C3bi-binding protein on Candida albicans: temperature-dependent expression and relationship to human complement receptor type 3.

We investigated in detail the previously described capacity of pseudohyphae of Candida albicans to bind C3-coated particles. We show that the expression of the C3bi receptor of C. albicans was dependent upon the growth temperature of the fungi. C. albicans grown at 30 degrees C bound strongly to EAC1423bi, whereas those cells grown at 38.5 degrees C were completely devoid of this capacity. The molecule responsible for the attachment of EAC1423bi was heat labile and trypsin sensitive. Several, but not all, monoclonal antibodies to the alpha-chain of human complement receptor type 3 (CR3) stained C. albicans, and this reactivity was expressed in parallel with the capacity of C. albicans to bind EAC1423bi, i.e., both were dependent on the growth temperature of the fungi and were trypsin sensitive. In contrast to CR3, the binding of EAC1423bi to C. albicans did not require the presence of divalent cations. Rabbit immunoglobulin G antibodies directed against C. albicans inhibited the binding of EAC1423bi to C. albicans but not to human CR3. These inhibiting IgG antibodies recognized antigens expressed on the surface of pseudohyphae but not those of yeast cells. OKM-1, a monoclonal antibody to human CR3 inhibited the attachment of EAC1423bi to CR3 and also to C. albicans. OKM-1 precipitated a 130-kilodalton band from solubilized 125I-labeled C. albicans. We conclude that the complement receptors on C. albicans and human CR3 were antigenically related but not identical and that they differed in their functional characteristics.     

Fungal colonization in neutropenic patients: a randomized study comparing itraconazole solution and amphotericin B solution

We assessed the impact of prophylaxis with the oral itraconazole solution and amphotericin B solution on fungal colonization and infection in a randomized study among patients with hematological malignancies and neutropenia. Infecting and colonizing Candida strains of patients suffering from candidiasis were genotyped by random amplification of polymorphic DNA (RAPD) analysis. A total of 106 patients were evaluated in this study: 52 patients in the itraconazole and 54 in the amphotericin B arm. During neutropenia fungal colonization in the oropharynx occurred in 11 (19.6%) and 24 (40.6%) and in the rectum in 11 (19.6%) and 23 (38.9%) courses in the itraconazole and amphotericin B groups (P<0.05), respectively. Candida albicans was the most prevalent species in both study groups. Mixed fungal colonization with Candida krusei and Candida glabrata was increased in the amphotericin B group, yet without clinical importance since infections were due to C. albicans. The occurrence of invasive candidiasis was significantly increased in multicolonized compared to monocolonized patients. In the amphotericin B group 20 and in the itraconazole group 2 neutropenic patients showed multicolonization with Candida spp. (P<0.05). Overall fungal infections were 3.8% in the itraconazole and 14.8% in the amphotericin B group (P<0.05). RAPD typing showed oropharynx strains involved in superficial infections in four of five patients. In all four patients with deep fungal infections, it appears that the colonizing rectum strains were identical to infecting strains of Candida spp. Itraconazole solution significantly reduced Candida colonization and infection compared to amphotericin B solution. Most patients remained infected with the colonized strains for the entire study period, irrespective of antifungal prophylaxis.     

Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC- IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph- , respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.     

Nilotinib as frontline and second-line therapy in chronic myeloid leukemia: open questions

Nilotinib is a second generation ABL tyrosine kinase inhibitor (TKI) that exerts major anti-leukemic effects in newly diagnosed patients with chronic myeloid leukemia (CML) as well as in most patients with imatinib-resistant CML. In freshly diagnosed patients, the anti-leukemic activity of nilotinib exceeds the efficacy of imatinib, and although long-term data for nilotinib are not available yet, the drug has recently been approved for firstline treatment of chronic phase CML in various countries. Still however, several questions concerning the optimal dose, follow-up parameters, long-term safety, and patient selection remain open. Likewise, it remains uncertain whether both Sokal low-risk and high-risk patients should receive nilotinib as frontline therapy in the future. Another question is whether nilotinib can completely eradicate CML in a subset of patients. Furthermore, it remains unclear whether and what comorbidity must be regarded as relative or absolute contra-indication for this TKI. To discuss these issues, the Austrian CML Working Group organized a series of meetings in 2010. In the current article, the outcomes from these discussions are summarized and presented together with recommendations for frontline use of TKIs in various groups of patients with CML. These recommendations should assist in daily practice as well as in the preparation and conduct of clinical trials.     

Impact of natural killer cell dose and donor killer-cell immunoglobulin-like receptor (KIR) genotype on outcome following human leucocyte antigen-identical haematopoietic stem cell transplantation

To define the role of quantitative graft composition and donor killer-cell immunoglobulin-like receptor (KIR) genotype in clinical outcome following unmanipulated peripheral blood stem cell transplantation (PBSCT) from human leucocyte antigen (HLA)-identical siblings, 43 consecutive transplants for haematological malignancies were analysed retrospectively. Twenty-four patients underwent myeloablative conditioning and 19 received busulphan/fludarabine-based reduced intensity conditioning (RIC). In patients with acute myelogenous leukaemia or myelodysplastic syndrome (AML/MDS; n = 18), no relapse occurred following transplants meeting both a high (above median) natural killer (NK) cell count and missing HLA-ligand(s) to donor's KIR(s), compared to all other AML/MDS patients (0% versus 44%; P = 0.049). Missing HLA-B and/or HLA-C ligand combined with missing HLA-A3/11 (KIR3DL2 unblocked) predicted for reduced relapse incidence regardless of diagnosis or conditioning type (P = 0.028). Moreover, in AML/MDS patients, this constellation predicted superior overall survival (OS) (P = 0.046). Transplants with more than two different activating donor KIRs were associated with an increased risk for non-relapse mortality (NRM), both by univariate and multivariate analysis. Quantitative graft composition had a significant impact exclusively in RIC transplants. Here, a trend towards reduced relapse incidence was found in patients receiving high numbers of NK cells (16% versus 54%; P = 0.09). In patients receiving high versus low T cell numbers, OS was superior (83% versus 37%; P = 0.01), due mainly to reduced NRM (0% versus 33%; P = 0.046). By multivariate analysis, relapse risk was decreased significantly in patients receiving high NK cell numbers (P = 0.039). These data suggest that both the number of transplanted NK cells and the donor KIR genotype play a role in graft-versus-malignancy mechanisms in HLA-identical PBSCT.     

Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.     

Anti‐leukemic activity of valproic acid and imatinib mesylate on human Ph+ ALL and CML cells in vitro

The armamentarium of anti‐leukemic drugs has increased substantially since anti‐leukemic activities were recently found for a variety of non‐classical cytostatic drugs, among them the histone deacetylase (HDAC) inhibitor valproic acid (VPA). This study investigated the effect of VPA on proliferation and apoptosis of human Philadelphia chromosome‐positive (Ph+) acute lymphatic (ALL) and chronic myeloid leukemia (CML) cells and on colony formation of human chronic‐phase CML progenitor cells. Strong anti‐proliferative and pro‐apoptotic effects of VPA were observed on human ALL and CML cell lines at concentrations achievable in vivo. These effects were most pronounced in ALL cell lines as well as in primary ALL cells. Notably, VPA revealed enhanced activity with imatinib mesylate, nilotinib, the farnesyl transferase inhibitor SCH66336, interferon‐alpha and cytosine arabinoside. VPA inhibited the growth of colony‐forming cells from 12 Ph+ chronic‐phase CML patients but also of those from normal healthy controls in a dose‐dependent fashion. HDAC‐inhibiting activity of VPA was confirmed on ALL and CML cells. In conclusion, VPA, whether alone or in combination with other non‐classical anti‐leukemic compounds, exerts significant anti‐leukemic effects on human ALL and CML cells.     

A2A antagonist prevents dopamine agonist-induced motor complications in animal models of Parkinson's disease

Adenosine A(2A) receptors, abundantly expressed on striatal medium spiny neurons, appear to activate signaling cascades implicated in the regulation of coexpressed ionotropic glutamatergic receptors. To evaluate the contribution of adenosinergic mechanisms to the pathogenesis of the response alterations induced by dopaminergic treatment, we studied the ability of the selective adenosine A(2A) receptor antagonist KW-6002 to prevent as well as palliate these syndromes in rodent and primate models of Parkinson's disease. In rats, KW-6002 reversed the shortened motor response produced by chronic levodopa treatment while reducing levodopa-induced hyperphosphorylation at S845 residues on AMPA receptor GluR1 subunits. In primates, KW-6002 evidenced modest antiparkinsonian activity when given alone. Once-daily coadministration of KW-6002 with apomorphine prevented the development of dyskinesias, which appeared in control animals 7-10 days after initiating apomorphine treatment. Animals initially given apomorphine plus KW-6002 for 3 weeks did not begin to manifest apomorphine-induced dyskinesias until 10-12 days after discontinuing the A(2A) antagonist. These results suggest that KW-6002 can attenuate the induction as well as the expression of motor response alterations to chronic dopaminergic stimulation in parkinsonian animals, possibly by blocking A(2A) receptor-stimulated signaling pathways. Our findings strengthen the rationale for developing A(2A) antagonists as an early treatment strategy for Parkinson's disease.     

Neuroprotection in the MPTP Parkinsonian C57BL/6 mouse model by a compound isolated from tobacco

Epidemiological evidence suggests a lower incidence of Parkinson's disease in smokers than in nonsmokers. This evidence, together with the lower levels of brain monoamine oxidase (MAO) activity in smokers and the potential neuroprotective properties of MAO inhibitors, prompted studies which led to the isolation and characterization of 2,3,6-trimethyl-1,4-naphthoquinone (TMN), an MAO-A and MAO-B inhibitor which is present in tobacco and tobacco smoke. Results of experiments reported here provide evidence that this compound protects against the MPTP-mediated depletion of neostriatal dopamine levels in the C57BL/6 mouse. These results support the hypothesis that the inhibition of MAO by constituents of tobacco smoke may be related to the decreased incidence of Parkinson's disease in smokers.