Fourth European Conference on Infections in Leukaemia (ECIL-4): guidelines for diagnosis, prevention, and treatment of invasive fungal diseases in paediatric patients with cancer or allogeneic haemopoietic stem-cell transplantation.

Invasive opportunistic fungal diseases (IFDs) are important causes of morbidity and mortality in paediatric patients with cancer and those who have had an allogeneic haemopoietic stem-cell transplantation (HSCT). Apart from differences in underlying disorders and comorbidities relative to those of adults, IFDs in infants, children, and adolescents are unique with respect to their epidemiology, the usefulness of diagnostic methods, the pharmacology and dosing of antifungal agents, and the absence of interventional phase 3 clinical trials for guidance of evidence-based decisions. To better define the state of knowledge on IFDs in paediatric patients with cancer and allogeneic HSCT and to improve IFD diagnosis, prevention, and management, the Fourth European Conference on Infections in Leukaemia (ECIL-4) in 2011 convened a group that reviewed the scientific literature on IFDs and graded the available quality of evidence according to the Infectious Diseases Society of America grading system. The final considerations and recommendations of the group are summarised in this manuscript.

Homeostasis limits the development of mature CD8+ but not CD4+ thymocytes.

The involvement of a variety of clonal selection processes during the development of T lymphocytes in the thymus has been well established. Less information, however, is available on how homeostatic mechanisms may regulate the generation and maturation of thymocytes. To investigate this question, mixed radiation bone marrow chimeras were established in which wild-type T cell precursors capable of full maturation were diluted with precursors deficient in maturation potential because of targeted mutations of the RAG1 or TCR-alpha genes. In chimeras in which the majority of thymocytes are blocked at the CD4- CD8- CD25+ stage (RAG1 deficient), and only a small proportion of T cell precursors are of wild-type origin, we observed no difference in the maturation of wild-type CD4- CD8- CD25+ cells to the CD4+ CD8+ stage as compared with control chimeras. Therefore, the number of cell divisions occurring during this transition is fixed and not subject to homeostatic regulation. In contrast, in mixed chimeras in which the majority of thymocytes are blocked at the CD4+ CD8+ stage (TCR-alpha deficient), an increased efficiency of development of wild-type mature CD8+ cells was observed. Surprisingly, the rate of generation of mature CD4+ thymocytes was not affected in these chimeras. Thus, the number of selectable CD8 lineage thymocytes apparently saturates the selection mechanism in normal mice while the development of CD4 lineage cells seems to be limited only by the expression of a suitable TCR. These data may open the way to the identification of homeostatic mechanisms regulating thymic output and CD4/CD8 lineage commitment, and the development of means to modulate it.

An adult thymic stromal-cell suspension model for in vitro positive selection.

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.

Follicular helper T cells: implications in neoplastic hematopathology.

A distinct subset of T helper cells, named follicular T helper cells (T(FH), has been recently described. T(FH) cells are characterized by their homing capacities in the germinal centers of B-cell follicles where they interact with B cells, supporting B-cell survival and antibody responses. T(FH) cells can be identified by the expression of several markers including the chemokine CXCL13, the costimulatory molecules PD1 and inducible costimulator, and the transcription factor BCL6. They appear to be relevant markers for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL) and have helped to recognize subsets of peripheral T-cell lymphoma, not otherwise specified, with nodal or cutaneous presentation expressing T(FH) antigens that might be related to AITL. In B-cell neoplasms, T(FH) cells are present within the microenvironment of nodular lymphocyte-predominant Hodgkin lymphoma and follicular lymphoma, where they likely support the growth of neoplastic germinal center-derived B cells. Interestingly, the amount of PD1+ cells in the neoplastic follicles might have a favorable impact on the outcome of follicular lymphoma patients. Altogether, the availability of antibodies directed to T(FH)-associated molecules has important diagnostic and prognostic implications in hematopathology. In addition, T(FH) cells could represent interesting targets in T(FH)-derived lymphomas such as AITL, or in some B-cell neoplasms where they act as part of the tumor microenvironment.

Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells.

Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1(-/)- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1(-/)- T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.

Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin

Lymphatic vascular morphogenesis in development, physiology, and disease.

The lymphatic vasculature constitutes a highly specialized part of the vascular system that is essential for the maintenance of interstitial fluid balance, uptake of dietary fat, and immune response. Recently, there has been an increased awareness of the importance of lymphatic vessels in many common pathological conditions, such as tumor cell dissemination and chronic inflammation. Studies of embryonic development and genetically engineered animal models coupled with the discovery of mutations underlying human lymphedema syndromes have contributed to our understanding of mechanisms regulating normal and pathological lymphatic morphogenesis. It is now crucial to use this knowledge for the development of novel therapies for human diseases.

A Multi-Center Phase II Study (SAKK 36/06) of Single Agent Everolimus (RAD001) In Patients with Relapsed or Refractory Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) accounts for 6% of all B-cell lymphomas and remains incurable for most patients. Those who relapse after first line therapy or hematopoietic stem cell transplantation have a dismal prognosis with short response duration after salvage therapy. On a molecular level, MCL is characterised by the translocation t[11;14] leading to Cyclin D1 overexpression. Cyclin D1 is downstream of the mammalian target of rapamycin (mTOR) kinase and can be effectively blocked by mTOR inhibitors such as temsirolimus. We set out to define the single agent activity of the orally available mTOR inhibitor everolimus (RAD001) in a prospective, multi-centre trial in patients with relapsed or refractory MCL (NCT00516412). The study was performed in collaboration with the EU-MCL network. Methods: Eligible patients with histologically/cytologically confirmed relapsed (not more than 3 prior lines of systemic treatment) or refractory MCL received everolimus 10 mg orally daily on day 1 - 28 of each cycle (4 weeks) for 6 cycles or until disease progression. The primary endpoint was the best objective response with adverse reactions, time to progression (TTP), time to treatment failure, response duration and molecular response as secondary endpoints. A response rate of ≤ 10% was considered uninteresting and, conversely, promising if ≥ 30%. The required sample size was 35 pts using the Simon's optimal two-stage design with 90% power and 5% significance. Results: A total of 36 patients with 35 evaluable patients from 19 centers were enrolled between August 2007 and January 2010. The median age was 69.4 years (range 40.1 to 84.9 years), with 22 males and 13 females. Thirty patients presented with relapsed and 5 with refractory MCL with a median of two prior therapies. Treatment was generally well tolerated with anemia (11%), thrombocytopenia (11%), neutropenia (8%), diarrhea (3%) and fatigue (3%) being the most frequent complications of CTC grade III or higher. Eighteen patients received 6 or more cycles of everolimus treatment. The objective response rate was 20% (95% CI: 8-37%) with 2 CR, 5 PR, 17 SD, and 11 PD. At a median follow-up of 6 months, TTP was 5.45 months (95% CI: 2.8-8.2 months) for the entire population and 10.6 months for the 18 patients receiving 6 or more cycles of treatment. Conclusion: This study demonstrates that single agent everolimus 10 mg once daily orally is well tolerated. The null hypothesis of inactivity could be rejected indicating a moderate anti-lymphoma activity in relapsed/refractory MCL. Further studies of either everolimus in combination with chemotherapy or as single agent for maintenance treatment are warranted in MCL.

Myeloid cells contribute to tumor lymphangiogenesis.

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation.

Cutting edge: thymic NK cells develop independently from T cell precursors.

Although NK cells in the mouse are thought to develop in the bone marrow, a small population of NK cells in the thymus has been shown to derive from a GATA3-dependent pathway. Characteristically, thymic NK cells express CD127 and few Ly49 molecules and lack CD11b. Because these NK cells develop in the thymus, the question of their relationship to the T cell lineage has been raised. Using several different mouse models, we find that unlike T cells, thymic NK cells are not the progeny of Rorc-expressing progenitors and do not express Rag2 or rearrange the TCRγ locus. We further demonstrate that thymic NK cells develop independently of the Notch signaling pathway, supporting the idea that thymic NK cells represent bona fide NK cells that can develop independently of all T cell precursors.

Immunogenetics of invasive aspergillosis.

Invasive aspergillosis is one of the most important infections in hematopoietic stem cell transplant recipients, with an incidence rate of 5-15% and an associated mortality of 30-60%. It remains unclear why certain patients develop invasive aspergillosis while others, undergoing identical transplant regimen and similar post transplant immunosuppression, do not. Over the last decade, pattern recognition receptors such as Toll-like receptors (TLRs) and the C-type lectin receptors (CLRs) have emerged as critical components of the innate immune system. By detecting specific molecular patterns from invading microbes and initiating inflammatory and subsequent adaptive immune responses, pattern recognition receptors are strategically located at the molecular interface of hosts and pathogens. Polymorphisms in pattern recognition receptors and downstream signaling molecules have been associated with increased or decreased susceptibility to infections, suggesting that their detection may have an increasing impact on the treatment and prevention of infectious diseases in the coming years. Infectious risk stratification may be particularly relevant for patients with hematologic malignancies, because of the high prevalence and severity of infections in this population. This review summarizes the innate immune mechanisms involved in Aspergillus fumigatus detection and the role of host genetic polymorphisms in susceptibility to invasive aspergillosis.

Influence of learning on range expansion and adaptation to novel habitats.

Learning has been postulated to 'drive' evolution, but its influence on adaptive evolution in heterogeneous environments has not been formally examined. We used a spatially explicit individual-based model to study the effect of learning on the expansion and adaptation of a species to a novel habitat. Fitness was mediated by a behavioural trait (resource preference), which in turn was determined by both the genotype and learning. Our findings indicate that learning substantially increases the range of parameters under which the species expands and adapts to the novel habitat, particularly if the two habitats are separated by a sharp ecotone (rather than a gradient). However, for a broad range of parameters, learning reduces the degree of genetically-based local adaptation following the expansion and facilitates maintenance of genetic variation within local populations. Thus, in heterogeneous environments learning may facilitate evolutionary range expansions and maintenance of the potential of local populations to respond to subsequent environmental changes.

Deciphering and reversing tumor immune suppression.

Generating an anti-tumor immune response is a multi-step process that is executed by effector T cells that can recognize and kill tumor targets. However, tumors employ multiple strategies to attenuate the effectiveness of T-cell-mediated attack. They achieve this by interfering with nearly every step required for effective immunity, from deregulation of antigen-presenting cells to establishment of a physical barrier at the vasculature that prevents homing of effector tumor-rejecting cells and the suppression of effector lymphocytes through the recruitment and activation of immunosuppressive cells such as myeloid-derived suppressor cells, tolerogenic monocytes, and T regulatory cells. Here, we review the ways in which tumors exert immune suppression and highlight the new therapies that seek to reverse this phenomenon and promote anti-tumor immunity. Understanding anti-tumor immunity, and how it becomes disabled by tumors, will ultimately lead to improved immune therapies and prolonged survival of patients.

Transitory glutathione deficit during brain development induces cognitive impairment in juvenile and adult rats: relevance to schizophrenia.

Glutathione (GSH) metabolism dysfunction is one risk factor in schizophrenia. A transitory brain GSH deficit was induced in Wistar (WIS) and mutant (ODS; lacking ascorbic acid synthesis) rats using BSO (l-buthionine-(S,R)-sulfoximine) from post-natal days 5-16. When GSH was re-established to physiological levels, juvenile BSO-ODS rats were impaired in the water maze task. Long after treatment cessation, adult BSO-WIS/-ODS rats showed impaired place discrimination in the homing board with distributed visual or olfactory cues. Their accuracy was restored when a single cue marked the trained position. Similarly, more working memory errors were made by adult BSO-WIS in the radial maze when several olfactory cues were present. These results reveal that BSO rats did not suffer simple sensory impairment. They were selectively impaired in spatial memory when the task required the integration of multimodal or olfactory cues. These results, in part, resemble some of the reported olfactory discrimination and cognitive impairment in schizophrenia.

Achievement of at least very good partial response is a simple and robust prognostic factor in patients with multiple myeloma treated with high-dose therapy: long-term analysis of the IFM 99-02 and 99-04 Trials.

PURPOSE: The prognostic impact of complete response (CR) achievement in multiple myeloma (MM) has been shown mostly in the context of autologous stem-cell transplantation. Other levels of response have been defined because, even with high-dose therapy, CR is a relatively rare event. The purpose of this study was to analyze the prognostic impact of very good partial response (VGPR) in patients treated with high-dose therapy. PATIENTS AND METHODS: All patients were included in the Intergroupe Francophone du Myelome 99-02 and 99-04 trials and treated with vincristine, doxorubicin, and dexamethasone (VAD) induction therapy followed by double autologous stem-cell transplantation (ASCT). Best post-ASCT response assessment was available for 802 patients. RESULTS: With a median follow-up of 67 months, median event-free survival (EFS) and 5-year EFS were 42 months and 34%, respectively, for 405 patients who achieved at least VGPR after ASCT versus 32 months and 26% in 288 patients who achieved only partial remission (P = .005). Five-year overall survival (OS) was significantly superior in patients achieving at least VGPR (74% v 61% P = .0017). In multivariate analysis, achievement of less than VGPR was an independent factor predicting shorter EFS and OS. Response to VAD had no impact on EFS and OS. The impact of VGPR achievement on EFS and OS was significant in patients with International Staging System stages 2 to 3 and for patients with poor-risk cytogenetics t(4;14) or del(17p). CONCLUSION: In the context of ASCT, achievement of at least VGPR is a simple prognostic factor that has importance in intermediate and high-risk MM and can be informative in more patients than CR.