•Borrelia garinii belongs to the B burgdorferi complex•Borrelia infections can be atypical in patients who are immunocompromised•Borrelia serology is often negative in patients treated with ...rituximab•Molecular biology is a valuable diagnostic tool in patients treated with rituximab•Borrelia infection may be associated with various type of lymphoma
We report an atypical Borrelia garinii infection in a patient who was immunocompromised. It was first suspected as a transformation of follicular lymphoma into high-grade lymphoma. Spirochetes were directly observed on a peripheral blood smear and the diagnosis was confirmed using molecular methods. The clinical presentation and the diagnosis are unique and contrast with the cases described in the literature in patients who are immunocompromised.
We determined the allelic frequency of the JAK2-V617F mutation in DNA and assessed the expression levels of the mutant and wild-type JAK2 mRNA in granulocytes from 60 patients with essential ...thrombocythemia (ET) and 62 patients with polycythemia vera (PV) at the time of diagnosis. Using allele-specific quantitative polymerase chain reaction (qPCR), we detected JAK2-V617F in 75% of ET and 97% of PV at diagnosis. The total JAK2 mRNA levels were elevated in ET, PV, and secondary and idiopathic erythrocytosis, suggesting that hyperactive hematopoiesis alters JAK2 expression. The expression levels of JAK2-V617F mRNA were variable but strongly correlated with the allelic ratio of JAK2-V617F determined in DNA. Thus, differences in JAK2-V617F expression, markedly lower in ET than in PV, reflected different percentages of granulocytes carrying the mutation. Moreover, allelic ratios higher than 50% JAK2-V617F, indicating the presence of granulocytes homozygous for JAK2-V617F, were found in 70% of PV at diagnosis but never in ET.
BACKGROUND: Controlled‐rate freezing and storage in nitrogen is the standard technique for cryopreservation of peripheral hematopoietic progenitor cells (PHPCs) but presents high cost and dimethyl ...sulfoxide (DMSO) toxicity. Cryopreservation at −80°C, by uncontrolled rate freezing with only 3.5% DMSO, preserves the functional capacities of PHPCs, produces successful engraftment, and reduces toxicity during infusion.
STUDY DESIGN AND METHODS: Long‐term hematopoietic and immunologic reconstitution for 342 autografts (311 adults, 31 children) after PHPCs were cryopreserved at −80°C was studied at 3, 6, and 12 months. The median (range) storage time of PHPCs cryopreserved was 1.7 (0.1‐5.99) months.
RESULTS: Hemoglobin (Hb), white blood cells, and platelets (PLTs) reach normal values to trilineage at 12 months for 39% patients. Multivariate analysis shows a significant impact on CD34+ infused and on conditioning regimen for PLTs. Hb was influenced by growth factor administration at 3 months. Long‐term recovery is also highly dependent on blood counts (Hb, PLT, and neutrophil) at start of high‐dose chemotherapy. Only 43% of patients had reached normal lymphocyte values at 12 months after transplant, and a profound CD4+ T‐lymphocyte deficit remained, as others reported.
CONCLUSION: Transplantation with PHPCs cryopreserved at −80°C for no more than 6 months is satisfactory for long‐term hematopoietic and immunologic reconstitution, even if a profound CD4+ T lymphocyte deficit persists at 1 year. This easier and cheaper cryopreservation method also leads to successful engraftment.
To understand the complex interactions between hematopoietic stem cells and the bone marrow niche, a human experimental model is needed. Our hypothesis is that hematons are an appropriate ex vivo ...model of human bone marrow. We analyzed the hierarchical hematopoietic cell content and the tissue organization of single hematons from healthy donors. Most (>90%) hematons contained precursors of all cell lineages, myeloid progenitors, and LTC-ICs without preferential commitment. Approximately, half of the hematons could generate significant levels of lympho-myeloid hematopoiesis after transplantation in an NSG mouse model, despite the low absolute numbers of transplanted CD34
cells. Mesenchymal STRO-1
and/or CD271
cells formed a critical network that preserved hematon cohesion, and STRO-1
cells colocalized with most hematopoietic CD34
cells (68%). We observed an influence of age and gender. These structures represent a particularly attractive model for studying the homeostasis of the bone marrow niche and pathological changes that occur during diseases.
Summary
The scarcity of mesenchymal stem cells (MSC) in bone marrow (BM) has justified their ex vivo expansion before therapeutic use, but a method to evaluate the quality of initial mesenchymal ...content and track the modifications induced by graft processing has not yet been proposed. The aim of this study was to establish such a procedure. Flow cytometric and functional assay methods were modified to count CD45− CD14−/CD73+ subsets containing all MSC and used them to study BM from spongy bone (SB) and iliac crest aspirate (ICA). These methods detected the target subsets in all BM suspensions derived from SB (n = 154) and ICA, (n = 44) with a satisfactory correlation between immuno‐phenotyping and functional tests by low‐density plating. We noted a higher overall MSC frequency in SB cell suspensions but a lower plating efficiency of CD45− CD14−/CD73+ SB cells under standard culture conditions.
We propose a cell quality control on un‐manipulated BM cell suspensions to quantify the mesenchymal compartment with regard to varying donor factors, such as age and sampling site, that influence expansion and define a therapeutic threshold value. Furthermore, we were able to confirm differences in plating efficiency and proliferative capacity between two BM origins.
Summary
One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of ...cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra‐cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)‐induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34+ and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC‐deficient CD34+ cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst‐forming units, granulocyte colony‐forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long‐term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.
Adult bone marrow (BM) mesenchymal stem/progenitor cells (MS/PC) are a potentially useful tool for cell therapy and tissue repair. However, the identification of cell subsets rich in MS/PC from fresh ...BM has not been described. We have developed a means of identifying such subsets from untouched bone marrow.
First, MS/PC were enriched by short-time adherence (D
1–3) before any cell division to evaluate the efficiency of CD73, CD105, CDw90, and CD49a antigens to select highly purified CD45
−CD14
− fluorescence-activated sorted subsets enriched in clonogenic mesenchymal cells. Then, we adapted this method to unmanipulated BM mononuclear cells (MNC).
Short-time (D
1–3) adherent CD45
−CD14
− cells expressing CD73 or CD49a antigens contained all the CFU-F, even though the CD105
+ and CDw90
+ subsets comprised less than half the total. In fresh unmanipulated BM MNC, CD73 and CD49a were also highly discriminative and allowed up to a 3 log enrichment of CFU-F when compared to BM MNC. Normal culture conditions upregulated most of the tested antigens.
The CD45
−CD14
−/CD73
+ and CD45
−CD14
−/CD49a
+ phenotypes identified subsets containing all the CFU-F and sufficiently enriched to detect them in fresh BM, enabling evaluation of mesenchymal content of BM collections for cell therapy.
The systematic localization of chronic lymphocytic leukemia (CLL) B-cells in the bone marrow (BM), together with the ex vivo protective effect of stromal cells on their spontaneous apoptosis, both ...indicate a specific role of the BM microenvironment. In vivo, the impact of CLL cells on mesenchymal stromal cells (MSCs) remains a source of debate. Here, we quantified and expanded colony forming unit-fibroblasts (CFU-Fs) from CLL-BM under standard conditions, analyzed the expression of selected genes, and studied secretion profiles. We observed failing of CLL-BM cultures in standard conditions (45.5% vs. <0.1%), and even after adding basic fibroblast growth factor (bFGF), there were fewer CFU-F than from normal BM (1.3 vs. 40/10(6) cells respectively; P<0.01). Furthermore, their polygonal aspect and low proliferative capacity, together with the expression of 384 selected genes and a secreted set of molecules related to senescence-associated secretory phenotype indicated a state of senescence, further confirmed by the higher proportion of senescence-associated β-galactosidase (SA-βGAL)-positive cells and p16INK4a overexpression. In our hands, hypoxic conditions (5% O2) did not rescue CFU-Fs. Given the role of MSC in BM tissue organization, we studied hematons that are generally considered to be elementary BM units. These structures were rare or had even disappeared completely. When hematons were present, we systematically observed nodular B-CLL cell invasion only. These data confirm that the B-CLL clone has a marked impact on MSC and disrupts BM organization in vivo, raising new questions about in vivo pathophysiology.
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