Phenotypic, transcriptomic, and genomic features of clonal plasma cells in light-chain amyloidosis.

Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs.

Phenotypic and genomic analysis of multiple myeloma minimal residual disease tumor cells: a new model to understand chemoresistance.

Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered atwww.clinicaltrials.gov as #NCT01237249.

Maturation-associated gene expression profiles along normal human bone marrow monopoiesis.

Human monopoiesis is a tightly coordinated process which starts in the bone marrow (BM) haematopoietic stem cell (HSC) compartment and leads to the production of circulating blood mature monocytes. Although mature monocytes/macrophages have been extensively studied in both normal or inflammatory conditions, monopoiesis has only been assessed in vitro and in vivo animal models, due to low frequency of the monocytic precursors in the normal human BM. Here we investigated the transcriptional profile along normal human BM monopoiesis. Five distinct maturation-associated stages of monocytic precursors were identified and isolated from (fresh) normal human BM through fluorescence-activated cell sorting, and the gene expression profile (GEP) of each monocytic precursor subset was analysed by DNA-oligonucleotide microarrays. Overall, >6000 genes (18% of the genes investigated) were expressed in ≥1 stage of BM monopoiesis at stable or variable amounts, showing early decrease in cell proliferation with increased levels of expression of genes linked with cell differentiation. The here-defined GEP of normal human BM monopoiesis might contribute to better understand monocytic differentiation and the identification of novel monocytic candidate markers, while also providing a frame of reference for the study of monocytic maturation in both neoplastic and non-neoplastic disease conditions involving monocytic precursor cells.

The cellular origin and malignant transformation of Waldenström macroglobulinemia.

Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25(-)CD22(+) B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25(+)CD22(+low)) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone.

Detailed characterization of multiple myeloma circulating tumor cells shows unique phenotypic, cytogenetic, functional, and circadian distribution profile.

Circulating myeloma tumor cells (CTCs) as defined by the presence of peripheral blood (PB) clonal plasma cells (PCs) are a powerful prognostic marker in multiple myeloma (MM). However, the biological features of CTCs and their pathophysiological role in MM remains unexplored. Here, we investigate the phenotypic, cytogenetic, and functional characteristics as well as the circadian distribution of CTCs vs paired bone marrow (BM) clonal PCs from MM patients. Our results show that CTCs typically represent a unique subpopulation of all BM clonal PCs, characterized by downregulation (P < .05) of integrins (CD11a/CD11c/CD29/CD49d/CD49e), adhesion (CD33/CD56/CD117/CD138), and activation molecules (CD28/CD38/CD81). Fluorescence in situ hybridization analysis of fluorescence-activated cell sorter-sorted CTCs also unraveled different cytogenetic profiles vs paired BM clonal PCs. Moreover, CTCs were mostly quiescent and associated with higher clonogenic potential when cocultured with BM stromal cells. Most interestingly, CTCs showed a circadian distribution which fluctuates in a similar pattern to that of CD34(+) cells, and opposite to stromal cell-derived factor 1 plasma levels and corresponding surface expression of CXC chemokine receptor 4 on clonal PCs, suggesting that in MM, CTCs may egress to PB to colonize/metastasize other sites in the BM during the patients' resting period.

Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance.

The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/µL (range: 0.0003-0.08 cells/µL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR-ABL1 fusion gene.

For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell(HPC) disorder. However, it remains to be determined whether BCR-ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR-ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR-ABL1 fusion gene in FACS-sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97%615%), basophils (95%612%), eosinophils (90%68%), CD341 precursors cells (90%67%),monocytes (84%630%), nucleated red blood cells (87%624%), and mast cells (77%633%). By contrast, MPC(30%634%), B-cells (15%627%), T-lymphocytes (50%626%), and NK-cells (35%634%) were involved at lower percentages. In 8/18 CML patients, 2 tumor BCR-ABL11 subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD341 cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR-ABL1 fusion gene inMPC, CD341 precursors, and other different BM hematopoietic myeloid cell lineages from CML patients,including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes.Interestingly, involvement of MPC was restricted to the ancestral BCR-ABL11 subclone.

Gene expression profile of highly purified bone marrow mast cells in systemic mastocytosis.

BACKGROUND: Despite the fact that a great majority (>90%) of patients with systemic mastocytosis (SM) carry a common genetic lesion, the D816V KIT mutation, little is known regarding the molecular and biological pathways underlying the clinical heterogeneity of the disease. OBJECTIVE: We sought to analyze the gene expression profile (GEP) of bone marrow mast cells (BMMCs) in patients with SM and its association with distinct clinical variants of the disease. METHODS: GEP analyses were performed by using DNA-oligonucleotide microarrays in highly purified BMMCs from patients with SM carrying the D816V KIT mutation (n=26) classified according to the diagnostic subtype of SM versus normal/reactive BMMCs (n=7). Validation of GEP results was performed with flow cytometry in the same set of samples and in an independent cohort of 176 subjects. RESULTS: Overall, 758 transcripts were significantly deregulated in patients with SM, with a common GEP (n=398 genes) for all subvariants of SM analyzed. These were characterized by upregulation of genes involved in the innate and inflammatory immune response, including interferon-induced genes and genes involved in cellular responses to viral antigens, together with complement inhibitory molecules and genes involved in lipid metabolism and protein processing. Interestingly, aggressive SM additionally showed deregulation of apoptosis and cell cycle-related genes, whereas patients with indolent SM displayed increased expression of adhesion-related molecules. CONCLUSION: BMMCs from patients with different clinical subtypes of SM display distinct GEPs, which might reflect new targetable pathways involved in the pathogenesis of the disease.

Can resistance prehabilitation training bring additional benefits in valvular cardiac surgery? protocol for a randomized controlled trial.

INTRODUCTION: Cardiovascular diseases (CVD) are a group of illnesses that include coronary heart disease, cerebrovascular disease, congenital heart disease and deep vein thrombosis. Major surgery is often chosen as the treatment of choice for CVD. The concept of fast-track rehabilitation after surgery appeared in the 1970s. Participation in these exercise-based prehabilitation programmes may decrease postoperative complications and length of hospital stay. The primary aim of the present study is to evaluate whether the implementation of an additional resistance training (RT) prehabilitation protocol within cardiac exercises based prehabilitation can reduce intensive care unit (ICU) length of stay, postoperative complications and hospital length of stay (LOS). METHODS: A protocol of a prospective, parallel, randomised clinical trial includes 96 adult patients diagnosed with valvular pathology and who have been scheduled for surgery. The participants will be randomly assigned to two groups of 48. Control group will be treated with ventilatory and strengthening of respiratory muscles, and aerobic exercise. Experimental group, in addition, will be treated with RT of peripheral muscles. Both hospital stay and ICU stay will be assessed as main variables. Other secondary variables such as exercise capacity, quality of life and respiratory values will also be assessed. Quantitative variables will be analysed with a T-Test or ANOVA, or Mann Witney if the distribution is non-parametric. RESULTS AND CONCLUSION: This will be the first controlled clinical study focused on adding strength exercise as an additional treatment during prehabilitation. The results of this study will focus on helping to improve rehabilitation and prehabilitation protocols, considering that it is essential to maintain pulmonary training, as well as the inclusion of peripheral exercises that help people with heart disease to be in a better physical condition in order to increase their participation and sense of quality of life.

Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells.

Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138(+) microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) -are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥ 98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that recurrent immunoglobulin heavy chain translocations might be absent in the primordial plasma cell clone in a significant proportion of patients with clonal plasma cell disorders carrying these cytogenetic alterations.

Impact of the COVID pandemic on vascular access creation for haemodialysis in 16 Spanish haemodialysis centres.

Background: The coronavirus disease (COVID) pandemic has resulted in a major disruption in healthcare that has affected several medical and surgical specialties. European and American Vascular Societies have proposed deferring the creation of an elective vascular access (VA) [autologous or prosthetic arteriovenous fistula (AVF) or arteriovenous graft (AVG)] in incident patients on haemodialysis (HD) in the era of the COVID pandemic. The aim of this study is to examine the impact of the COVID pandemic on VA creation and the central venous catheter (CVC)-related hospitalizations and complications in HD patients dialyzed in 16 Spanish HD units of three different regions. Methods: We compared retrospectively two periods of time: the pre-COVID (1 January 2019-11 March 2020) and the COVID era (12 March 2020-30 June 2021) in all HD patients (prevalent and incident) dialyzed in our 16 HD centres. The variables analysed were type of VA (CVC, AVF and AVG) created, percentage of CVC in incident and prevalent HD patients, CVC-related hospitalizations and complications (infection, extrusion, disfunction, catheter removal) and percentage of CVC HD sessions that did not reach the goal of Kt (>45) as a marker of HD adequacy. Results: A total of 1791 VAs for HD were created and 905 patients started HD during the study period. Patients who underwent vascular access surgery during the COVID period compared with pre-COVID period were significantly younger, with a significant decrease in surgical activity to create AVFs and AVGs in older HD patients (>75 and >85 years of age). There was a significant increase in CVC placement (from 59.7% to 69.5%; P < 0.001) from the pre-COVID to the COVID period. During the COVID pandemic, a significantly higher number of patients started HD through a CVC (80.3% versus 69.1%; P < 0.001). The percentage of CVC in prevalent HD patients has not decreased in the 19 months since the start of the pandemic [414 CVC/1058 prevalent patients (39.4%)]. No significant changes were detected in CVC-related hospitalizations between the pre-COVID and COVID periods. In the COVID period, a significant increase in catheter replacement and the percentage of HD session that did not reach the HD dose objective (Kt > 45) was observed. Conclusions: COVID has presented a public health system crisis that has influenced VA for HD, with an increase in CVCs relative to AVFs. A decrease in HD sessions that did not reach the HD dose objective was observed in the COVID period compared with a pre-COVID period.

Evaluation of the WHO criteria for the classification of patients with mastocytosis.

Diagnosis and classification of mastocytosis is currently based on the World Health Organization (WHO) criteria. Here, we evaluate the utility of the WHO criteria for the diagnosis and classification of a large series of mastocytosis patients (n=133), and propose a new algorithm that could be routinely applied for refined diagnosis and classification of the disease. Our results confirm the utility of the WHO criteria and provide evidence for the need of additional information for (1) a more precise diagnosis of mastocytosis, (2) specific identification of new forms of the disease, (3) the differential diagnosis between cutaneous mastocytosis vs systemic mastocytosis, and (4) improved distinction between indolent systemic mastocytosis and aggressive systemic mastocytosis. Based on our results, a new algorithm is proposed for a better diagnostic definition and prognostic classification of mastocytosis, as confirmed prospectively in an independent validation series of 117 mastocytosis patients.

Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach.

Monoclonal B-cell lymphocytosis (MBL) indicates the presence of less than 5 x 10(9)/L circulating monoclonal B cells in otherwise healthy subjects. Recently, it has been reported that circulating chronic lymphocytic leukemia (CLL)-like B cells can be detected using 4- or 5-multicolor flow cytometry in 5% to 7% of adults with normal lymphocyte counts. We investigated the frequency of circulating monoclonal B cells in 608 healthy subjects older than 40 years with normal blood counts, using a highly sensitive 8-color flow cytometry approach and systematic screening for total PB leukocyte count higher than 5 x 10(6). We show that the frequency of PB monoclonal B cells is markedly higher than previously reported (12% for CLL-like B cells, found at frequencies of 0.17 +/- 0.13 x 10(9) cells/L), the incidence progressively increasing with age. Most cases (62%) showed clonal B-cell levels below the maximum sensitivity of the techniques described by others (< 0.01%), supporting the notion that detection of MBL may largely depend on the sensitivity of the flow cytometry approach used.

Students' Perceptions of Instructional Rubrics in Neurological Physical Therapy and Their Effects on Students' Engagement and Course Satisfaction.

One of the main challenges faced by physical therapy (PT) students is to learn the practical skills involved in neurological physical therapy (PT). To help them to acquire these skills, a set of rubrics were designed for formative purposes. This paper presents the process followed in the creation of these rubrics and their application in the classroom, noting that students perceived them as valid, reliable, and highly useful for learning. The perception of the validity and usefulness of the rubrics has different closely related dimensions, showing homogeneous values across the students´ sociodemographic and educational variables, with the exception of dedication to studying, which showed a significant relationship with schoolwork engagement and course satisfaction. The adequacy of the hypothesized structural model of the relationships among the variables was confirmed. Direct effects of the perception of the rubrics' validity and engagement on course satisfaction were found, as well as direct effects of the assessment of the usefulness of the rubrics on schoolwork engagement and indirect effects on course satisfaction through this latter variable. The results are discussed taking into account the conclusions of previous research and different instructional implications.

Validity of the International Physical Activity Questionnaire Long Form for Assessing Physical Activity and Sedentary Behavior in Subjects with Chronic Stroke.

Validation studies of questionnaires used to assess physical activity (PA) and sedentary behavior (SB) in stroke survivors are scarce. This cross-sectional study aimed to examine the validity of the International Physical Activity Questionnaire long-form (IPAQ-LF) in community living adults with post-stroke sequelae (≥6 months) and preserved ambulation. Participants' functional mobility, lower limb strength, ambulatory level, stroke severity, and disability were assessed. An accelerometer (ActiGraph GT3X+) was worn for ≥7 consecutive days. Subsequently, the IPAQ-LF was interview-administered. Fifty-six participants (58.1 ± 11.1 years, 66.1% male) were included. A strong correlation between the two methods was found for total PA time (ρ = 0.55, p < 0.001). According to the Bland-Altman analyses, over-reporting moderate-to-vigorous PA and under-reporting total PA in the IPAQ-LF were found in those participants with higher PA levels. Both methods measured sedentary time similarly, though random error was observed between them. Moderate-strong correlations were found between the IPAQ-LF and physical function (ρ = 0.29-0.60, p < 0.05). In conclusion, in people with chronic stroke, the IPAQ-LF presented acceptable levels of validity for estimating total PA time in those who are insufficiently active. Therefore, it could be a useful tool to screen for inactive individuals with chronic stroke who can benefit from PA interventions addressed to implement healthier lifestyles.

Mediterranean diet and health: A systematic review of epidemiological studies and intervention trials.

Diet is a crucial variable for a healthy life. A rapidly growing number of studies in recent years support the hypothesis that the Mediterranean diet (MedDiet) has a beneficial effect on certain body systems, but the highly varied objectives and quality of these publications warrants an updated assessment. In the present review we performed a comprehensive evaluation of current evidence on the impact of the MedDiet on human health, assessing its effect on the incidence or progression of the main non-communicable diseases and their intermediate outcomes and risk factors. We scrutinised the clinical evidence from observational studies and randomised controlled trials. Cardiovascular disease was the condition with most information. The MedDiet showed a general preventive effect, which was reproduced to varying degrees for certain intermediate cardiovascular outcomes such as blood pressure, lipids, obesity, metabolic syndrome and diabetes. Benefits were also found for several types of cancer, brain function (including cognition, mood and to a lesser extent Parkinson's disease) and mortality. The quality of the published evidence was, however, generally moderate or low. In conclusion, the MedDiet shows a favourable impact on health. General adoption of a MedDiet is concordant with current policies promoting healthy and sustainable nutrition worldwide. Nonetheless, more high-quality research is needed to improve the consistency of the findings.

The Mediterranean diet: A historical perspective on food for health.

The Mediterranean diet (MedDiet) represents the crystallisation of the centuries-old cooking legacies of different civilisations. The association of the MedDiet with longevity and low cardiovascular risk prompted the Seven Countries Study, which provided epidemiological evidence on the health effects of diet. This led to further studies, both epidemiological and interventional. Scales to measure adherence as well as studies of food components have consolidated a body of knowledge that is of great interest to institutions and governmental agencies. The recognition of its benefits for health has made the widespread introduction of the MedDiet an urgent challenge. Parallel efforts are being made to extend research through experimental and clinical studies. The purpose of this review is to present the historical background of the MedDiet, the main steps leading to the recognition of its health benefits, and the current strategy to facilitate its global implementation.

Immunophenotypical, morphologic, and functional characterization of maturation-associated plasmacytoid dendritic cell subsets in normal adult human bone marrow.

BACKGROUND: Information about maturation of plasmacytoid dendritic cell precursors (pre-pDCs) in normal bone marrow (BM) remains limited. STUDY DESIGN AND METHODS: Immunophenotypical, morphologic, and functional changes associated with maturation of pre-pDCs were analyzed in adult normal human BM (n = 45). RESULTS: Three pre-pDC maturation stages, with an increasingly higher degree of maturity, were systematically identified: CD34++/HLA-DR++/+++/CD123++/CD45+/++ (Stage I), CD34+/HLA-DR+/++/CD123++/+++/CD45+/++ (Stage II), and CD34-/HLA-DR++/CD123++/+++/CD45++ (Stage III) cells. Lymphoid- and early myeloid-associated molecules, as well as CD86, were coexpressed in Stage I pre-pDCs, being down regulated afterward. CD304 and CD85j appeared in Stage I, progressively increasing their levels thereafter. Interestingly, cutaneous lymphocyte-associated antigen was heterogeneously expressed throughout the maturation, particularly in Stage I pre-pDCs. The morphologic appearance of Stage I pre-pDCs was consistent with their undifferentiated stage, while Stage II/III cells showed morphologic features of more differentiated cells. From the functional point of view, only Stage II and Stage III pre-pDCs were capable of inducing allogeneic T-cell proliferation, the later subset also showing interferon-g secretion; in contrast, Stage I pre-pDCs showed the highest endocytic ability. CONCLUSION: In summary, three maturation stages of pre-pDCs can be identified in adult normal BM, which show unique phenotypical, morphologic, and functional characteristics; these results provide a frame of reference for a better understanding of pDC malignancies.

Maturation-associated gene expression profiles during normal human bone marrow erythropoiesis.

Erythropoiesis has been extensively studied using in vitro and in vivo animal models. Despite this, there is still limited data about the gene expression profiles (GEP) of primary (ex vivo) normal human bone marrow (BM) erythroid maturation. We investigated the GEP of nucleated red blood cell (NRBC) precursors during normal human BM erythropoiesis. Three maturation-associated populations of NRBC were identified and purified from (fresh) normal human BM by flow cytometry and the GEP of each purified cell population directly analyzed using DNA-oligonucleotide microarrays. Overall, 6569 genes (19% of the genes investigated) were expressed in ≥1 stage of BM erythropoiesis at stable (e.g., genes involved in DNA process, cell signaling, protein organization and hemoglobin production) or variable amounts (e.g., genes related to cell differentiation, apoptosis, metabolism), the latter showing a tendency to either decrease from stage 1 to 3 (genes associated with regulation of erythroid differentiation and survival, e.g., SPI1, STAT5A) or increase from stage 2 to stage 3 (genes associated with autophagy, erythroid functions such as heme production, e.g., ALAS1, ALAS2), iron metabolism (e.g., ISCA1, SLC11A2), protection from oxidative stress (e.g., UCP2, PARK7), and NRBC enucleation (e.g., ID2, RB1). Interestingly, genes involved in apoptosis (e.g., CASP8, P2RX1) and immune response (e.g., FOXO3, TRAF6) were also upregulated in the last stage (stage 3) of maturation of NRBC precursors. Our results confirm and extend on previous observations and providing a frame of reference for better understanding the critical steps of human erythroid maturation and its potential alteration in patients with different clonal and non-clonal erythropoietic disorders.

Cross-sectional study of quadriceps properties and postural stability in patients with chronic stroke and limited vs. non-limited community ambulation.

Background: Changes in the paretic-side metabolism post-stroke and quadriceps muscle mechanical properties favour muscle wasting, affecting postural instability and walking impairment. Further clarification is needed in subjects post-stroke who show limited or non-limited community ambulation. Objectives: To analyze between-limb differences in quadriceps muscle thickness, strength and thigh cutaneous temperature, as well as investigate postural stability in subjects with chronic stroke and limited vs. non-limited community ambulation and compared against healthy controls. Methods: In this controlled cross-sectional study, 26 participants with chronic hemiparesis post-stroke (divided in a slow gait group (SG<0.8 m/s) (n = 13) and a fast gait group with full community ambulation speed (FG≥0.8 m/s)) and 18 healthy people were recruited. Thigh surface temperature, rectus femoris (RF) and vastus intermedius (VI) muscles thickness, quadriceps' isometric maximal voluntary contraction and postural stability were measured. Results: The SG presented significantly lower RF (P = .019) and VI (P = .006) muscle thickness, less peak force (P < .001) and lower temperature (P = .002) in the paretic vs the non-paretic limb. The FG showed significantly lower VI thickness (P = .036) and peak force (P < .001) in the paretic vs the non-paretic limb. Regarding balance, all indices were worse in the SG versus the FG and CG. Conclusions: Subjects of the FG, despite showing full community ambulation speed, had less quadriceps strength and VI muscle thickness but not RF muscle wasting in the paretic limb. The paretic VI muscle wasting may be an important factor to reach normal walking. The SG showed between-limb differences in all the studied variables and the worst postural stability.