Italian Cooperative Study for the treatment of children and young adults with localized Ewing sarcoma of bone: a preliminary report of 6 years of experience.

BACKGROUND: In 1991, the Italian Association for Pediatric Hematology-Oncology and the National Council of Research (CNR) initiated an Italian Cooperative Study (SE 91-CNR Protocol) with the main objective of improving the overall survival (SUR) and the event free survival (EFS) of children and young adults with localized Ewing sarcoma and primitive neuroectodermal tumors of bone compared with a previous study (IOR/Ew2 Protocol). METHODS: Between November 1991 and November 1997, 165 patients were enrolled in this study, 160 of whom were evaluable. The patients were treated with a multimodal approach characterized by intensified chemotherapy, hyperfractionated and accelerated radiation therapy, and the addition of ifosfamide and etoposide to standard chemotherapy with vincristine, actinomycin-D, doxorubicin, and cyclophosphamide. RESULTS: After a median follow-up of 37 months, 126 of the 160 evaluable patients remained free of disease recurrence. Thirty-one patients developed a disease recurrence (20 with disseminated disease). CONCLUSIONS: The 3-year SUR and EFS rates found in the current study (83.6% and 77.8%, respectively) may be considered satisfactory. Only age at diagnosis < or =14 years and a good histologic response appeared to affect the outcome of patients with localized Ewing sarcoma positively. These results appear to demonstrate the efficacy of the addition of ifosfamide in induction chemotherapy to four-drug standard combination chemotherapy, as confirmed by the improved outcome in terms of 3-year EFS reported in the SE 91-CNR Protocol compared with the IOR/Ew2 Protocol (77.8% vs. 60.7%). In addition, the better outcome also could be explained by the change in treatment strategy with a trend toward the use of more surgery than radiation therapy compared with the authors' previous protocol.     

High-dose cyclophosphamide followed by autografting can improve the outcome of relapsed or resistant non-Hodgkin's lymphomas with involved or hypoplastic bone marrow

We report our experience of high-dose cyclophosphamide (HDCY) followed by high-dose therapy (HDT) and peripheral blood progenitor cell (PBPC) autografting in patients with diffuse, intermediate and high-grade non-Hodgkin's lymphomas who have failed conventional treatment. From 1991 to 1996, 54 consecutive patients pre-treated with a median of two chemotherapy lines entered the study. Eighteen patients (33%) were still responders to conventional chemotherapy (sensitive relapse), and 20 patients (37%) were in partial response (PR) after chemotherapy (CT). Sixteen patients (30%) were resistant to conventional CT either at presentation (non responder) or in relapse (resistant relapse). Thirty-nine patients had bone marrow involved by disease and fifteen had an hypoplastic marrow following conventional treatment. Patients received HDCY (7gr/m2) and G-CSF or GM-CSF in order to collect PBPC. Median collected CD34+ cells was 12.3 x 10(6)/Kg (range 0.7-197). After HDT (BEAM or Melphalan + TBI) 50 patients underwent PBPC autografting. According to intention to treat, 44 (81%) of 54 patients achieved complete remission (CR) (50% after HDCY and 31% after HDT). Procedure related death occurred in 6 patients (11%), one after HDCY and 5 after autografting. Twenty-nine (66%) of 44 patients are still in CR, 7 to 63 months (median 27 months) after the procedure. Three-year probability of survival, disease-free survival and progression-free survival are 63%, 64% and 52% respectively. In conclusion, HDCY is an effective procedure not only in mobilizing PBPC, but also in reducing tumour burden. HDT with PBPC support may further improve the outcome in this category of high-risk non-Hodgkin's lymphomas.     

Options for Inactivation,Adjuvant, and Route of Topical Administration of a Killed,Unencapsulated Pneumococcal Whole-Cell Vaccine

We previously reported that ethanol-killed cells of a noncapsulated strain of Streptococcus pneumoniae, given intranasally with cholera toxin as an adjuvant, protect rats against pneumonia and mice against colonization of the nasopharynx and middle ear by capsulated pneumococci of various serotypes. The acceleration of pneumococcal clearance from the nasopharynx in mice is CD4⁺ T cell-dependent and interleukin 17A (IL-17A) mediated and can be antibody independent. Here, anticipating human studies, we have demonstrated protection with a new vaccine strain expressing a nonhemolytic derivative of pneumolysin and grown in bovine-free culture medium. Killing the cells with chloroform, trichloroethylene, or beta-propiolactone—all used without postinactivation washing—produced more-potent immunogens than ethanol, and retention of soluble components released from the cells contributed to protection. Two sequential intranasal administrations of as little as 1 μg of protein (total of cellular and soluble combined) protected mice against nasopharyngeal challenge with pneumococci. Nontoxic single and double mutants of Escherichia coli heat-labile toxin were effective as mucosal adjuvants. Protection was induced by the sublingual and buccal routes, albeit requiring larger doses than when given intranasally. Protection was likewise induced transdermally with sonicates of the killed-cell preparation. Thus, this whole-cell antigen can be made and administered in a variety of ways to suit the manufacturer and the vaccination program and is potentially a solution to the need for a low-cost vaccine to reduce the burden of childhood pneumococcal disease in low-income countries.Streptococcus pneumoniae (pneumococcus) imparts a major disease burden among children in low-income countries (27). Capsular polysaccharide conjugate vaccine provides type-specific protection but has the disadvantages of limited serotype coverage, serotype replacement, and high cost of production, storage, and injection (12, 29). Therefore, potentially more economical serotype-independent vaccines based upon species-common protein antigens are being pursued (32). In one such approach, we have studied killed cells of noncapsulated pneumococci, with the intent of maximizing the exposure of species-common subcapsular antigens. This antigen preparation, designated “whole-cell vaccine” (WCV), when formulated with a suitable adjuvant, is intended for mucosal administration to reduce pneumococcal colonization. Killing the cells with 70% (vol/vol) ethanol at 4°C produces a more immunogenic antigen (WCE) than traditional methods of inactivation such as heat, formalin, or UV radiation (21) (unpublished data). Intranasal (i.n.) application had been examined initially, since this route is effective for inducing both systemic and mucosal immunity. Vaccination i.n. with WCE plus cholera toxin (CT) as a mucosal adjuvant prevents fatal serotype 3 pneumonia in rats and reduces nasopharygeal (NP) and middle ear colonization in mice by strains of serotype 6B or 23F (22, 23). Although the levels of serum antibodies are raised by the i.n. vaccination, protection against colonization is induced in mice in the absence of antibodies by a CD4⁺ T-cell-dependent, interleukin 17A (IL-17A)-mediated mechanism (20, 24). As few as 10⁷ cells (ca. 10 μg of protein) of WCE, given thrice sequentially, are protective in the colonization model (34). This potency and the potential low cost of manufacture were motivation for a partnership with PATH and Instituto Butantan (São Paolo, Brazil) for further development of the WCV.Previous studies used strain Rx1AL expressing native pneumolysin, a potent cytolysin. Here, anticipating human studies, a derivative of pneumolysin (PdT), with mutations W433F, D385N, and C428G (which render the molecule nonhemolytic and unable to activate complement [5] but maintain its TLR4 agonistic properties [21]), was used. Previously, cells were grown in Todd-Hewitt-yeast broth, which contains beef heart infusion. Here, to avoid any hazard of bovine components, cells grown in a soy-based medium (19) were examined for protection. The challenge of safe handling and disposal of large volumes of ethanol led to the evaluation of three alternative agents of inactivation that are bactericidal at low concentration: chloroform, trichloroethylene, and beta-propiolactone. Since chloroform and trichloroethylene are both highly volatile and beta-propiolactone is inactivated by warming, these agents may be removed without postinactivation washing, which permitted convenient examination of soluble components released from the killed cells. Soluble components produced by sonication also were examined.The possible side effects of enterotoxins as adjuvants (25) and other problems with intranasal vaccination prompted the consideration of genetically detoxified enterotoxin derivatives and of the buccal and sublingual routes of administration. Transdermal immunization was also examined with sonicated antigen preparations. These varied immunization procedures were surveyed for protection against colonization, evident as acceleration of nasopharyngeal clearance after intranasal challenge with a strain of serotype 6B (20). The results indicate that the cells can be inactivated by several agents to generate a potent whole-cell antigen that could be given in a variety of ways to accommodate preferences of a particular vaccination program.     

Transcutaneous immunization with heat-labile enterotoxin: development of a needle-free vaccine patch

The skin is an attractive target for vaccine delivery. Adjuvants and antigens delivered into the skin can result in potent immune responses and an unmatched safety profile. The heat-labile enterotoxin (LT) from Escherichia coli, which acts both as antigen and adjuvant, has been shown to be delivered to human skin efficiently when used in a patch, resulting in strong immune responses. Iomai scientists have capitalized on these observations to develop late-stage products based on LT. This has encouraged commercial-level product development of a delivery system that is efficient, user-friendly and designed to address important medical needs. Over the past 2 years, extensive clinical testing and optimization has allowed the patch to evolve to a late-stage product. As a strategy for approval of a revolutionary vaccine-delivery system, the singular focus on optimization of LT delivery has enabled technical progress to extend patch-vaccine product development beyond LT. The field efficacy of the LT-based travelers' diarrhea vaccine has validated this approach. The discussion of transcutaneous immunization is unique, in that any consideration of the adjuvant must also include delivery, and the significant advances in a commercial patch application system are described. In this review, we integrate these concepts, update the clinical data and look to the future.     

Recognition of stage-specific mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis

Mycobacterium tuberculosis is estimated to infect 80 to 100 million people annually, the majority of whom do not develop clinical tuberculosis (TB) but instead maintain the infection in a latent state. These individuals generally become positive in response to a tuberculin skin test and may develop clinical TB at a later date, particularly if their immune systems are compromised. Latently infected individuals are interesting for two reasons. First, they are an important reservoir of M. tuberculosis, which needs to be considered for TB control. Second, if detected prior to recrudescence of the disease, they represent a human population that is making a protective immune response to M. tuberculosis, which is very important for defining correlates of protective immunity. In this study, we show that while responsiveness to early secretory antigenic target 6 is a good marker for M. tuberculosis infection, a strong response to the 16-kDa Rv2031c antigen (HspX or alpha-crystallin) is largely restricted to latently infected individuals, offering the possibility of differential immunodiagnosis of, or therapeutic vaccination against, TB.     

The sigma-1 receptor antagonist PB212 reduces the Ca²⁺-release through the inositol (1, 4, 5)-trisphosphate receptor in SK-N-SH cells

Sigma-1 receptors are specifically located at the endoplasmic reticulum-mitochondrion interface, but upon stimulation by ligands or under prolonged cellular stress, they translocate to other areas of the cell. Sigma-1 receptors are involved in the regulation of intracellular [Ca(2+)] by affecting the Ca(2+)-influx or the release from intracellular stores. In SK-N-SH cells, we measured the affinity of 4-methyl-1-[4-(6-methoxynaphthalen-1-yl)butyl]piperidine (PB212) at sigma-1 receptor by using a competition binding assay with specific radioligand; we obtained a K(i) value=316 ± 19 nM. PB212 also showed an antiproliferative effect in SK-N-SH cells (EC(50)=32 ± 4 μM) but had no effect in MCF7 cells, which only express sigma-2 receptor; these findings suggest that PB212 behaves as a sigma-1 receptor antagonist. We have studied the effect of PB212 on Ca(2+) homeostasis of the SK-N-SH cell line with the fluorescent probe Fura-2. 100 μM PB212 induced a Ca(2+)-efflux from the endoplasmic reticulum through the inositol (1, 4, 5)-trisphosphate (IP(3)) receptor. Moreover, [PB212] ranging from 1 to 100μM reduced the Ca(2+)-response, triggered by carbachol or bradykinin that engage the phospholipase C/IP(3) pathway; such a response is generally increased by sigma-1 receptor agonists. On the other hand, PB212 did not reduce the Ca(2+)-response mediated by IP(3) in LoVo cells, which do not express neither sigma-1 nor sigma-2 receptors, and in MCF7 cells. The fact that the activity of the sigma-1 receptor can be experimentally modulated by agonists and antagonists supports the intriguing hypothesis that some endogenous molecules, unknown at the moment, modulate the sigma-1 receptor and its cellular targets.     

A Mentor-Based Portfolio Program to Evaluate Pharmacy Students’ Self-Assessment Skills

Objective. To evaluate pharmacy students'' self-assessment skills with an electronic portfolio program using mentor evaluators.Design. First-year (P1) and second-year (P2) pharmacy students used online portfolios that required self-assessments of specific graded class assignments. Using a rubric, faculty and alumni mentors evaluated students'' self-assessments and provided feedback. Assessment. Eighty-four P1 students, 74 P2 students, and 59 mentors participated in the portfolio program during 2010-2011. Both student groups performed well overall, with only a small number of resubmissions required. P1 students showed significant improvements across semesters for 2 of the self-assessment questions; P2 students'' scores did not differ significantly. The P1 scores were significantly higher than P2 scores for 3 questions during spring 2011. Mentors and students had similar levels of agreement with the extent to which students put forth their best effort on the self-assessments.Conclusion. An electronic portfolio using mentors based inside and outside the school provided students with many opportunities to practice their self-assessment skills. This system represents a useful method of incorporating self-assessments into the curriculum that allows for feedback to be provided to the students.     

Frailty of older age: the role of the endocrine--immune interaction

The so-called demographic transition has changed the age structure of the population worldwide, with profound effects on societal organization. The growing number and percentage of old and very old people has compelled the scientific community to focus on age related diseases and peculiar consequences of aging itself such as disability and frailty. Understanding the pathophysiology of frailty, a syndrome characterized by a reduced functional reserve and impaired adaptive capacity that results from cumulative declines of multiple subsystems, and causes increased vulnerability to adverse outcomes, is a major topic in aging research. Aging processes induce multiple changes in the hormones network (menopause, andropause, somatopause and adrenopause), in the immune system, and can modulate their efficiency and effectiveness in determining a response to stressors. These triggering events can unmask frailty in older people. Starting from these assumptions, we analyzed the relationship of the endocrine and immune networks in aging and in the different domains that are characteristically associated with the frailty syndrome, such as disability and sarcopenia, as well as in diseases related to aging such as Alzheimer's dementia and Congestive Heart Failure.     

Ethanol exposure during late gestation and nursing in the rat: effects upon maternal care, ethanol metabolism and infantile milk intake

Ethanol experiences, during late gestation as well as during nursing, modify the behavioral dynamics of the dam/pup dyad, and leads to heightened ethanol intake in the offspring. This study focuses on: a) behavioral and metabolic changes in intoxicated dams with previous exposure to ethanol during pregnancy and b) infantile consumption of milk when the dam is either under the effects of ethanol or sober. Pregnant rats received water, 1.0 or 2.0 g/kg ethanol, and were administered with water or ethanol during the postpartum period. Intoxication during nursing disrupted the capability of the dam to retrieve the pups and to adopt a crouching posture. These disruptions were attenuated when dams had exposure to ethanol during pregnancy. Ethanol experiences during gestation did not affect pharmacokinetic processes during nursing, whereas progressive postpartum ethanol experience resulted in metabolic tolerance. Pups suckling from intoxicated dams, with previous ethanol experiences, ingested more milk than did infants suckling from ethanol-intoxicated dams without such experience. Ethanol gestational experience results in subsequent resistance to the drug's disruptions in maternal care. Consequently, better maternal care by an intoxicated dam with ethanol experience during gestation facilitates access of pups to milk which could be contaminated with ethanol.