Search

Western Ghats are designated as world heritage sites and global biodiversity hotspots. Microbial diversity in this forest area has been largely neglected and is getting attention since the end of the last millennium. In this review, we have studied and organized various microorganisms from Western Ghats and their diversity, important characteristics and potential biotechnological applications. Microorganisms from Western Ghats have been explored individually for potential bioactive molecules. While most of the microorganisms were analyzed for antimicrobial activities, there have been studies on microbial promotion of plant growth. The microbes analyzed included from aquatics, soil, rhizosphere, phyllosphere and even as endophytes. There have been microorganisms which have shown antioxidant and anti-cancerous activities. There are microorganisms capable of degrading plant wastes and also xenobiotics. Microorganisms capable of producing industrially important enzymes have also been reported. The present review explores largely neglected microbial diversity with respect to their ability to produce potential bioactive biomolecules.

The present study was carried out to characterize six Pasteurella multocida isolates obtained from sheep, duck, pig, cow, poultry, and P. multocida P52 vaccine strain by multilocus sequence typing (MLST). All the isolates were first confirmed positive for P. multocida by culturally first and then by a biochemical method. The Deoxyribonucleic acid (DNAs) were extracted from the isolates and PM-PCR was carried out, which confirmed all the isolates as P. multocida, and further studied for capsular typing. Among them three isolates of P. multocida were typed as capsular type A (sheep, duck and poultry), and the other two were typed as capsular type B (cow and pig). Specific primers and protocols described in the Rural Industries Research and Development Corporation (RIRDC) MLST scheme for P. multocida were used to amplify the seven house-keeping genes, viz., adk, est, pmi, zwf, mdh, gdh, and pgi. The purified PCR products were subjected to sequencing by Sanger's method. The total length sequences of genes were trimmed to the length of 466 bp, 535 bp, 602 bp, 500 bp, 521 bp, 530 bp, and 560 bp for adk, est, pmi, zwf, mdh, gdh and pgi genes, respectively, as described in the RIRDC MLST scheme. Isolates of sheep, duck, pig, cattle, poultry, PAS-506/06, PAD-914/11, PAP-721/09, PAC-93/15, PAP-87/13 and P52 vaccine strain were grouped to sequence type 122, 307, 308, 309, 129 and 122, respectively.

Acute myeloid leukemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. With increasing age, tissues accumulate senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of a set of proinflammatory cytokines, chemokines, and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). Here, we report that AML blasts induce a senescent phenotype in the stromal cells within the BM microenvironment and that the BM stromal cell senescence is driven by p16INK4a expression. The p16INK4a-expressing senescent stromal cells then feed back to promote AML blast survival and proliferation via the SASP. Importantly, selective elimination of p16INK4a+ senescent BM stromal cells in vivo improved the survival of mice with leukemia. Next, we find that the leukemia-driven senescent tumor microenvironment is caused by AML-induced NOX2-derived superoxide. Finally, using the p16-3MR mouse model, we show that by targeting NOX2 we reduced BM stromal cell senescence and consequently reduced AML proliferation. Together, these data identify leukemia-generated NOX2-derived superoxide as a driver of protumoral p16INK4a-dependent senescence in BM stromal cells. Our findings reveal the importance of a senescent microenvironment for the pathophysiology of leukemia. These data now open the door to investigate drugs that specifically target the “benign” senescent cells that surround and support AML.

S100P has been shown to be a marker for carcinogenesis where its expression in solid tumours correlates with metastasis and a poor patient prognosis. This protein’s role in any physiological process is, however, unknown. Here we first show that S100P is expressed both in trophoblasts in vivo as well as in some corresponding cell lines in culture. We demonstrate that S100P is predominantly expressed during the early stage of placental formation with its highest expression levels occurring during the first trimester of gestation, particularly in the invading columns and anchoring villi. Using gain or loss of function studies through overexpression or knockdown of S100P expression respectively, our work shows that S100P stimulates both cell motility and cellular invasion in different trophoblastic and first trimester EVT cell lines. Interestingly, cell invasion was seen to be more dramatically affected than cell migration. Our results suggest that S100P may be acting as an important regulator of trophoblast invasion during placentation. This finding sheds new light on a hitherto uncharacterized molecular mechanism which may, in turn, lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation.

The burden of infections is known to increase with age. Not only is ageing associated with greater susceptibility to infections but also an increase in subsequent morbidity and mortality. The bone marrow (BM) niche is essential for the body's response to infection. Haematopoietic stem and progenitor cells (HSPCs) heavily rely on their supporting BM microenvironment to effectively expand and differentiate in response to stress and infection (1). The role of senescent cells has been explored in a number of age-related diseases including acute myeloid leukaemia (2). Here we explore the role of senescence during natural ageing in the BM microenvironment, the mechanism which drives this and how this impacts on the metabolic health of HSPCs. BM was isolated from aged (18-24 months) and young (8-12 weeks) C57Bl/6 mice and flow cytometry was used to compare mitochondrial membrane potential (ΔΨm) and mitochondrial ROS in HSPCs. Results show that HSPCs from aged animals increase in numbers and accumulate mitochondria with low membrane potential with lower mitochondrial ROS. Next, young and aged mice were treated with lipopolycaccharide (LPS). Metabolic analysis revealed that HSPCs from young mice increase metabolism of mitochondrial TCA cycle substrates in response to LPS whereas aged HSPCs continued to rely on glycolysis. When HSPCs from aged C57Bl/6 mice (CD45.2+) were FACS purified and adoptively transferred into young PepCboy (CD45.1+) mice, thus removing them from the aged BM microenvironment, they were able to recover their mitochondrial health and showed an improved metabolic response to treatment with LPS. Furthermore, qRT-PCR analysis of p16 and p21 expression in HSPCs and mesenchymal stromal cells (MSC) showed that MSCs, but not HSPCs, acquire a senescent phenotype in aged mice, and depletion of senescent cells in the p16-3MR mouse model (3) allowed recovery of HSPC mitochondrial function and response to LPS. Mechanistically, we found a significant upregulation of the anti-apoptotic protein BCL-XL in MSCs of aged mice. This has previously been described to drive the senescent phenotype and prevent apoptosis in senescent cells. By over-expressing GFP-tagged BCL-XL in MSCs and then co-culturing them with LSKs we were able to show that BCL-XL is transferred from MSCs to HSPCs in vitro. Finally, we demonstrated that targeting BCL-XL in vivo, using the senolytic drug ABT-263, in aged mice can restore the HSPC metabolic response to stress resulting in upregulation of TCA cycle metabolism. In conclusion, we show that the aged BM microenvironment is responsible for the HSPC metabolic shortfall resulting in impaired response to stress. Targeting the senescent cells in the environment restored the HSPC metabolic function and their response to infection in aged mice. This suggests that manipulation of the ageing BM microenvironment can help to improve the body's response to infection. 1. Mistry JJ, Marlein CR, Moore JA, Hellmich C, Wojtowicz EE, Smith JGW, et al. ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection. Proc Natl Acad Sci U S A. 2019;116(49):24610-9. 2. Abdul-Aziz AM, Sun Y, Hellmich C, Marlein CR, Mistry J, Forde E, et al. Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment. Blood. 2019;133(5):446-56. 3. Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell. 2014;31(6):722-33. Disclosures No relevant conflicts of interest to declare.

Acute infection is known to promote rapid differentiation of hematopoietic stem cells (HSC) and expansion of leukocytes. Metabolic changes in the HSC underpin the mammalian response to pathogenic stimuli however, knowledge of how this occurs is not fully understood. Here we investigate the immunometabolic changes which facilitate HSC expansion in response to acute infection. We created a novel in vivo transplant model to understand if the immune response to infection involves the acquisition of free fatty acids (FFA) by HSCs in the bone marrow (BM). We transduced CD45.1 lineage negative, c-kit positive cells with firefly luciferase and transplanted into CD45.2 animals. Post engraftment animals were treated with lipopolysaccharide (LPS). Live animal imaging confirmed activation of luciferase in the BM, demonstrating in vivo, uptake of long chain FFA by haematopoietic cells in response to LPS. To confirm the specific haematopoietic stem and/or progenitor cells (HSPC) with increased lipids during infection, mice were treated with S.typhimurium for 72h then sacrificed Analysis of LSK, MPP and HSC populations (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) showed increased intracellular BODIPY 493/503 neutral lipid dye and uptake of Bodipy FL-C12 (FFA linked to bodipy) at 72 h compared to control non-infected animals. Together, these experiments show that HSC, multipotent progenitor (MPP) and LSK (LN-, CD117+, Sca1+) cells all acquire FFA in response to bacterial infection. Seahorse mitochondrial stress test confirmed increased oxygen consumption levels in HSC from LPS (16 hours) and S.typhimurium (72 hours) treated C57BL/6. LSK metabolic activity for different substrates was assessed LSKs from LPS treated animals had an increased dependency on β-oxidation when compared to control cells. Moreover, the β-oxidation inhibitor etomoxir blocks LSK basal metabolism and reduced HSC expansion as measured by Ki-67 staining in LPS and S.typhimurium treated animals. To understand the importance of β-oxidation in HSC expansion in response to infection we first measured CPT1A expression in HSC. RNA analysis confirmed CPT1A was increased in sorted HSCs in response to LPS. Next we transplanted WTor CPT1A KD cells into WT animals, and treated with LPS. HSC expansion as measured by Ki-67 staining was inhibited in the CPT1A KD cells in response to LPS, thus confirming the importance of β-oxidation based metabolism in HSC expansion. To determine how FFA are transported into the HSC we analysed the expression of known genes to be involved in lipid uptake. HSC sorted from LPS and S.typhimurium treated animals had increased CD36 mRNA expression compared to control. C57BL/6 mice were pre-treated with the CD36 inhibitor sulfosuccinimidyl oleate (SSO) before injection with LPS. HSCs had had less lipids, lower maximal respiration and reduced cycling when compared to animals treated with LPS alone. In a similar way CD36 KO animals were treated with LPS for 16 h, no increased FFA uptake or increased lipid content or HSC cycling was observed. Seahorse mitochondrial stress test confirmed CD36-/- LSK have increased basal ECAR but no change in basal OCR in response to S.typhimurium infection. Moreover, CD36-/- LSK have reduced dependency on β-oxidation. To determine if the uptake of FFA response is specific to HSC we transplanted CD36+/+ (CD45.1) LK into CD36-/- (CD45.2) animals. We found that CD36 expression was elevated in CD36+/+ HSC transplanted into CD36-/- treated with LPS, normal uptake of FFA, an increase in lipid content and cycling of HSCs. In addition, transplanted WT CD36+/+ (CD45.1) LSK into CD36-/- (CD45.2) animals reversed the metabolic phenotype observed in CD36-/- in response to LPS. We transplanted WT(CD36+/+) or CD36KO (WT(CD36-/-)) LSK into WT animals infected with S. typhimurium for 4 days. WT(CD36-/-) transplanted animals showed enhanced mortality, increased weight loss and increased liver injury. We also observed less HSC cycling and a reduced lipid content in HSC from WT(CD36-/-) compared to WT(CD36+/+). These findings expand the mechanistic understanding of the interplay between HSC and the bone marrow microenvironment, and in doing so explains how immunometabolic reprogramming of HSCs during infection supports the metabolic demands of HSCs in response to a pathogenic challenge without which results in increased susceptibility to infection. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:AbbVie: Research Funding; Janssen: Research Funding.

Introduction The burden of age-related diseases is steadily increasing as our population ages. A better understanding of the physiological changes that occur with age may provide an insight into the processes that promote disease development which in turn may help to identify potential targets for the management of these diseases. Haematopoietic stem cells (HSCs) play a vital role in the bone marrow response to infection and previous work has shown that emergency granulopoiesis is supported by increased mitochondrial function in HSCs (1). HSC numbers have previously been shown to increase with age whilst mitochondrial function is known to decline with age. Here we investigate how mitochondrial health changes in ageing HSCs and how this impacts on HSC function and their ability to respond to stress. Methods C57Bl/6 mice were aged to 18-24 months and 8-12-week-old mice were used as controls. Both aged and young mice were sacrificed, and their BM was isolated. Flow cytometry was used to measure numbers of HSCs and mitochondrial content and function using MitoTracker Green and TMRM. Seahorse metabolic flux analysis was used to assess metabolic changes in young and aged HSC populations. Next young and aged C57Bl/6 mice were treated with lipopolycaccharide (LPS) and sacrificed after 16 hours. Their BM was isolated HSC populations were quantified and mitochondrial health was assessed using flow cytometry. Finally, ABT-263 was used to deplete senescent cells in the BM microenvironment. C57Bl/6 mice were treated with 100mg/kg of ABT-263 or vehicle control by oral gavage for 17 days, they were then treated with 0.4mg/kg of LPS and sacrificed after 12 hours. They BM was isolated and analysed by flow cytometry as described above. Results In aged mice HSC numbers were increase and this was associated with an increase in mitochondrial content but a decrease in mitochondrial function. Levels of oxidative phosphorylation (OXPHOS) or glycolysis were not significantly changed in aged HSCs in a steady state. In response to infection, however, in aged mice HSC mitochondrial content and function was not shown to increase following LPS treatment when compared to HSCs from young mice. Moreover, LPS significantly increased OXPHOS in young HSCs whilst aged HSCs were shown to favour glycolysis. Finally, aged HSCs were shown to have increased expression of the anti-apoptotic protein BCL-XL but not BCL-2. In vitro analysis of aged HSC showed the BCL-XL inhibitor ABT-263 but not the BCL-2 inhibitor ABT-199 induced HSC apoptosis. Finally, to determine the effect of targeting BCL-XL on HSCs in vivo we treated aged animals with ABT-263 daily for 17 days and then treated these animals with LPS. Results show that treating aged animals with ABT-263 allowed the HSC to use OXPHOS instead of glycolysis in response to stress. Conclusion Aged HSCs have altered mitochondrial content and function and this impairs their ability to respond to infection through changes in OXPHOS. Instead aged HSC have increased reliance on glycolysis in response to LPS treatment, which is less efficient, and this may impact on HSC function. Targeting BCL-XL with ABT-263 allows elimination of aged HSCs within the BM, reverses some of the age-related mitochondrial changes observed and improves the HSCP ability to respond to stress. References 1. Mistry JJ, Marlein CR, Moore JA, Hellmich C, Wojtowicz EE, Smith JGW, et al. ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection. Proc Natl Acad Sci U S A. 2019;116(49):24610-9. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to 1-methylnicotinamide. We have previously shown that NNMT is significantly overexpressed in the brains of patients who have died of Parkinson's disease, and others have shown that NNMT is significantly overexpressed in a variety of diseases ranging from cancer to hepatic cirrhosis. In vitro overexpression has revealed many cytoprotective effects of NNMT, in particular increased complex I activity and ATP synthesis. Although this appears to be mediated by an increase in 1-methylnicotinamide production, the molecular mechanisms involved remain unclear. In the present study, we have investigated the role that sirtuins 1, 2 and 3, class III DNA deacetylase enzymes known to regulate mitochondrial energy production and cell cycle, have in mediating the effects of NNMT upon complex I activity. Expression of NNMT in SH-SY5Y human neuroblastoma cells, which have no endogenous expression of NNMT, significantly increased the expression of all three sirtuins. siRNA-mediated silencing of sirtuin 3 expression decreased complex I activity in NNMT-expressing SH-SY5Y cells to that observed in wild-type SH-SY5Y, and significantly reduced cellular ATP content also. These results demonstrate that sirtuin 3 is a key mediator of NNMT-induced complex I activity and ATP synthesis. These results further reinforce a central role for NNMT in the regulation of energy homeostasis and provide further mechanistic insight into the consequences of enhanced NNMT expression.

Acute myeloid leukaemia (AML) causes 85,000 global deaths per year, which is estimated to double by 2040. AML has been shown to be highly dependent on the bone marrow (BM) microenvironment. Mesenchymal stromal cells (MSC) specifically, play an important pro-tumoral role. We previously demonstrated AML blasts rely on higher mitochondrial content and are more dependent on oxidative phosphorylation compared to non-malignant unstimulated CD34+ progenitor cells. To achieve this high metabolic demand, functional mitochondria are transferred from the MSC to the AML blasts. Mitochondrial transfer has also been demonstrated in myeloma in a pro-tumoral process regulated by CD38. Taken together, these data indicate that mitochondria are a biologically plausible and attractive drug target in the treatment of AML.We investigated the role mitochondrial transfer and the subsequent metabolic consequences of inhibiting CD38 using daratumumab on the AML BM microenvironment.Primary AML blasts and MSC were isolated from patient's BM. Mitochondrial transfer was assessed by coculture of AML and MSC in vitro using both qPCR DNA analysis and flow cytometry analysis. We used an NSG xenograft mouse model of AML, we transplanted OCI-AML3-luc, and treated the animals with either vehicle control (PBS) or daratumumab (5mg/kg) on day 9 and 16 followed by bioluminescence imaging. Post transplantation, AML mitochondrial transfer was assessed by murine mitochondrial DNA in human AML blasts by species specific PCR analysis. Post transplantation mitochondrial health and function was measured by TMRM and Seahorse analysis.Targeting CD38 using daratumumab inhibits the transfer of mitochondria from MSC to AML in vitro. In vivo, treatment with daratumumab significantly reduced tumor burden and improved survival compared to untreated controls. Additionally, we found two doses of daratumumab resulted in reduced mitochondrial potential, mitochondrial content and oxygen consumption rate in the AML cells sorted from the human xenograft mouse model. Finally, analysis of human AML cells sorted from NSG mouse BM showed reduced levels of mouse mitochondrial DNA in the human AML blasts from daratumumab treated mice compared to mice with AML treated with vehicle control.CD38 inhibition by daratumumab treatment inhibits mitochondrial transfer from MSC to AML blasts in the BM microenvironment. This results in a reduction in oxidative phosphorylation in AML blasts and subsequent reduced leukemia growth, which in turn results in improved NSG/AML animal survival. Whilst it is probable that daratumumab has a number of mechanisms of action, here we demonstrate inhibition of mitochondrial transfer is an addition to the list for this drug in AML. These data support the further clinical investigation of daratumumab based chemotherapeutic strategies as a therapeutic approach for the treatment AML. Citation Format: Jayna J. Mistry, Jamie A. Moore, Christopher R. Marlein, Charlotte Hellmich, Genevra Pillinger, Federica Di Palma, Angela Collins, Kristian M. Bowles, Stuart A. Rushworth. Targeting CD38 inhibits metabolic capacity of acute myeloid leukemia in the tumour microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2974.

Haematopoietic stem and progenitor cells (HSPC) are tightly regulated in a balance between self-renewal and differentiation and undergo rapid expansion in response to infection. In the context of acute bacterial infection, the hematopoietic system needs energy to drive the immune response necessary for host survival. In an adult human, bone marrow adipose tissue accounts for up to 70% of bone marrow and we have previously shown that free fatty acids are transferred from adipocytes to AML blasts providing the AML blasts more energy for proliferation and survival (Shafat, Blood 2017). Therefore, we hypothesis that during an acute bacterial infection, the HSPC population uptakes free fatty acids (FFA) to support and sustain the rapid response to bacterial infection. To determine if HSPC acquired FFA we infected C57BL/6 with Salmonella Typhimurium (S.typhimurium) or treated C57BL/6 with lipopolysaccharide (LPS) for 2 and 16 hours. Using both models we show that the HSPC populations (HSC (haematopoietic stem cell), MPP (multipotent progenitor) and LSK (LN-, CD117+, Sca1+)) all accumulate FFA as measured by staining with neutral lipid-specific BODIPY 493/503 dye. To determine if this was uptake or accumulation of fatty acid within the HSPC of animals infected with S.typhimurium we used BODIPY®-dodecanoic acid fluorescent fatty acid to trace FFA uptake. HSPC populations from animals treated with LPS had a higher uptake of BODIPY®-dodecanoic acid when compared to the control treated mice. To assess metabolic changes within HSC population in response to infection, cells were sorted (LN-, CD117+, Sca1+, CD48-, CD150+ and CD34+) and analysed for gene expression changes. In HSC, increases were observed in several metabolic genes including FABP4, CD36 and CPT1 in response to S.typhimurium as well as LPS at 16 hours post stimulation. Moreover, following cell sorting, the LSK (LN-, CD117+, Sca1+) population were assessed for metabolic activity for different substrates. The LSK population from LPS treated animals had an increased dependency on beta oxidation when compared to the control. Moreover, etoximir (a beta-oxidation inhibitor) inhibited LSK basal metabolism and reduced LSK expansion (as measured by colony forming cell assay and Ki-67 staining) in animals treated with LPS. Our data indicates that infection drives uptake of FFA in different HSPC populations. The FFA are metabolised by beta oxidation which provides the energy for the expansion of leukocytes. Disclosures Bowles: Abbvie: Research Funding; Janssen: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

This article is a commentary on the role of sports cardiologists in the athletic arena and the beneficial impact they offer sports medicine in the comprehensive care of competitive athletes. The focus is a dialogue on current recommendations for primary prevention of sudden cardiac arrest (SCA), incorporating elements of the preparticipation evaluation and continuing care of athletes with diagnosed heart disease (HD). The feasibility and potential advantages of implementing well-designed preparticipation cardiovascular screening programs and the role of sports cardiologists to educate primary care team physicians on secondary prevention of SCA and proper treatment of underlying HD are discussed.

Introduction The bone marrow microenvironment regulates the production of both hematopoietic and non-hematopoietic cells for the maintenance of blood production under normal and stress conditions. Intercellular mitochondrial transfer has recently been reported in an acute myeloid leukemia as well as models of lung inflammation. In the context of acute bacterial infection the hematopoietic system needs to drive the granulocytic response necessary for host survival. Therefore, we hypothesis that under stressed hematopoiesis, mitochondria move from the non-hematopoietic cells of the bone marrow microenvironment to the hematopoietic stem/progenitor cells (HSPC) to rapidly support and sustain the host response to bacterial infection. Methods C57/BL6 mice were injected with lipopolysaccharide (LPS) for 16 hours or infected with salmonella for 72 hours. The mice were sacrificed after 16 hours and the BM harvested and analyzed using flow cytometry for HSPC mitochondrial content using MitoTracker Green FM. Human cord blood CD34+ cells were engrafted into 3 to 4 week old NSG mice (hu-NSG). After 3 months engraftment of human cord blood CD34+ cells was verified by flow cytometry. Hu-NSG mice were then injected with LPS. HSPC populations were isolated by cell sorting for human CD34, CD38, CD45RA, CD90 and CD49f. Quantification of mitochondrial DNA (mtDNA) transfer was undertaken using Taqman qPCR with species or strain specific probes. Human CD34+ cells obtained from patient cord blood, with informed consent and under approval from the United Kingdom (UK) National Health Service Health Research Authority. Animal experiments were conducted with approval from the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board Results First, we quantified mitochondria content in the HSPC populations before and after LPS treatment or salmonella infection and quantified multipotent progenitor cells (MPP), total hematopoietic stem cell (HSC), long-term (LT-HSC) and short-term (ST-HSC) hematopoietic stem cells. MPP, HSC and ST HSC, but not the LT-HSC population, have increased mitochondria content in response to LPS treatment or salmonella infection. This corresponded with an increase frequency of MPP, HSC, and ST HSC. Next, we quantified levels of mitochondrial transfer within the bone marrow by analyzing the percentage of mouse mitochondria in the human HSC and MPP population in response to LPS treatment. Results show that mouse mtDNA was detected in the human HSC and human MPP populations of hu-NSG animals treated with LPS. No mouse mtDNA was detected in human HSC and human MPP populations in untreated hu-NSG animals. Conclusion Here we show that acute bacterial infection and LPS drive mitochondrial transfer from the bone marrow microenvironment to HSPC populations. We do not observe this occurring in comparator baseline unstressed hematopoiesis. Disclosures No relevant conflicts of interest to declare.

This article addresses programmatic cardiovascular screening and evaluation of the elite athlete at the intercollegiate, national team, professional, and Olympic levels. Although much of this content may apply to high-school and recreational sports at large, it is not specifically designed to address athletes participating in all sports activities.

Background Electrocardiographic screening of intercollegiate athletes is controversial because the costs and yield are not well defined. Both the American Heart Association (AHA) and the European Society of Cardiology (ESC) have different criteria for screening, partly because the populations being screened are different. Objective The purpose of this study was to determine the cost and yield of a 5-year ECG screening program at a United States Division I college. Methods At the University of Virginia, all 1,473 competitive athletes over the course of 5 years were screened with history and physical and with ECGs using ESC guidelines with follow-up testing as dictated by clinical symptoms and ECG findings. Results History and physical alone uncovered five significant cardiac abnormalities. ECGs were abnormal in 275 (19%), resulting in 359 additional tests. Additional testing confirmed eight significant cardiac abnormalities that were not found by history and physical: 1 bicuspid aortic valve, 4 rapidly conducting accessory pathways, 1 long QT patient, 1 with frequent premature ventricular contractions and low ejection fraction, and 1 with frequent premature ventricular contractions but normal ejection fraction. No cases of hypertrophic cardiomyopathy were found. Total cost of the program was US $894,870. Cost of history and physical screening alone was $343,725 or $68,745 per finding. The marginal cost of adding ECG screening, including resulting tests and procedures. was US$551,145 or US$68,893 per additional finding. Conclusion ECG screening of U.S. college athletes can uncover significant cardiac pathology not discovered by history and physical alone. Although ECG screening also results in many false positives resulting in additional tests, the overall cost per diagnosis of adding ECG screening is similar to that of history and physical screening alone.

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or "track" formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm(2). Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm(2). AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.

Aim: Study was undertaken to analyze the frequency of anti-viral citrullinated peptide (anti-VCP) antibodies in sera from patients with early rheumatoid arthritis (ERA). Materials and Methods: Viral citrullinated peptide (VCP) and Epstein-Barr nuclear antigen (EBNA-1) peptide were commercially prepared and antibodies to these were determined in 25 patients of ERA, 40 disease control patients constituting 25 rheumatoid arthritis (RA), 7 systemic lupus erythematosus (SLE), 2 scleroderma, 1 spondyloarthritis (SpA), 1 juvenile rheumatoid arthritis (JRA), 1 osteoarthritis (OA), 1 psoriatic arthritis (PsA), 1 undifferentiated arthritis (UA), and 1 gout and 25 healthy controls (HCs) were taken for comparison. In-house ELISA was established for both the antibodies while cyclic citrullinated peptide (CCP) antibody was detected by commercial ELISA kit. Results: Significant increase in VCP antibody by ERA and disease controls than healthy normal was observed. VCP IgM antibody was significantly increased in RA patients than HC. The presence of VCP antibody signifies a good marker for ERA. We observed significant difference in the VCP IgG and IgM antibody when compared to EBNA-1. In-house ELISA established for EBNA-1 and VCP antibodies showed low sensitivity but 96% specificity. Conclusions: We observed that sera from early RA patients reacted to the deiminated protein encoded by Epstain Barr Virus (EBV). Thus a possible role of virus in inducing an anti-citrullinated peptide antibody (ACPA) response reveals viral etiology in this disease.

Objective: Recent evidence suggest that allergen type 2 helper T cells (Th2) play a triggering role in the activation/recruitment of IgE antibody producing B cells, mast cells and eosinophils. Reduced microbial exposure in early life is responsible for a shift of Th1/Th2 balance in the immune system towards the pre-allergic Th2 response. The Th1 predominantly produce IFNg and delayed type hypersensitivity while Th2 secrete IL-4, IL-5, IL-6, IL-13 and regulate B cell and eosinophil mediated responses. To assess regulatory changes in the immune system, in patients with allergy and asthma, we studied the cytokine profile in serum in comparison with normal healthy controls. Patients and Methods: A total of 170 patients with various allergies and asthmatic conditions were studied, for cytokines in the serum by ELISA using kits from Immunotech, and analyzed to identify the triggering factors or main contributors towards allergy and asthma. Results: Our study showed increase in the levels of IL-4, IL-5 and IL-6 in all groups which were non- significant. But the levels of IL-10, IL-13 and TNF α were highly significant. Besides, we found correlation of GM-CSF with IL-10. Significant correlation with different cytokines was observed. Most of these patients showed increase in IgE levels. Conclusions: This study gives a better understanding of how cytokines are the mediators of balance of Th1 and Th2 immune responses and IgE synthesis is controlled by cytokines. Further studies will eventually lead to improved treatment strategies in the clinical management of IgE mediated allergy.

year old female patient presented with complaint of persistent weakness and pedal oedema.Her hemo- globin level was fluctuating between 8-10gm% which was not improved even after blood transfusion , iron and folic acid supplements.We ruled out all possible causes of pedal oedema.Total Protein ,Albumin , Globulin and A/G ratio was done. Globulin was 6.3gm%(increased).A/G ratio was reversed.ESR was markedly elevated.Protein electrophoresis was done which showed M-Band.Bone marrow showed 22% of plasma cells.Trephine biopsy was done which showed interstitial infiltrate of about 20% of plasma cells.S.creatinine ,S.calcium were normal.X-ray skull,pelvis and hip joint were normal.Diagnosis of Smoldering multiple my- eloma was put. Patient was put on Dexamethasone and pamidronate.She was followed after 3 months with protein electrophoresis and other biochemical reports.

Myotonic muscular dystrophy (DM) is characterized by abnormal skeletal muscle Na channel gating and reduced levels of myotonic dystrophy protein kinase (DMPK). Electrophysiological measurements show that mice deficient in Dmpk have reduced Na currents in muscle. We now find that the Na channel expression level is normal in mouse muscle partially or completely deficient in Dmpk. Reduced current amplitudes are not changed by age or gene dose, and the reduction is not due to changes in macroscopic or microscopic gating kinetics. The mechanism of abnormal membrane excitability in DM may in part be silencing of muscle Na channels due to Dmpk deficiency.

Stathmin is a microtubule-destabilizing phosphoprotein that plays a critical role in the regulation of mitosis. The microtubule-depolymerizing activity of stathmin is lost upon phosphorylation in mitosis. Although the role of phosphorylation of stathmin by p34cdc2 kinase in the assembly of the mitotic spindle is well established, the role of dephosphorylation of stathmin in mitosis is unknown. In this study, we tested the hypothesis that dephosphorylation of stathmin may be critically important for the depolymerization of the mitotic spindle and the exit from mitosis. We compared the effects of okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, on different parameters of mitotic progression in the presence or absence of stathmin deficiency. Because okadaic acid prevents dephosphorylation of stathmin and results in accumulation of the inactive phosphorylated form, exposure to okadaic acid would be expected to have a more profound effect on mitosis in the presence of relative stathmin deficiency. We found that inhibition of stathmin expression results in increased sensitivity to the antimitotic effects of okadaic acid. This was reflected by increased growth inhibition associated with mitotic arrest. A vast majority of the stathmin-inhibited cells were found to be arrested in late metaphase/anaphase and had severe mitotic spindle abnormalities. Exposure to okadaic acid also resulted in a bigger ratio of polymerized/unpolymerized tubulin in stathmin-inhibited cells relative to control cells. Because the only difference between the control and the stathmin-inhibited cells is the deficiency of stathmin in the latter, the increased susceptibility of the stathmin-inhibited cells to okadaic acid-induced mitotic arrest implies a role for stathmin in the later stages of mitosis.

The hormonal actions of 1a,25-dihydroxyvitamin D3 [1,25(OH)2D3] are mediated by its cognate receptor protein, the vitamin D receptor (VDR). Despite the growing importance of the VDR system as a modulator of cell growth and differentiation, convenient assays for quantitative measurement of VDR are not readily available, and [ 3 H]1,25(OH)2D3 ligand binding assays remain the standard method. In this paper, we present data to validate and characterize the usefulness of a new VDR enzyme-linked immunosorbant assay (ELISA) kit developed for the measurement of VDR in biological samples. In this assay, samples are added to microtitration wells coated with anti-VDR antibody and incubated with a second anti-VDR antibody that is biotinylated. The antibody receptor complex is then detected with streptavidin-labeled horseradish peroxidase followed by incubation with a chromogenic substrate, tetramethylbenzidine. The assay was found to be sensitive and accurate for measurements of VDR and compared favorably with the conventional radioligand binding assay (RBA). The interassay variation ranged from 5% to 25% and the intraassay variation was less than 5%. The ELISA presents several advantages over existing methodology, including the use of nonradioactive detection systems, lower protein and sample volume requirements, as well as convenience and speed. The assay can be completed in as short a time as 3 h, avoiding overnight incubations. Data are also presented to demonstrate the ability of the ELISA to detect both occupied and unoccupied VDR, making it a valuable research tool in settings where 1,25(OH)2D3 is present. However, the ELISA, as currently formulated, is only useful for the detection of human VDR. (Bone 28:319 ‐326; 2001) © 2001 by Elsevier Science Inc. All rights reserved.

Apolipoprotein J/clusterin (apoJ/clusterin), an intriguing protein with unknown function, is induced in myocarditis and numerous other inflammatory injuries. To test its ability to modify myosin-induced autoimmune myocarditis, we generated apoJ-deficient mice. ApoJ-deficient and wild-type mice exhibited similar initial onset of myocarditis, as evidenced by the induction of two early markers of the T cell-mediated immune response, MHC-II and TNF receptor p55. Furthermore, autoantibodies against the primary antigen cardiac myosin were induced to the same extent. Although the same proportion of challenged animals exhibited some degree of inflammatory infiltrate, inflammation was more severe in apoJ-deficient animals. Inflammatory lesions were more diffuse and extensive in apoJ-deficient mice, particularly in females. In marked contrast to wild-type animals, the development of a strong generalized secondary response against cardiac antigens in apoJ-deficient mice was predictive of severe myocarditis. Wild-type mice with a strong Ab response to secondary antigens appeared to be protected from severe inflammation. After resolution of inflammation, apoJ-deficient, but not wild-type, mice exhibited cardiac function impairment and severe myocardial scarring. These results suggest that apoJ limits progression of autoimmune myocarditis and protects the heart from postinflammatory tissue destruction.

The acid-labile subunit (ALS) of the high molecular weight insulin-like growth factor binding protein complex is a liver-derived glycoprotein which is regulated by growth hormone and serves as a serum marker of growth hormone action. We have compared the measurement of ALS by four immunoassay methods (two RIAs, two ELISAs) utilizing various polyclonal and monoclonal antibodies raised against natural or recombinant human ALS, or synthetic ALS peptides. Despite the variety of methodologies and reagents, results obtained by the four methods were highly correlated for 125 sera from various patient groups, and when compared for individual groups of sera from healthy children and adults, growth hormone-deficient children and adults, and subjects with acromegaly. Some weaker correlations among methods were seen when measuring ALS levels in groups of sera from pregnant subjects and subjects with chronic renal failure. An assay using antibodies raised against recombinant ALS yielded lower apparent values than the other methods in patient sera, the discrepancy probably being attributable to a difference in standardization. We conclude that a variety of assay formats and reagents can yield serum ALS values of potential clinical utility.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Metastatic prostate cancer has limited therapeutic options and has remained a major clinical challenge. Historically, prostate cancer has been widely recognized as a chemotherapy-resistant disease. However, clinical studies with anti-microtubule agents over the past decade have shown important efficacy in improving survival in patients with advanced disease. The favorable outcomes with microtubule-targeted agents have thus rekindled interest in such therapies for the clinical management of prostate cancer. Microtubules are dynamic polymers of tubulin molecules that play diverse roles within the cell. The dynamic property of microtubules is responsible for forming the bipolar mitotic apparatus, the mitotic spindle, that functions to precisely segregate the chromosomes during cell division. Thus, owing to the pivotal role that they play in the orchestration of mitotic events, microtubules provide excellent targets for anti-cancer therapy. Recent evidence also suggests that microtubules play a crucial role in the regulation of endocrine signaling pathways. Interestingly, microtubule-targeted agents such as taxanes not only inhibit cell division but also impair endocrine receptor signaling in prostate cancer. Herein, we provide an overview of the current status of microtubule-targeted therapies that are used in the treatment of prostate cancer and discuss novel mechanisms by which such therapies modulate endocrine signaling in prostate cancer. We also address the emerging roles of microtubule regulatory proteins in prostate carcinogenesis that could serve as attractive targets for prostate cancer therapy and might also serve as predictive biomarkers to identify patients who may benefit from endocrine and/or chemotherapy. This may have important implications in designing mechanism-based and targeted-therapeutic strategies for prostate cancer. Mol Cancer Ther; 12(5); 555–66. ©2013 AACR.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that have a critical role in the regulation of mitosis. Stathmin is expressed at high levels in breast cancer and its overexpression is linked to disease progression. Although there is a large body of evidence to support a role for stathmin in breast cancer progression, the validity of stathmin as a viable therapeutic target for breast cancer has not been investigated. Here, we used a bicistronic adenoviral vector that co-expresses green fluorescent protein and a ribozyme that targets stathmin messenger RNA in preclinical breast cancer models with different estrogen receptor (ER) status. We examined the effects of anti-stathmin ribozyme on the malignant phenotype of breast cancer cells in vitro and in xenograft models in vivo both as a single agent and in combination with chemotherapeutic agents. Adenovirus-mediated gene transfer of anti-stathmin ribozyme resulted in a dose-dependent inhibition of proliferation and clonogenicity associated with a G2/M arrest and increase in apoptosis in both ER-positive and ER-negative breast cancer cell lines. This inhibition was markedly enhanced when stathmin-inhibited breast cancer cells were exposed to low concentrations of taxol, which resulted in virtually complete loss of the malignant phenotype. Interestingly, breast cancer xenografts treated with low doses of anti-stathmin therapy and taxol showed regression in a majority of tumors, while some tumors stopped growing completely. In contrast, combination of anti-stathmin ribozyme and adriamycin resulted in only a modest inhibition of growth in vitro and in breast cancer xenografts in vivo. Although inhibition of tumor growth was observed in both the combination treatment groups compared with groups treated with single agent alone, combination of anti-stathmin therapy and taxol had a more profound inhibition of tumorigenicity, as both agents target the microtubule pathway. Clinically, these findings are highly relevant because taxol is one of the most active chemotherapeutic agents in breast cancer. These studies provide the proof-of-principle that stathmin provides an attractive molecular target, which could serve as a primary focus of novel approaches to breast cancer.

Measurement of serum insulin-like growth factor I (IGF-I) is important in assessing the growth hormone/IGF axis. Competitive immunoassay methods for IGF-I are complicated by substantial interference from IGF-binding proteins (IGFBPs) due to the relatively low ratio of specific antibody to IGFBPs in the sample, even after standard acid-ethanol sample extraction. We report of development of a noncompetitive ELISA for human IGF-I that avoids this problem by utilizing excess amounts of capture and detection antibodies. Serum samples were prepared by using an abbreviated acid-ethanol extraction method. Neutralized supernatant was added to microwells coated with IGF-I capture antibody; horseradish peroxidase-labeled detection antibody was then added, incubated for 2 h, and then developed. Compared with the "gold standard" method of acid-column chromatography, the simplified acid-ethanol extraction yields a mean +/- SD recovery of 103% +/- 5.5% despite the presence of residual IGFBPs in the extracted sample. Comparisons with a centrifugal filtration sample extraction method are also shown. The ELISA is specific for IGF-I with an absolute sensitivity of 0.03 microgram/L and inter- and intraassay CVs of 3.9-8.8% and 2.6-6.7%, respectively. The availability of a rapid IGF-I ELISA combined with a simple and reliable sample preparation procedure should facilitate clinical and research studies of this important growth factor.

Apolipoprotein E (apoE), a lipid transport protein important in cholesterol homeostasis, inhibits the proliferation of interleukin-dependent lymphocytes. Growth factor-responsive cells are blocked in the G1A phase of the cell cycle. Suppression by apoE is independent of growth factor, as evidenced by the fact that interleukin-2 (IL2)- and IL4-dependent proliferation of HT-2 T lymphocytes is equally inhibited. apoE has no effect on IL2-augmented killing of target cells by cytotoxic T cells, indicating that it has no direct effect on signaling via interleukin receptors. The data are consistent with inhibition by apoE of an event or pathway distal to receptor signaling and required for G1A transition, or G1B entry.

Apolipoprotein E (apoE), but not apoAI or apoCIII, suppresses mitogen-activated T lymphocyte proliferation, independent of the type of activation signal. Both CD4 and CD8 T cells are inhibited. ApoE inhibits T cell proliferation, in part, by reducing the production of bioactive interleukin 2 (IL2). IL2 activity is reduced by approximately 50-65% in cultures of mitogen-stimulated T cells when apoE is present. ApoE does not significantly alter levels of IL2 mRNA or the mass of secreted IL2 protein, quantitated by enzyme immunoassay. Reduced IL2 activity was not a consequence of induction of IL2 inhibitors in response to apoE or effects of apoE on the bioassay. These results suggest that apoE antagonizes post-translational events in mitogen-activated T lymphocytes that are required for the secretion of a bioactive IL2 protein.

p18 is a phosphoprotein that is expressed at very high levels in leukemic cells, at moderately high levels in proliferating normal lymphocytes, and at low levels in quiescent lymphocytes. Induction of terminal differentiation of leukemic cells in culture results in a decrease in cellular proliferation. These phenotypic changes are associated with rapid phosphorylation of p18, followed by a more gradual decrease in the level of its mRNA expression. More than 12 different phosphorylation products of p18 have been identified in different cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Previous studies have suggested that p18 may be a substrate for protein kinase C in some cellular processes and protein kinase A in others. In this report, we show that the phosphorylation of p18 increases as cells progress toward the G2-M phases of the cell cycle in proliferating leukemic cells. We have examined the hypothesis that the putative role of p18 in cellular proliferation may be mediated by its involvement in the p34cdc2 kinase signal transduction pathway. We have produced recombinant p18 in bacterial cells and shown that it can be phosphorylated in vitro by purified p34cdc2 kinase with a stoichiometry of 0.86 mol of PO4/mol of substrate. We have used site-directed mutagenesis to demonstrate that the site of p34cdc2 phosphorylation is the serine at position 38. This same site has previously been shown to be phosphorylated in vivo in bovine brain along with another serine at position 25. The observation that p18 gets phosphorylated in the G2-M phases of the cell cycle and the demonstration that p18 is phosphorylated efficiently by p34cdc2 kinase in vitro at a residue that is also phosphorylated in vivo support the hypothesis that p18 may be a physiologic substrate for p34cdc2 kinase in vivo. We have also examined the effect of inhibiting the expression of p18 on cell cycle progression. These experiments demonstrated that antisense inhibition of the expression of p18 in K562 erythroleukemia cells is associated with a decrease in cellular proliferation and accumulation of cells in the G2-M phases of the cycle. The implications of these findings to the proposed role of p18 in the regulation of cellular proliferation are discussed.

Chest pain remains one of the most concerning complaints among athletes and active individuals. Fortunately, the vast majority of chest pain in this group is benign, noncardiac, and rarely associated with catastrophic manifestations such as sudden death [1]. [2] A thorough assessment requires a full understanding of the differential of chest pain, a detailed history designed to elicit the nuances of each general category of chest pain, a directed physical examination, and prudent choices of ancillary testing as clinically warranted.

Background: This study examines the impact of dietary fatty acids on regulation of gene expression in mammary epithelial cells before and during puberty. Methods: Diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Diet-induced changes in gene expression were examined in laser capture microdissected mammary ductal epithelial cells at day of weaning and end of puberty. PCNA immunohistochemistry analysis compared proliferation rates between diets. Results: Genes differentially expressed between each test diets and the reference diet were significantly enriched by cell cycle genes. Some of these genes were involved in activation of the cell cycle pathway or the G2/M check point pathway. Although there were some differences in the level of differential expression, all diets showed qualitatively the same pattern of differential expression compared to the reference diet. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. Conclusion: Fatty acid-enriched diets significantly upregulated proliferation above normal physiological levels during puberty. Higher cellular proliferation during puberty caused by enriched fatty acid diets poses a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle rat genes are included in a human breast cancer cluster of 45 cell cycle genes, further emphasizing the importance of our findings in the rat model.

The retinoblastoma gene product (RB) regulates cell cycle, quiescence, and survival in a cell type–dependent and environment-dependent manner. RB function is critical in the pulmonary epithelium, as evidenced by nearly universal RB inactivation in lung cancer and increased lung cancer risk in persons with germline RB gene mutations. Lung carcinomas occur in the context of epithelial remodeling induced by cytotoxic damage. Whereas the role of RB in development and normal organ homeostasis has been extensively studied, RB function in the context of cellular injury and repair has remained largely unexplored. In the current studies, the RB gene was selectively deleted in the respiratory epithelium of the mouse. Although RB was not required for establishing or maintaining quiescence during lung homeostasis, RB was essential for establishing quiescence during epithelial repair after injury. Notably, aberrant cell cycle progression was sustained for 9 months after injury in RB-deficient lungs. Prenatal and postnatal RB ablation had similar effects, providing evidence that timing of RB loss was not critical to the outcome and that the injury-induced phenotype was not secondary to compensatory alterations occurring during development. These data show that RB is essential for repair of the respiratory epithelium after cytotoxic damage and support a critical unique role for RB in the context of epithelial remodeling after injury. Because human cancers are associated with chronic cellular damage, these findings have important new implications for RB-mediated tumor suppression. [Cancer Res 2008;68(11):4068–76]

Limitations of prostate cancer therapy may be overcome by combinations of chemotherapeutic agents with gene therapy directed against specific proteins critical for disease progression. Stathmin is overexpressed in many types of human cancer, including prostate cancer. Stathmin is one of the key regulators of the microtubule network and the mitotic spindle and provides an attractive therapeutic target in cancer therapy. We recently showed that adenovirus-mediated gene transfer of anti-stathmin ribozyme could suppress the malignant phenotype of prostate cancer cells in vitro. In the current studies, we asked whether the therapeutic effects of stathmin inhibition could be further enhanced by exposure to different chemotherapeutic agents. Exposure of uninfected LNCaP human prostate cancer cells or cells infected with a control adenovirus to Taxol, etoposide, 5-fluorouracil (5-FU), or Adriamycin resulted in modest decrease in proliferation and clonogenicity. Interestingly, exposure of cells infected with an anti-stathmin adenovirus to Taxol or etoposide resulted in a complete loss of proliferation and clonogenicity, whereas exposure of the same cells to 5-FU or Adriamycin potentiated the growth-inhibitory effects of the anti-stathmin ribozyme, but the cells continued to proliferate. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling analysis of uninfected cells or cells infected with a control adenovirus showed modest induction of apoptosis in the presence of different drugs. In contrast, cells infected with the anti-stathmin adenovirus showed a marked increase in apoptosis on exposure to Taxol or etoposide and a modest increase on exposure to 5-FU or Adriamycin. Overall, the effects of combinations of anti-stathmin ribozyme with Taxol or etoposide were synergistic, whereas the effects of combinations of anti-stathmin ribozyme with 5-FU or Adriamycin were additive. Moreover, triple combination of anti-stathmin ribozyme with low noninhibitory concentrations of Taxol and etoposide resulted in a profound synergistic inhibition of proliferation, clonogenicity, and marked induction of apoptosis. This synergy might be very relevant for the treatment of prostate cancer because Taxol and etoposide are two of the most effective agents in this disease. Thus, this combination may provide a novel form of prostate cancer therapy that would avoid toxicities associated with the use of multiple chemotherapeutic agents at full therapeutic doses. [Mol Cancer Ther 2006;5(12):3248–57]

Stathmin is the founding member of a family of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin is expressed at high levels in a variety of human cancers and provides an attractive molecule to target in cancer therapies that disrupt the mitotic apparatus. We developed replication-deficient bicistronic adenoviral vectors that coexpress green fluorescent protein and ribozymes that target stathmin mRNA. The therapeutic potential of these recombinant adenoviruses was tested in an experimental androgen-independent LNCaP prostate cancer model. Adenovirus-mediated transfer of anti-stathmin ribozymes resulted in efficient transduction and marked inhibition of stathmin expression in these cells. Cells that were transduced with the anti-stathmin adenoviruses showed a dramatic dose-dependent growth inhibition. This was associated with accumulation of LNCaP cells in the G2-M phases of the cell cycle. A similar dose-dependent inhibition of clonogenic potential was also observed in cells infected with anti-stathmin adenoviruses. Morphologic and biochemical analysis of infected cells showed a marked increase in apoptosis characterized by detachment of the cells, increased chromatin condensation, activation of caspase-3, and fragmentation of internucleosomal DNA. If these findings are confirmed in vivo, it may provide an effective approach for the treatment of prostate cancer. [Mol Cancer Ther 2005;4(12):1821–9]

Previously, we reported that glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1) was a novel target for prolactin (PRL) in the mouse mammary gland. However, the signaling pathway by which PRL regulates GlyCAM 1 expression has not been specified. In the present study, we showed that PRL induced GlyCAM 1 expression in primary mammary epithelial cells of mice through the Janus kinase 2/signal transducer and activator of transcription 5 (Stat5) pathway. Deletion and site-directed mutagenesis analyses of the GlyCAM 1 promoter demonstrated that the two tandemly linked Stat5 binding sites [interferon-gamma-activated sequence 1 and -2 (GAS1 and GAS2)] in the proximal promoter region were crucial and synergistically responded to PRL. GAS2, a consensus GAS site, was essential and, by itself, weakly responded to PRL, whereas GAS1, a nonconsensus site, failed to respond to PRL but was indispensable for the maximal activity of the GlyCAM 1 promoter. Gel shift assays showed that probe containing GAS1 and GAS2 bound two Stat5 complexes, which represent Stat5 dimer and tetramer, respectively, while GAS2, by itself, bound Stat5 as a dimer only, and GAS1 showed no apparent binding activity. Interruption of tetramer formation by mutation of a tryptophan to alanine (W37A), and a leucine to serine (L83S) in the N terminus of Stat5A attenuated the synergistic effect between the two tandemly linked GAS sites. Overexpression of W37A and L83S mutants in primary mammary epithelial cells suppressed endogenous GlyCAM 1 expression.

DMPK is a serine/threonine kinase implicated in the human disease myotonic muscular dystrophy (DM). Skeletal muscle Na channels exhibit late reopenings in Dmpk-deficient mice and peak current density is reduced, implicating DMPK in regulation of membrane excitability. Since complete heart block and sudden cardiac death occur in the disease, we tested the hypothesis that cardiac Na channels also exhibit abnormal gating in Dmpk-deficient mice. We made whole cell and cell-attached patch clamp recordings of ventricular cardiomyocytes enzymatically isolated from wild-type, Dmpk+/−, and Dmpk−/− mice. Recordings from membrane patches containing one or a few Na channels revealed multiple Na channel reopenings occurring after the macroscopic Na current had subsided in both Dmpk+/− and Dmpk−/− muscle, but only rare reopenings in wild-type muscle (>3-fold difference, P < 0.05). This resulted in a plateau of non-inactivating Na current in Dmpk-deficient muscle. The magnitude of this plateau current was independent on the magnitude of the test potential from −40 to 0 mV and was also independent of gene dose. Macroscopic Na current density was similar in wild-type and Dmpk-deficient muscle, as was steady-state Na channel gating. Decay of macroscopic currents was slowed in Dmpk−/− muscle, but not in Dmpk+/− or wild-type muscle. Entry into, and recovery from, inactivation were similar at multiple test potentials in wild-type and Dmpk-deficient muscle. Resting membrane potential was depolarized, and action potential duration was significantly prolonged in Dmpk-deficient muscle. Thus in cardiac muscle, Dmpk deficiency results in multiple late reopenings of Na channels similar to those seen in Dmpk-deficient skeletal muscle. This is reflected in a plateau of non-inactivating macroscopic Na current and prolongation of cardiac action potentials.

Myotonic dystrophy results from a trinucleotide repeat expansion between the myotonic dystrophy protein kinase gene ( Dmpk), which encodes a serine-threonine protein kinase, and the Six5 gene, which encodes a homeodomain protein. The disease is characterized by late bursts of skeletal muscle Na channel openings, and this is recapitulated in Dmpk −/− and Dmpk +/− murine skeletal muscle. To test whether deficiency of the nearby Six5 gene also affected Na channel gating in murine skeletal muscle, we measured Na currents from cell-attached patches in Six5 +/− mice and age-matched wild-type and Dmpk +/− mice. Late bursts of Na channel activity were defined as an opening probability >10% measured from 10 to 110 ms after depolarization. There was no significant difference in the occurrence of late Na channel bursts in wild-type and Six5 +/− muscle, whereas in Dmpk +/− muscle there was greater than fivefold increase in late bursts ( P < 0.001). Compared with wild-type mice, Na current amplitude was unchanged in Six5 +/− muscle, whereas in Dmpk +/− muscle it was 36% reduced ( P < 0.05). Thus, since Six5 +/− mice do not exhibit the Na channel gating abnormality of Dmpk deficiency, we conclude that Six5 deficiency does not contribute to the Na channel gating abnormality seen in dystrophia myotonica patients.

Stathmin is a major cytosolic phosphoprotein that plays an important role in the control of cellular proliferation by regulating the dynamics of the microtubules that make up the mitotic spindle. Because stathmin is expressed at high levels in all human cancers, it is an attractive molecular target for anticancer interventions. We had shown previously that antisense stathmin inhibition results in marked abrogation of the transformed phenotype of leukemic cells in vitro and in vivo. Unlike the antisense approach, ribozymes can catalytically cleave several molecules of target RNA. This may provide a more efficient strategy for downregulating genes, such as stathmin, that are expressed at very high levels in cancer cells. We designed several antistathmin hammerhead ribozymes and tested their cleavage activity against short synthetic stathmin RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of stathmin RNA that was dependent on ribozyme concentration and duration of exposure to ribozyme. The most active antistathmin ribozyme was capable of cleaving >90% stathmin RNA in a catalytic manner, cleaving multiple substrate molecules per ribozyme molecule. We also demonstrated that the designed antistathmin ribozymes are capable of selectively cleaving native stathmin RNA in a mixture of total RNA isolated from leukemic cells. These antistathmin ribozymes may provide a novel and effective form of gene therapy that may be applicable to a wide variety of human cancers.

Stathmin is a major cytosolic phosphoprotein that plays an important role in the regulation of microtubule dynamics during cell cycle progression. It has recently been proposed that the major function of stathmin is to promote depolymerization of the microtubules that make up the mitotic spindle. In this report, we tested the prediction that a deficiency in stathmin expression would result in constitutive stabilization of microtubules and lead to abnormalities in the organization of the mitotic spindle. Our studies demonstrate that antisense inhibition of stathmin expression in K562 erythroleukemic cells results in increased ratio of polymerized to depolymerized tubulin. These changes are associated with phenotypic abnormalities of the mitotic spindle and difficulty in completing mitosis. These studies also showed that inhibition of stathmin expression results in increased susceptibility of K562 leukemic cells to the pharmacological agents, like taxol, which are known to stabilize the mitotic spindle. In contrast, stathmin inhibition results in decreased sensitivity to vinblastine, an agent that destabilizes the mitotic spindle. Thus, our experimental findings are supportive of the model that stathmin is a microtubule-destabilizing factor that plays an important role in the regulation of the mitotic spindle. We also suggest a potential therapeutic approach for cancer based on the combination of stathmin inhibition with pharmacologic agents that stabilize the mitotic spindle.

Stathmin is a major cytosolic phosphoprotein that regulates microtubule dynamics during the assembly of the mitotic spindle. The activity of stathmin itself is regulated by changes in its state of phosphorylation during the transition from interphase to metaphase. For a better understanding of the regulation of stathmin activity during the cell cycle, we explored the mechanism(s) responsible for the decrease in the level of phosphorylation of stathmin as cells complete mitosis and enter a new G1 phase. We show that stathmin mRNA and protein are expressed constitutively throughout the different phases of the cell cycle. This suggests that the non-phosphorylated stathmin that predominates during G1 is not generated by degradation of phosphorylated stathmin in mitosis and synthesis of new unphosphorylated stathmin as cells enter a new G1 phase. This suggested that protein phosphatases might be responsible for dephosphorylating stathmin as cells enter a new cell cycle. Okadaic acid-mediated inhibition of protein phosphatases in vivoshowed a major increase in the level of phosphorylation of stathmin. Dephosphorylation studies in vitro showed differential patterns of site-specific dephosphorylaton of stathmin to protein phosphatase type 1, protein phosphatase type 2A and protein phosphatase type 2B. Thus stathmin might be a target for okadaic acid-sensitive protein phosphatase(s), and its activity in eukaryotic cells might be modulated by the sequential activity of specific protein kinases and phosphatases.

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

Background: Stathmin is a major cytosolic protein that promotes microtubule depolymerization. Recent crystallographic data indicate that both stathmin & the chemotherapeutic agent, vinblastine, act together by binding simultaneously on free tubulin. Stathmin was shown to mediate vinblastine activity by increasing the affinity of vinblastine for tubulin. We had previously demonstrated that intracellular delivery of short stathmin-like peptide(s) could inhibit the malignant proliferation of breast cancer (BC) cells by disrupting microtubule assembly. Here, we asked whether the anti-proliferative effects of stathmin-like peptides could be further enhanced by vinblastine. Methods: The peptide(s) that we used consisted of a short stathmin-like domain that was fused to a TAT transduction domain & a hemagglutinin epitope tag to track the peptide intracellularly. Since stathmin could be phosphorylated & inactivated intracellularly by p34cdc2 kinase, we also used a mutant peptide in which Ser-16 was mutated to an alanine to prevent its inactivation by phosphorylation. As a control, we used a similar peptide in which the stathmin-like domain was scrambled. Results: We tested the effects of these peptides in combination with vinblastine in different BC cell lines (MDA-MB-231, T47D, & SKBR-3). The rate of proliferation of untreated cells or cells exposed to control peptide was not affected by 2 nM concentration of vinblastine & was modestly inhibited at 5 nM concentration. When cells were exposed to wild type (WT) stathmin-like peptide & vinblastine, the rate of proliferation was further decreased at 2 nM concentration & nearly suppressed at 5 nM concentration of vinblastine. In contrast, cells exposed to mutant (MT) peptide showed complete cessation of growth when combined with 2 nM or 5 nM vinblastine. Thus, exposure to MT peptide seemed to result in greater sensitization of BC cells to the growth inhibitory effects of vinblastine. Similarly, cells exposed to either WT or MT peptide showed a 22% & 39% reduction in the colony formation respectively. When cells were exposed to either WT or MT peptide in combination with vinblastine, the clonogenicity was further reduced to 47% & 73%, respectively. This suggests that the peptides can inhibit anchorage independent growth, which is markedly enhanced by vinblastine. We next analyzed the ability of the designed peptides to induce apoptosis either as a single agent or in combination with vinblastine. TUNEL analysis of control cells showed essentially no apoptosis. When the cells were exposed to either WT or MT peptides, there was a 13% & 24% increase in the fraction of apoptosis respectively. However, combination of vinblastine with either WT or MT peptide significantly increased the fraction of apoptotic cells to 54% & 76%, respectively. These data suggest that the anti-tumor activity of stathmin-like peptides is markedly enhanced by vinblastine in BC cells in vitro. Citation Format: Ishan Purani, Arlene Tejada, Sucharita J. Mistry. Effects of combination of microtubule depolymerizing peptides and vinblastine in breast cancer. [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 3275. doi:10.1158/1538-7445.AM2013-3275

Development of an ultrasensitive immunoassay for serum PSA involving conventional detection probes.The assay involves a polyclonal antibody immobilized in microtitration wells and a monoclonal antibody labeled with horseradish peroxidase. In a one-step assay, the enzymatic activity of the bound detection antibody is monitored by the addition of hydrogen peroxide/tetramethylbenzidine substrate reagent followed by spectrophotometric quantification of the conversion product.The assay has a lower detection limit of 0.003 micrograms/L, biological detection limit of 0.009 micrograms/L, and intra- and interassay CVs of 8.2% and 10.5% at PSA concentrations of 0.022 and 0.065 micrograms/L, respectively. The recovery of the assay averaged 104% and it demonstrated a dilution linearity down to at least 0.01 micrograms of PSA/L. Results of comparison data correlated well with those obtained by a well established enzyme immunoassay. The serum PSA concentrations were0.012 micrograms/L in the majority of patients (53.8%) who had undergone radical prostatectomy.This assay is well suited for post-surgical monitoring of PSA in patients with prostate cancer.

Background and Objectives: Stathmin is a founding member of a family of microtubule-destabilizing proteins and is one of the key regulators of the microtubule cytoskeleton. Interestingly, stathmin is expressed at high levels in breast cancer and its overexpression is linked with disease progression. Recent studies identified numerous short peptides from the N-terminal region of stathmin family that were shown to impede tubulin polymerization. Here, we tested the hypothesis whether intracellular delivery of stathmin-like peptide(s) would inhibit the malignant proliferation of breast cancer cells by disrupting microtubule assembly in in vitro models of human breast cancer. Methods and Results: The peptide(s) that we designed consisted of a short stathmin-like domain that was fused to a TAT transduction domain, which served as a carrier to facilitate entry into breast cancer cells. We also included a hemagglutinin (HA) epitope tag to track the peptide intracellularly. Since stathmin could be phosphorylated & inactivated intracellularly by p34cdc2 kinase, we also designed another peptide in which Ser-16 was mutated to an alanine to prevent its inactivation by phosphorylation. Previous studies had shown that this substitution results in a more potent form of stathmin that cannot be inactivated by phosphorylation. As a control, we had generated a similar peptide in which the stathmin-like domain was scrambled. We tested the effects of these peptides in different breast cancer cell lines (MDA-MB-231, T47D, & SKBR-3). Immunostaining of breast cancer cells exposed to different peptides showed a vast majority of cells (>95%) positive for anti-HA staining suggesting that the designed peptides can be efficiently taken up by the cells. Proliferation assays in different breast cancer cell lines showed growth inhibitory effects in cells exposed to both, wild type or mutant peptides compared to cells exposed to a control peptide. However, the mutant peptide mediated a more profound anti-proliferative effect as expected. Immunofluorescence analysis of the microtubule organization of breast cancer cells exposed to different peptides using an anti-tubulin antibody showed a marked reduction in the density of microtubules. We also quantified the levels of polymerized and unpolymerized tubulin in breast cancer cells exposed to the different peptides using a sensitive biochemical assay. Immunoblot analysis showed a 1.8 fold decrease in the ratio of polymerized to soluble tubulin in cells exposed to wild-type peptide relative to control cells. The ratio of polymerized to soluble tubulin was further decreased by 3.7 fold in cells exposed to the mutant peptide. This suggests that the observed anti-proliferative effect is likely a result of inhibition of microtubule assembly. Thus, peptide(s) could be used to modulate stathmin function and inhibit malignant proliferation of breast cancer cells by interfering with the microtubule equilibrium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2792. doi:1538-7445.AM2012-2792

AIM: Stathmin, a founding member of a family of microtubule-destabilizing proteins, is expressed at high levels in breast cancer (BC) and provides an attractive target for BC therapy. We previously showed that stathmin inhibition could suppress the malignant phenotype of BC cells in vitro and in vivo. Here, we tested whether the therapeutic efficacy of stathmin inhibition could be further enhanced by combining it with standard chemotherapeutic agents in vivo. METHODS: We used a bicistronic adenoviral (Ad) vector that coexpresses green fluorescent protein (GFP) and a ribozyme (Rz) that targets human stathmin mRNA. Thirty-five female nude mice were injected orthotopically in the mammary fat pad of two of the mammary glands (# 4), each with 5×10e6 MDA-MB-231 cells/gland. We initiated treatment after the mice developed tumors of 0.3-0.4 cm in size. Mice bearing established MDA-MB-231 xenografts were divided into 7 groups of 5 mice each. Group I was injected with PBS alone. Groups 2, 3 & 4 were injected with control Ad.GFP (1×10e8 vp/injection). At the same time, adriamycin (5 mg/kg/injection) & taxol (5 mg/kg/injection) was co-administered in groups 3 & 4 respectively. Similarly groups 5, 6 & 7 were injected with Ad.Rz.GFP (1×10e8 vp/injection) anti-stathmin adenovirus. Groups 6 & 7 were also given adriamycin & taxol respectively at the same doses. The adenovirus & the drugs were administered at subtherapeutic doses that were selected based on dose response experiments. All drugs were injected i.p. & the adenoviruses were injected intratumorally once a week for a total of 3 injections. Changes in tumor growth were monitored in the different groups for another 6-8 weeks. RESULTS: Tumors in animals injected with PBS (group 1) or the control Ad.GFP (group 2) continued to grow to larger sizes. The growth of the tumors in animals treated with low doses of drugs alone (groups 3 & 4) or Ad.Rz.GFP alone (group 5) was only modestly slowed compared to control groups. When animals were treated with a combination of low doses of anti-stathmin therapy & adriamycin (group 6), the rate of tumor growth was much slower than animals treated with drug alone or anti-stathmin Rz alone. Interestingly, when animals were treated with a combination of low doses of anti-stathmin therapy & taxol (group 7), tumor regression (greater than or equal to 40%) was observed in 60% of the tumors & 40% of the tumors were stabilized. Although inhibition of tumor growth was observed in both the combination treatment groups compared to groups treated with single agent alone, combination of anti-stathmin therapy & taxol had a more profound inhibition of tumorigenicity, since both agents target the same microtubule pathway. In contrast, the activity of adriamycin does not result from direct interactions with microtubules & the mitotic spindle. These findings are highly relevant for the treatment of BC, since taxol is one of the most active chemotherapeutic agents used in BC care. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2011-1654