Proteomic analysis of osteogenic sarcoma: association of tumour necrosis factor with poor prognosis

A significant proportion of patients with osteogenic sarcoma die from lung metastasis within 5 years of diagnosis. Molecular signatures that predict pulmonary metastasis from primary osteogenic sarcoma and identify those patients at risk would be clinically useful as prognostic markers. Protein expression profiles of two clonally related murine osteogenic sarcoma cell lines with low (K12) and high (K7M2) metastatic potential were compared using two different proteomic technologies, two‐dimensional difference gel electrophoresis and cell profiling by matrix‐assisted laser desorption/ionization mass spectrometry. Interrogation of a molecular pathways network database suggested several additional candidate molecules that potentially predict metastatic potential of primary osteogenic sarcoma. Two such proteins, macrophage migration inhibitory factor and tumour necrosis factor were selected for further validation studies. Western blots confirmed increased expression of both cytokines in K7M2 cells compared to K12 cells. Levels of migration inhibitory factor and tumour necrosis factor were semi‐quantitatively measured in human osteogenic sarcoma samples by immunohistochemistry and were correlated with clinicopathologic parameters and patient outcomes. Multivariate survival analysis demonstrated that tumour necrosis factor expression in chemotherapy naïve osteogenic sarcoma is an independent prognostic factor for overall and metastasis‐free survival. No significant differences in adverse outcomes were observed based on macrophage migration inhibitory factor expression.     

Increased Allergic Immune Response to Sarcoptes scabiei Antigens in Crusted versus Ordinary Scabies

Scabies, a parasitic skin infestation by the burrowing “itch” mite Sarcoptes scabiei, causes significant health problems for children and adults worldwide. Crusted scabies is a particularly severe form of scabies in which mites multiply into the millions, causing extensive skin crusting. The symptoms and signs of scabies suggest host immunity to the scabies mite, but the specific resistant response in humans remains largely uncharacterized. We used 4 scabies mite recombinant proteins with sequence homology to extensively studied house dust mite allergens to investigate a differential immune response between ordinary scabies and the debilitating crusted form of the disease. Subjects with either disease form showed serum IgE against recombinant S. scabiei cysteine and serine proteases and apolipoprotein, whereas naive subjects showed minimal IgE reactivity. Significantly (P < 0.05) greater serum IgE and IgG4 binding to mite apolipoprotein occurred in subjects with crusted scabies than in those with ordinary scabies. Both subject groups showed strong proliferative responses (peripheral blood mononuclear cells) to the scabies antigens, but the crusted scabies group showed increased secretion of the Th2 cytokines interleukin 5 (IL-5) and IL-13 and decreased Th1 cytokine gamma interferon (IFN-γ) in response to the active cysteine protease. These data confirm that a nonprotective allergic response occurs in the crusted disease form and demonstrate that clinical severity is associated with differences in the type and magnitude of the antibody and cellular responses to scabies proteins. A quantitative IgE inhibition assay identified IgE immunoreactivity of scabies mite antigens distinct from that of house dust mite antigens, which is potentially important for specific scabies diagnosis and therapy.Scabies is a disease of the skin caused by the burrowing “itch” mite Sarcoptes scabiei. The predominant disease manifestations are mediated through inflammatory and allergic-like reactions to mite products, leading to intensely pruritic skin lesions. A spectrum of disease is recognized, from the more common ordinary scabies (OS), with an average infestation of 10 to 15 mites per person, to a rare and severely debilitating form of the disease termed crusted (Norwegian) scabies (CS). In this form, mite infestations can number in the millions, and hyperkeratotic skin crusts develop (41, 50). Due to the extreme burden of mites, CS is considerably more infectious, and infectivity persists far longer, as eradicating mites from heavily crusted skin is extremely difficult.Crusted scabies is an important public health problem, not only for the individuals concerned, but also for their families and communities, in which sufferers may act as “core transmitters” who continue to reinfect others. In many remote Aboriginal communities in northern Australia, scabies prevalence is high: up to 65% in children, with first presentation peaking at 2 months of age (13).Adult female mites reside in burrows within the stratum granulosum of the epidermis. The clinical rash and itch present as papules or vesicles that may contain individual mites, eggs, egg cases, mite fecal pellets, and debris present in the burrow. A secondary, more generalized papular immune response also occurs. The associated underlying inflammatory response varies in intensity, with combinations of lymphocytes, histiocytes, and polymorphonuclear leukocytes (10, 47). Due to difficulties in isolating sarcoptic mites on the host in OS and to the clinical symptoms imitating those of other skin diseases, scabies is a challenging disease to diagnose (48). To date, limited investigations of humoral immunity to the scabies mite (SM) in patients have yielded contradictory results and have used scabietic extracts from other hosts, such as dogs (37). Interpretation of these studies is complex, as scabies mites are highly host specific and generally produce only a transient, self-limiting reaction in the nonpermissive host.In Darwin, Northern Territory, Australia, patients with CS were noted to have extremely high levels of total IgE and IgG in serum (41). Western blotting with plasma from these patients demonstrated that 6 of 7 individuals had strong IgE binding to S. scabiei var. canis protein extracts (2). Immunochemical studies have previously demonstrated that sera from rabbits infested with S. scabiei var. canis bind to house dust mite (HDM) extracts and, conversely, that sera from rabbits immunized with HDM extract bind with S. scabiei var. canis whole-mite protein extract (4-6, 18, 20, 36). Allergens from HDMs are recognized as major causes of allergic respiratory disease in humans (17, 43). As SMs and HDMs are phylogenetically related arthropods with similar nutritional requirements, it is not surprising that these mites or their excreta have homologous allergens. However, it is likely that only a few of these allergens are cross-reactive. For example, Der p 5 from the HDM Dermatophagoides pteronyssinus and Blo t 5 from the storage mite Blomia tropicalis have been studied extensively, and although they have 43% amino acid identity, they are not IgE cross-reactive (31). The identities of the specific cross-reactive molecules between S. scabiei and D. pteronyssinus remain undefined but may be glycan related (33).The recent development of S. scabiei cDNA libraries and expressed sequence tag (EST) databases (21, 29) allows more precise characterization of the specific antigens responsible for the immune reactions to the SM. The cDNAs encoding S. scabiei var. hominis cysteine proteases (27), serine proteases (26), glutathione S-transferase (GST) (16), and apolipoprotein (25) show homology to the D. pteronyssinus HDM group 1, 3, 8, and 14 allergens, respectively (43). As with HDMs, the availability of recombinant proteins and identification of key immunoreactive allergens for the SM would facilitate development of refined diagnosis and potential immunotherapy. Thus, more effective control of mite infestations at both an individual and a community level may be possible. We report here the characterization of specific antibody binding profiles and cellular immune responses of subjects with clinical scabies by using purified S. scabiei var. hominis recombinant proteins. Quantitative IgE inhibition analysis of cross-reactivity with HDM allergens identified IgE epitopes of scabies mite proteins distinct from HDM epitopes, a prerequisite for using purified allergens in scabies diagnosis and therapy.     

Dendritic cell immaturity during infancy restricts the capacity to express vaccine-specific T-cell memory

The capacity of the immune system in infants to develop stable T-cell memory in response to vaccination is attenuated, and the mechanism(s) underlying this developmental deficiency in humans is poorly understood. The present study focuses on the capacity for expression of in vitro recall responses to tetanus and diphtheria antigens in lymphocytes from 12-month-old infants vaccinated during the first 6 months of life. We demonstrate that supplementation of infant lymphocytes with "matured" dendritic cells (DC) cultured from autologous CD14+ precursors unmasks previously covert cellular immunity in the form of Th2-skewed cytokine production. Supplementation of adult lymphocytes with comparable prematured autologous DC also boosted vaccine-specific T-cell memory expression, but in contrast to the case for the infants, these cytokine responses were heavily Th1 skewed. Compared to adults, infants had significantly fewer circulating myeloid DC (P < 0.0001) and plasmacytoid DC (P < 0.0001) as a proportion of peripheral blood mononuclear cells. These findings suggest that deficiencies in the numbers of antigen-presenting cells and their functional competence at 12 months of age limit the capacity to express effector memory responses and are potentially a key factor in reduced vaccine responsiveness in infants.     

Thrombin induces osteosarcoma growth, a function inhibited by low molecular weight heparin in vitro and in vivo: procoagulant nature of osteosarcoma

BACKGROUND:

Procoagulant states, leading to activation of the coagulation protease thrombin, are common in cancer and portend a poor clinical outcome. Although procoagulant states in osteosarcoma patients have been described, studies exploring osteosarcoma cells' ability to directly contribute to procoagulant activity have not been reported. This study explores the hypothesis that osteosarcoma can regulate thrombin generation and proliferate in response to thrombin, and that attenuating thrombin generation with anticoagulants can slow tumor growth.

METHODS:

Pathologic analysis of osteosarcoma with adjacent venous thrombus was performed. In vitro proliferation assays, cell‐based coagulant activity assays, and quantification of coagulation cofactor expression were performed on human and murine osteosarcoma cell lines with varying aggressiveness. The efficacy of low molecular weight heparin (LMWH) attenuation of tumor‐dependent thrombin generation and growth in vitro and in vivo was determined.

RESULTS:

Venous thrombi adjacent to osteosarcoma were found to harbor tumor surrounded by fibrin expressing coagulation cofactors, a finding associated with poor clinical outcome. More aggressive osteosarcoma cell lines had greater surface expression of procoagulant factors and generated more thrombin than less aggressive cell lines and were found to proliferate in response to thrombin. Treatment with LMWH reduced in vitro osteosarcoma proliferation and procoagulant activity as well as tumor growth in vivo.

CONCLUSIONS:

These findings suggest that elements of the coagulation cascade may play a role in and represent a pharmaceutical target to disrupt osteosarcoma growth. They also have broader implications, as they suggest that, to be effective, dosing of anticoagulants must take into account an individual tumor's capacity to generate thrombin. Cancer 2012. © 2011 American Cancer Society.     

Epidemiology competency development and application to training for local and regional public health practitioners

In 2002, the Northwest Center for Public Health Practice (NWCPHP) at the University of Washington initiated the Epidemiology Competencies Project, with the goal of developing competency-based epidemiology training for non-epidemiologist public health practitioners in the northwestern United States. An advisory committee consisting of epidemiology faculty and experienced public health practitioners developed the epidemiology competencies. NWCPHP used the competencies to guide the development of in-person trainings, a series of online epidemiology modules, and a Web-based repository of epidemiology teaching materials. The epidemiology competencies provided a framework for collaborative work between NWCPHP and local and regional public health partners to develop trainings that best met the needs of a particular public health organization. Evaluation surveys indicated a high level of satisfaction with the online epidemiology modules developed from the epidemiology competencies. However, measuring the effectiveness of competency-based epidemiology training for expanding epidemiology knowledge and skills of the public health workforce remains a challenge.     

Microbiologic and immunologic characteristics of periodontal disease in Hispanic americans with type 2 diabetes

BACKGROUND: The microbiology of periodontitis in type 1 diabetes has been reported, but less is known about type 2 diabetes. Moreover, these data have not linked microbial colonization, host response, and clinical presentation in type 1 or type 2 diabetes. The objectives of this study were to relate periodontal status, periodontal microorganisms, and host-response characteristics in Hispanic Americans with type 2 diabetes. METHODS: Plaque and serum samples were obtained from 63 Hispanic American subjects with and without type 2 diabetes. The microbiology of subgingival plaque samples was evaluated using DNA checkerboard hybridization, and serum antibody to a battery of oral microorganisms was determined using an enzyme-linked immunosorbent assay. RESULTS: In general, similar pathogens were present in periodontitis sites from subjects with and without type 2 diabetes, although the periodontitis sites in diabetes showed a higher frequency of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), and Campylobacter spp. Serum antibody to Campylobacter rectus was elevated in type 2 diabetes, whereas antibody to P. gingivalis and C. rectus were elevated in subjects with periodontitis, irrespective of diabetes status. Stratification of the population based upon antibody to P. gingivalis or C. rectus suggested a linkage between elevated antibody to P. gingivalis, increased frequency of diabetes, and significantly worse periodontitis. CONCLUSION: The increased severity of periodontal disease with type 2 diabetes may reflect an alteration of the pathogenic potential of periodontal bacteria and/or a modification of the characteristics of the host's inflammatory response that may contribute to a breakdown in the homeostasis of the periodontium.