The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.
Rett syndrome (RTT) is an inborn neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 gene (MECP2). Besides mental retardation, most patients suffer from potentially life-threatening breathing arrhythmia. To study its pathophysiology, we performed comparative analyses of the breathing phenotype of Mecp2 −/y knockout (KO) and C57BL/6J wild-type mice using the perfused working heart-brainstem preparation (WHBP). We simultaneously recorded phrenic and efferent vagal nerve activities to analyse the motor pattern of respiration, discriminating between inspiration, postinspiration and late expiration. Our results revealed respiratory disturbances in KO preparations that were similar to those reported from in vivo measurements in KO mice and also to those seen in RTT patients. The main finding was a highly variable postinspiratory activity in KO mice that correlated closely with breathing arrhythmias leading to repetitive apnoeas even under undisturbed control conditions. Analysis of the pontine and peripheral sensory regulation of postinspiratory activity in KO preparations revealed: (i) prolonged apnoeas associated with enhanced postinspiratory activity after glutamate-induced activation of the pontine Kölliker-Fuse nucleus; and (ii) prolonged apnoeas and lack of reflex desensitization in response to repetitive vagal stimulations. We conclude that impaired network and sensory mediated synaptic control of postinspiration induces severe breathing dysfunctions in Mecp2 −/y KO preparations. As postinspiration is particularly important for the control of laryngeal adductors, the finding might explain the upper airway-related clinical problems of patients with RTT such as apnoeas, loss of speech and weak coordination of breathing and swallowing.
Summary:Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells. Keywords: chimerism; short-tandem repeats (STR); quantification; PCR; fluorescence detection Allogeneic blood stem cell transplantation is frequently performed in patients with nonmalignant and malignant hematological diseases such as severe aplastic anemia (SAA), severe combined immunodeficiency (SCID), acute and chronic leukemia and lymphoma. Detection of the degree of chimerism after transplantation is an important method for monitoring the engraftment of donor cells and allows early detection of graft failure. This seems to be especially important in patients at risk for graft failure, ie patients Correspondence: C Thiede, Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus der Technischen Universität, Fetscherstrasse 74, 01307 Dresden, Germany Received 12 September 1998; accepted 4 January 1999 receiving T cell-depleted stem cell grafts from unrelated donors.1 In addition, novel therapeutic approaches like nonmyeloablative stem cell transplantation 2,3 are dependent on rapid information on the degree of mixed chimerism to schedule therapeutic interventions, eg donor lymphocyte infusion (DLI).Several approaches have been published for the detection of chimerism. In sex-mismatched transplantation settings, information on the ratio between donor and recipient can be obtained efficiently and rapidly by using fluorescent in situ hybridization (FISH) with probes specific for X-and Y-chromosome.4,5 PCR-based amplification of a single variable number of tandem repeat (VNTR) or short tandem repeat (STR) markers is another frequently perf...
The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dosedependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [ 125 I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.
Follicular T-helper (T FH ) cells cooperate with GL7 + CD95 + germinal center (GC) B cells to induce antibody maturation. Herein, we identify the transcription factor IRF4 as a T-cell intrinsic precondition for T FH cell differentiation and GC formation. After immunization with protein or infection with the protozoon Leishmania major , draining lymph nodes (LNs) of IFN-regulatory factor-4 ( Irf4 −/− ) mice lacked GCs and GC B cells despite developing normal initial hyperplasia. GCs were also absent in Peyer’s patches of naive Irf4 −/− mice. Accordingly, CD4 + T cells within the LNs and Peyer’s patches failed to express the T FH key transcription factor B-cell lymphoma-6 and other T FH -related molecules. During chronic leishmaniasis, the draining Irf4 −/− LNs disappeared because of massive cell death. Adoptive transfer of WT CD4 + T cells or few L. major primed WT T FH cells reconstituted GC formation, GC B-cell differentiation, and LN cell survival. In support of a T-cell intrinsic IRF4 activity, Irf4 −/− T FH cell differentiation was not rescued by close neighborhood to transferred WT T FH cells. Together with its known B lineage-specific roles during plasma cell maturation and class switch, our study places IRF4 in the center of antibody production toward T-cell–dependent antigens.
Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1-8) compared to 7 (range 1-9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34 + cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34 + Kasumi cell in 40 000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7-9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCRdetectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods. Leukemia (2001) 15, 293-302.
IntroductionImatinib mesylate, or IM (Gleevec; Novartis Pharma, Basel, Switzerland), has demonstrated a remarkable efficacy in the treatment of BCR/ABL ϩ leukemias, with most patients in early chronic phase achieving complete cytogenetic remissions (CCRs). 1 Despite this, BCR/ABL ϩ disease remains detectable in essentially all patients with chronic-phase chronic myelogenous leukemia (CML), and CML always recurs after cessation of IM treatment. 2 This reflects disease persistence under IM therapy. Mechanisms of stem and progenitor cell persistence are presumably progenitor cell quiescence, 3,4 BCR/ABL overexpression, 5 BCR/ABL kinase mutations, 6 and influx and efflux pump expression 7,8 regulating intracellular concentrations of IM. However, additional aberrations may be required to cause a fully drug-resistant phenotype. 9 Clinically manifest IM resistance primarily occurs in advanced stages of CML and in BCR/ABL ϩ acute lymphoblastic leukemia (ALL), and is frequently associated with mutations 10-17 in the causative oncogene of CML, BCR/ABL. 18,19 BCR/ABL kinase mutations are today the best-characterized IM resistance mechanism.To overcome mutation-dependent IM resistance, more selective second-generation ABL kinase inhibitors such as nilotinib (NI; AMN107) 20 and dasatinib (DA), 21 a dual-kinase inhibitor of Abl and Src kinases, were developed and recently also introduced into clinical practice. 22,23 However, DA and NI failed to achieve sustained responses in IM-resistant CML blast crisis and ALL. [22][23][24] Interestingly, neither the response nor the depth of response to NI (hematologic or cytogenetic) depended on the presence or absence of kinase mutations in IM-resistant CML and BCR/ABL ϩ ALL 22 . This implies that BCR/ABL-independent resistance mechanisms 25,26 contribute to IM and NI resistance in a major cohort of patients.Using clonal populations of LAMA cells, we have previously shown that a BCR/ABL-independent activation of the PI3K/Akt signaling cascade precedes kinase mutation-dependent IM resistance development in vitro, 27 and that this autonomous pathway activation mediates early BCR/ABL-independent IM resistance. Others have shown a growth factor-dependent, BCR/ABLindependent activation of the Ras-signaling pathway in CD34 ϩ CML progenitors under IM exposure. 28 Here we were seeking to identify putative factors present in the culture supernatant of IM-exposed BCR/ABL ϩ cells that confer IM resistance. Materials and methods Drugs and reagentsIM and NI was kindly supplied by Drs E. Buchdunger and P. Manley (both of Novartis Pharma). NI was dissolved in dimethyl sulfoxide (DMSO) as a For personal use only. on May 11, 2018. by guest www.bloodjournal.org From 10-mM stock solution and stored in aliquots at Ϫ20°C; IM was stored at Ϫ20°C at 10 mM in distilled water. AG490 29 was obtained from CalbiochemNovabiochem (Bad Soden, Germany). Stock solutions (50 mM) were prepared in DMSO and stored in aliquots at Ϫ20°C. Fresh working solutions were prepared in RPMI-1640 for each experiment. The recombinant ...
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