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The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways.

LF Amorosa,1 CH Lee,2 AB Aydemir,1 S Nizami,1 A Hsu,1 NR Patel,1 TR Gardner,1 A Navalgund,3 D-G Kim,3 SH Park,4 JJ Mao,2 FY Lee11Center for Orthopaedic Research, Columbia University Medical Center, New York, NY, 2College of Dental Medicine, Columbia University Medical Center, New York, NY, 3Orthodontics, College of Dentistry, Ohio State University, Columbus, OH, 4Department of Orthopaedic Surgery, University of California at Los Angeles Medical Center, Los Angeles, CA, USABackground: To address the challenge of treating critical sized intercalary defects, we hypothesized that under physiologic cyclic loading, autografts, allografts, and scaffolds loaded with and without human mesenchymal stem cells (hMSCs) would have different biomechanical characteristics.Methods: Using a rat femoral defect model, 46 rats were assigned to four groups, ie, autograft (n = 12), allograft (n = 10), scaffold (n = 13), and scaffold with hMSCs (n = 11). The scaffold groups used a 5 mm segment of scaffold composed of 80% poly-ε-caprolactone and 20% hydroxyapatite. Rats were sacrificed 4 months postoperatively, and the repairs were assessed radiographically and biomechanically.Results: Autograft and allograft groups exhibited the most bridging callus, while the scaffold/hMSCs group had more callus than the scaffold repairs. Although signs of radiographic healing did not accurately reflect restoration of mechanical properties, addition of hMSCs on the scaffold enhanced bone formation. The scaffold alone group had significantly lower elastic and viscous stiffness and higher phase angles than other repairs and the contralateral controls. Addition of hMSCs increased the elastic and viscous stiffness of the repair, while decreasing the phase angle.Conclusion: Further comparative analysis is needed to optimize clinical use of scaffolds and hMSCs for critical sized defect repairs. However, our results suggest that addition of hMSCs to scaffolds enhances mechanical simulation of native host bone.Keywords: fracture healing, scaffolds, human mesenchymal stem cells, tissue engineering

The SCALOP trial tested the safety and efficacy of gemcitabine (Gem) versus capecitabine (Cap) based CRT following induction chemotherapy and showed that GemRT was associated with greater toxicity and worse survival1. The evaluation of investigator-delineated volumes and plan assessment from the pre-trial RTQA program using a single benchmark case showed considerable variation in gross tumour volume (GTV) outlining but no major deviations in other aspects of RT planning.

Influenza viral passaging through pre-vaccinated mice shows that emergent antigenic site mutations on the viral hemagglutinin (HA) impact host receptor-binding affinity and, therefore, the evolution of fitter influenza strains. To understand this phenomenon, we computed the Significant Interactions Network (SIN) for each residue and mapped the networks of antigenic site residues on a representative H1N1 HA. Specific antigenic site residues are ‘linked’ to receptor-binding site (RBS) residues via their SIN and mutations within “RBS-linked” antigenic residues can significantly influence receptor-binding affinity by impacting the SIN of key RBS residues. In contrast, other antigenic site residues do not have such “RBS-links” and do not impact receptor-binding affinity upon mutation. Thus, a potential mechanism emerges for how immunologic pressure on RBS-linked antigenic residues can contribute to evolution of fitter influenza strains by modulating the host receptor-binding affinity., National Institutes of Health (U.S.) (grant GM R37 GM057073-13), Singapore–MIT Alliance for Research and Technology, National Institutes of Health (U.S.) (grant AI058113)

The increasing complexity of cancer chemotherapy makes it mandatory that pharmacists be familiar with these highly toxic agents. This column reviews various issues related to the preparation, dispensing, and administration of cancer chemotherapy, both commercially available and investigational.

The separate meningeal layers and extraaxial spaces are complex and can only be differentiated by pathologic processes on imaging. Differentiation of the location of such processes can be achieved using different imaging modalities. In this pictorial review we address the imaging techniques, enhancement and location patterns, and disease spread that will promote accurate localization of the pathology, thus improving accuracy of diagnosis. Typical and unusual magnetic resonance (MR), computed tomography (CT), and ultrasound imaging findings of many conditions affecting these layers and spaces are described.

Introduction and objective. We examined patient waiting times, physician utilization, and exam room utilization in order to identify process improvements that may improve patient satisfaction. Methods. Time patient arrived to clinic, time patient was placed in the exam room, time the physician arrived in the exam room, and time physician discharged the patient from the exam room were prospectively recorded for 226 outpatient visits. Results. Overall, 63.2% of patients were on time for their scheduled appointment with 14.8% patient “no-shows.” On-time patients were found to have a longer wait time once in the exam room for the physician than those that were late ( 1 4 . 8 ± 9 . 2 minutes versus 1 1 . 0 ± 8 . 4 minutes, 𝑃 = . 0 0 5 ); however, those patients spent a significantly longer time with the physician ( 1 0 . 7 ± 6 . 0 minutes versus 8 . 9 ± 5 . 8 minutes, 𝑃 = . 0 4 1 ). Exam room utilization was lower for late patients (28.9% versus 44.7%, 𝑃 = . 0 3 ) with physician utilization lower in clinics with 3 or more late patients when compared to clinics with 2 or fewer (59.7% versus 68.7%, 𝑃 = . 0 0 4 ). Conclusions. Late patients had significantly less time with the physician than on-time patients. Late patients also decreased the overall efficiency of the clinic; therefore, measures to reduce late patients are vital to improve clinic efficiency.

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 × 106 donor CD8+ T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8+ T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-γ and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-α, perforin, or TRAIL. Recipients of IFN-γ−/− CD8+ T cells are unable to reject RENCA compared with recipients of wild-type CD8+ T cells and, importantly, neither group develops severe GVHD. IFN-γ−/− CD8+ T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-γ enhances class I and FasL expression and rescues the lytic capacity of IFN-γ−/− CD8+ T cells. These results demonstrate that the addition of low numbers of selected donor CD8+ T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-γ and FasL.

There is growing interest in the anticancer effect of the diabetes drug, metformin, and several clinical studies are underway worldwide. Metformin's anticancer effects are thought to be mediated by its activation of AMPK and subsequent downstream catabolic effects on protein and lipid metabolism pathways that are commonly dysregulated in cancer cells. We have used mass spectrometry techniques and stable isotopes to investigate the effects of metformin on breast cancer cell lipid metabolism. There was great variability in lipid metabolism response to 2mM metformin treatment in a panel breast cancer cell lines (MCF7, BT474, ZR-75-1, T47D MDA-MB-157, MDA-MB-468 and MDA-MB-231). Levels of stearic acid (p=0.019), palmitoleic acid (p=0.01), oleic acid (p=0.002), cis-vaccenic acid (p=0.003) increased 3-4 fold with metformin treatment in MCF7 cells but there was no significant change for MDA-MB-231 cells. 2mM Metformin decreased fatty acid β-oxidation of oleate 4-fold (p=0.03). Accumulation of de novo lipogenesis derived (p=0.004) and exogenous fatty acid (p=0.02) occurred in MCF7 cells but siRNA knockdown of AMPK did not abrogate these effects. A 3-fold decrease in the diglyceride to triglyceride ratio (p=0.02) suggested reduced lipolysis and inhibited adipose triglyceride lipase activity. Reduced incorporation of exogenous fatty acid into phospholipid was evident (p=0.002). Heterogeneity of lipid metabolism response correlated with effects on proliferation, oxygen consumption and mitochondrial morphology. Our data shows that metformin has marked effects on accumulation of fatty acid in breast cancer cells most likely due to reduced lipolysis and β-oxidation and these effects cannot be explained as a result of AMPK activation. This work will contribute to developing biomarkers for metformin's metabolic anticancer effect and our understanding of how to combine it with other therapies to exploit synthetic lethality. We have initiated a multicenter, phase II, single arm clinical study, to further characterise metformin influenced, cancer relevant metabolic pathways. We will look to assess the correlation between metabolic biomarkers, including the use of mass spectrometry and gas chromatography analysis of tissue samples, and metabolic response using 18F-FDG PET-CT scans. Citation Format: Simon Lord, Jennifer Collins, Barbara Fielding, Neel Patel, Fergus Gleeson, Adrian Harris, Fredrik Karpe. Metformin treatment leads to the accumulation of lipid in breast cancer cell lines - a potential new biomarker of response and mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1897. doi:10.1158/1538-7445.AM2013-1897

Several studies have suggested that race might play a role in outcome of patients following acute coronary syndrome (ACS) admission. To examine the association between patients’ race and long-term outcome after discharge following ACS admission from our hospital ACAP-PAIN registry. The study

Clinical Question: A 67-year-old Chinese man presented with pain and widespread skin erythema and detachment. A clinical diagnosis of toxic epidermal necrolysis (TEN) was made, which was supported by skin biopsy findings. The TEN was thought to be due to recent initiation of lamotrigine for mild epilepsy, and the drug was stopped immediately. The severity of TEN, measured by the Severityof-Illness Score for Toxic Epidermal Necrolysis (SCORTEN) scale, was 3 on admission owing to renal impairment, indicating a mortality risk of 35%. The patient was transferred to the burns unit for close monitoring of temperature, fluid balance, and appropriate dressings for his affected skin. A debate ensued among the dermatology, plastic surgery, and intensive care teams responsible for his care about whether intravenous immunoglobulin (IVIg) should be added to his treatment because improvement was slow with conservative treatment.

Accepted tools for early cancer detection run the gamut from Pap staining to detect cervical cancer detection to colonoscopy and biopsy for colorectal cancer detection to imaging (mammogram and, in high risk women, magnetic resonance imaging) and biopsy for breast cancer detection. These modalities use standard cytopathologic assessment to determine if disease is present. There are few biologic (DNA, RNA, protein or carbohydrate) markers (biomarkers) that are in general use for the detection of any cancer, or to identify individuals at increased cancer risk. Biomarkers have been identified that provide information to physicians on disease prognosis. Panels of biomarkers are being developed to predict response to treatment in individuals with known cancer, and some are currently in use. Nonethless, there is a great need to identify accurate biomarkers for the early detection of a new cancer, to identify individuals at increased cancer risk, and among those with cancer, to determine their likelihood of responding to a given treatment, risk of disease relapse and death. Micro (mi)RNAs hold promise as biomarkers for determining at risk individuals, for early cancer detection, and among those with cancer, to assess how likely a person is to respond a given treatment, their risk of disease recurrence and death.

Graft-versus-host-disease (GVHD) is a serious complication of allogeneic bone marrow transplantation (allo-BMT). The physiology of GVHD is dominated by alloactivated donor T cells, yet current treatments are often nonspecific and offer limited efficacy with relatively high toxicities. We screened for novel drug targets in alloactivated T cells in a murine allo-BMT model using a flow cytometric technique for the in vivo analysis of intracellular signaling. We defined the signaling profile of normal T cells and alloreactive T cells during GVHD, focusing on pathways involved in T cell receptor (TCR), costimulatory, and cytokine signaling. This analysis revealed that although proteins in multiple pathways (MAP kinases, PI3K, Jak/STAT signaling) were all heavily phosphorylated in alloactivated T cells, phosphorylation of STAT-3 and ERK1/2 were particularly prominently increased in donor alloactivated CD4 T cells. We further analyzed the importance of STAT-3 and ERK1/2 signaling in alloactivated T cells via the use of small-molecule inhibitors of STAT-3 (curcurbitacin E/I) and ERK1/2 phosphorylation (SL327). Treatment with these inhibitors attenuated T cell proliferation in response to anti-CD3+CD28 stimulation and in mixed leukocyte reactions in vitro in a dose-dependent fashion (figure 1). Figure 1 Figure 1. Pre-incubation of donor splenocytes with cucurbitacin E significanly reduced T cell activation (CD25, CD69) at 24 hours in adoptive transfer experiments in vivo (p Figure 2 Figure 2. We conclude that flow cytometric analysis of signaling pathways in single cells represents a novel methodology to assess the in vivo signaling profiles of specific cell populations in order to select drug targets for further study. STAT-3 and ERK1/2 phosphorylation may also represent potential targets to selectively inhibit donor T cell alloactivation and proliferation in GVHD.

Post-transplant peripheral T cell apoptosis is an important (but ill-defined) contributor to the delay in T cell recovery after an allogeneic bone marrow transplantation (BMT). Using murine bone marrow transplantation (BMT) models to analyze post-transplant peripheral T cell apoptosis, we found that donor BM-derived T cells had a higher percentage of apoptotic cells, with decreased BCL-2 levels in young (3-month old) and old (20-month old) recipients of T cell-depleted (TCD) and T cell-repleted allogeneic BMT. Allogeneic recipients of donor BM deficient of Fas, BAX and TRAIL, or with overexpression of BCL-2 and AKT showed no decrease in donor-derived apoptotic T cells, suggesting that these molecules are not directly responsible for post-transplant peripheral T cell apoptosis. However, we noted that both CD4+ and CD8+ Memory T cells (CD44high) exhibited increased apoptosis when compared to naive T cells post-BMT and therefore used RAG-2-eGFP transgenic mice to study whether T cell maturation affects peripheral T cell apoptosis. We found increased numbers of apoptotic cells in mature (eGFPlow) T cells compared to non-divided (eGFPhigh) recent thymic emigrants in RAG-2-eGFP transgenic mice and in recipients of RAG-2-eGFP transgenic (Tg) BM. Interestingly, most of the apoptotic T cells were of eGFPlowCD44high memory phenotype in both CD4+ and CD8+ T cells, while eGFPlowCD44low naive T cells showed no sign of enhanced apoptosis. Moreover, we found similar increased levels of apoptosis of homeostatically proliferating T cells (CD44high-memory-like cells) in recipients of lineage-depleted allogeneic RAG2-eGFP Tg BM or B6 BM. We then tested the effects of homeostatic T cell proliferation on peripheral T cell apoptosis in recipients of syngeneic CFSE-labeled T cells and found again increased levels of apoptosis in the fast proliferating populations of both CD4+ and CD8+ T cells. These cells expressed high levels of CD44 but down-regulated CD62L on their surface and in contrast to allogeneic proliferation exhibited low levels of CD25 expression and high level of IL-7Rα expression (75%). In addition, BCL-2 expression was down-regulated in the fast-proliferating cell population in both CD4+ and CD8+ T cells. We conclude that newly generated donor BM-derived T cells have increased peripheral T cell apoptosis, which is associated with homeostatic proliferation and does not involve Fas, BAX, TRAIL, and AKT. This mechanism of peripheral T cell apoptosis, which has not been recognized previously, plays a significant role in post-transplant T cell deficiency and represents a promising target for novel strategies to overcome post-transplant immune deficiency in BMT recipients.

We examine the problem of assigning plots of land to prospective buyers who prefer living next to their friends. In this setting, each agent's utility depends on the plot she receives and the identities of the agents who receive the adjacent plots. We are interested in mechanisms without money that guarantee truthful reporting of both land values and friendships, as well as Pareto optimality and computational efficiency. We explore several modifications of the Random Serial Dictatorship (RSD) mechanism, and identify one that performs well according to these criteria, We also study the expected social welfare of the assignments produced by our mechanisms when agents' values for the land plots are binary; it turns out that we can achieve good approximations to the optimal social welfare, but only if the agents value the friendships highly.