protrusions in this population. We evaluated the cervical and lumbar spine with magnetic resonance imaging (MRI) to assess the prevalence of degenerative changes in three subpopulations of pilots. METHODS: Fighter pilots (FP), transport pilots (TP), and HP (10 pilots in each group) underwent cervical and lumbar MRI. Degenerative pathologic changes (disk herniation, cord compression, foraminal stenosis, and the presence of osteophytes) were evaluated in each group by two independent experienced radiologists. RESULTS: Cervical spine degenerative changes seemed to be associated with older age rather then aircraft type, affecting the older group of TP (8/10 pilots) more than the younger FP group who were exposed to high +Gz (3/10 pilots). In contrast, for lumbar spine degenerative changes, we found an uncommon pattern of lumbar spine degeneration in HP, affecting the upper part of the lumbar spine (10/13 disks found at L1-L4). CONCLUSIONS: The results of this study suggest that HP may have detectable degenerative lumbar findings. More research is needed to validate these findings as well as to explore the possible pathophysiological link between occupational exposures and the specific involvement of the upper lumbar spine.

Smoking cessation therapy and the return of aviators to flying duty.

Smoking cessation is an important part of every primary care physician's work. The importance of smoking cessation in the reduction of cardiovascular morbidity and mortality and the reduction of cancer incidence cannot be overstated. Various treatments have been established to encourage smoking cessation; these include group and individual psychological therapy, nicotine replacement in various forms, and drug therapy. The best-known drug used for smoking cessation is bupropion SR (Zyban). Smoking in aviators is not different than in the general population in terms of prevalence. Thus it is important for flight surgeons worldwide to be familiar with the magnitude of the problem and the available treatment options. Yet, it is also important for this community to become familiar with the relevance of this treatment to aviation and to recognize the limitations pertinent to flying personnel who are attempting to quit smoking. We present treatment options for smoking cessations and their limitations on flying personnel.

MicroRNAs in mutagenesis, genomic instability, and DNA repair.

MicroRNAs (miRNAs) are aiding our understanding of cancer biology, and are now coming close to therapeutic use as well. Here, we focus specifically on the interaction between miRNAs and genomic instability. MiRNA regulation is essential to many cellular processes, and escape from this regulatory network seems to be a common characteristic of malignant transformation. Genomic instability may preferentially target miRNAs either because of selective pressure or because of inherent vulnerability related to their location near fragile sites. Furthermore, disruption of miRNA processing elements affords a more global release from miRNA regulation. Finally, we review how miRNAs function as both effectors and modulators of the DNA damage response, intricately weaved with traditional elements such as ATM, P53, and MMR. Thus, miRNAs are important substrates for genomic instability and play a crucial role in cellular DNA sensing and repair mechanisms.