Co-designing a Canadian adaptation of a lifestyle-oriented intervention aimed to improve daily functioning of individuals living with chronic pain: a multi-method study protocol of REVEAL(OT) Canada.

Background: Living with chronic pain (CP) often implies major lifestyle changes, including modifications of daily routines and work. Surprisingly, few validated and effective interventions specifically target functional outcomes in this population. Redesign your Everyday Activities and Lifestyle with Occupational Therapy [REVEAL(OT)] is a lifestyle-oriented intervention led by occupational therapists that directly targets the daily functional challenges of living with CP. The intervention was initially developed and studied as an add-on to standard treatment delivered by Danish multidisciplinary specialized pain clinics. Adapting, implementing, and evaluating REVEAL(OT) within the Canadian healthcare system will contribute to broadening the scope of treatments offered in specialized pain clinics that do not yet include occupational therapy. Objective: The proposed study aims to define and refine REVEAL(OT)/CA with partners (authors of original intervention, people with lived experience, clinicians, managers). Methods: This participatory action research will use a multi-method design and follow the ORBIT model for developing behavioral treatments for chronic diseases. A process of co-construction with partners and an advisory committee will take place in two Montreal specialized pain clinics. It consists of two related work packages (WPs). In WP1, a first series of focus groups with partners (n = 86) and workshops with the advisory committee will be conducted to co-develop the hypothetical pathway describing intervention components and their potential mechanisms of action on targeted outcomes, as well as the first version of the adapted intervention manual. WP2 will co-refine REVEAL(OT)/CA by exploring its acceptability, feasibility and mechanisms of action through intervention deliveries (at least twice in each of two specialized pain clinics; n ≥ 60 patients) and focus groups and/or individual interviews with participating patients and partners. At the end of this study, the intervention manual will be generated both in French and English. Discussion: This study will set the stage for subsequent implementation and effectiveness assessment projects and be an important step towards the deployment of interventions aiming to improve engagement in meaningful daily activities among adults living with CP. Registration: OSF Registries, osf.io/8gksa. Registered 3 August 2023, https://osf.io/8gksa.

Pilot Study of French-Canadian Lifestyle Redesign® for Chronic Pain Management.

As chronic pain (CP) interferes with an individual's lifestyle by limiting meaningful activities and health-related quality of life (HRQoL), occupational therapy (OT) plays an important role in CP management interventions. This pilot study aimed to explore the influence of a 13-week French-Canadian Lifestyle Redesign® for CP. A mixed-methods research design including a preexperimental quantitative component pre-/posttest was used with 15 participants with fibromyalgia. Although pain remained unchanged after the intervention, improvements were observed in participants' engagement in meaningful activities (p < .01), life balance (p < .01), mental components of HRQoL (p < .01), depressive symptoms (p = .047), and pain self-efficacy (p < .01). After the intervention, phone interviews (n = 6) highlighted the participants' appreciation of the focus being placed on their daily routines and the development of a sense of belonging throughout the intervention. This study suggests the potential feasibility and benefits of an occupation-based approach in CP management.

An observational cohort study on antimicrobial usage on dairy farms in Quebec, Canada.

Quantification of antimicrobial usage (AMU) is crucial to measure the effect of intervention programs, to determine associations between usage and resistance, to compare populations, and for benchmarking purposes. The primary objective of the study was to describe quantitatively the AMU on Quebec dairy farms over 1 yr: (1) the total AMU, (2) the AMU per administration route (intramammary, injectable, oral, intrauterine), and (3) the AMU per antimicrobial class and according to the categorizations of Health Canada and the World Health Organization. The secondary objective was to assess the effect of several characteristics (herd size, level of milk production, and incidence rate of common infectious diseases) on AMU rate. The AMU data were obtained for 101 dairy farms randomly selected in 3 important Quebec dairy regions by collecting and recording all empty drug packaging and invoices for medicated feed (spring 2017 to spring 2018). The AMU rate was reported in number of Canadian defined course doses for cattle per 100 cow-years. The average herd size was 67 cows per farm, and 2/101 farms were certified organic. Overall, an estimated mean of 537 Canadian defined course doses for cattle/100 cow-years was observed. The intramammary route during lactation was the most frequently observed, followed, in decreasing order of usage, by oral route in the feed, intramammary route at drying-off, and injectable route. Oral (other than in animal feed) and intrauterine formulations were infrequently collected from the garbage cans. The 5 most frequently observed antimicrobial classes were, by decreasing order of usage, ionophores, penicillins, aminocoumarins, aminoglycosides, and polymyxins. Highest priority critically important antimicrobials as defined by the World Health Organization were mainly collected from intramammary formulations during lactation followed by injectable and drying-off intramammary formulations. The herd size was positively associated with the total AMU rate but not with the usage rate of highest priority critically important antimicrobials. Incidence of diseases along with preventive use of antimicrobials (drying-off and medicated feed with antimicrobials) explained 48% of the variance in total AMU rate.

Review: Climate Finance readiness of the animal protein sector: overview of experience in linking the sector to Climate Finance, and options to address bottlenecks.

Despite the importance of the role of Climate Finance to comply with the United Nations Framework Convention on Climate Change 1.5°C objective, there is no consensus on the definition of Climate Finance and the estimated assessment of its aggregated flows and effects remains challenging. Despite being a major emitter and having a significant and cost-effective mitigation potential, the livestock sector has so far only received a marginal share of Climate Finance. As demand for animal protein products continues to increase (68% between 2010 and 2050), there is a compelling case for channeling more Climate Finance investments into the sector to incentivize greenhouse gas emissions reduction at scale. Bottlenecks in linking the livestock sector to Climate Finance include the insufficient capacity to assess the cost-benefit of projects, high upfront cost and risk perception of investors, the informality of the sector, non-existence of Climate Finance instruments dedicated to the livestock sector and lack of cost-efficient Monitoring, Reporting and Verification systems. Nevertheless, recent developments provide avenues to increase the access of the animal protein sector to Climate Finance.

Data on soil physicochemical properties and chemical composition of rainfall and of throughfall and stemflow generated by Turkey oak trees (Quercus cerris L.) in acid and sub-alkaline soils.

We report data on the physicochemical properties of soils collected in two adjacent areas, one acid and one sub-alkaline, both developed on sequential beds of Plio-pleistocene marine sediments, and on the chemical composition of ecological solutions (rainfall, throughfall and stemflow) separately collected in the two areas. Throughfall and stemflow were generated by Turkey oak trees (Quercus cerris L.), which was the dominant tree species in both study areas. These data are related to the original article "Soil affects throughfall and stemflow under Turkey oak (Quercus cerris L.)" (Corti et al., 2019) [1].

Colonization pressure as a risk factor of ICU-acquired multidrug resistant bacteria: a prospective observational study.

The primary objective of this study was to evaluate the impact of colonization pressure on intensive care unit (ICU)-acquired multidrug resistant bacteria (MDRB). All patients hospitalized for more than 48 h in the ICU were included in this prospective observational study. MDRB were defined as methicillin resistant Staphylococcus aureus, Pseudomonas aeruginosa resistant to ceftazidime or imipenem, Gram-negative bacilli producing extended-spectrum beta-lactamases (ESBL), and all strains of Acinetobacter baumannii and Stenotrophomonas maltophilia. Colonization pressure was daily calculated in the three participating ICUs. Univariate and multivariate analyses were used to determine risk factors for ICU-acquired MDRB. Two hundreds and four (34%) of the 593 included patients acquired an MDRB during their ICU stay. Multivariate analysis identified colonization pressure as an independent risk factor for ICU-acquired MDRB (OR (95% CI) 4.18 (1.03-17.01), p = 0.046). Other independent risk factors for ICU-acquired MDRB were mechanical ventilation (3.08 (1.28-7.38), p = 0.012), and arterial catheter use (OR, 3.04 (1.38-6.68), p = 0.006). ICU-acquired MDRB were associated with increased mortality, duration of mechanical ventilation, and ICU stay. However, ICU-acquired MDRB was not independently associated with ICU-mortality. Colonization pressure is an independent risk factor for acquiring MDRB in the ICU.

Screening for pre-eclampsia early in pregnancy: performance of a multivariable model combining clinical characteristics and biochemical markers.

OBJECTIVE: To investigate the performance of a multivariable model combining a priori clinical characteristics and biomarkers to detect, early in pregnancy, women at higher risk of developing pre-eclampsia (PE). DESIGN: Nested case-control study. SETTING: University medical centre, Quebec, Canada (CHU de Québec). POPULATION: A total of 7929 pregnant women recruited between 10 and 18 weeks of gestation. In all, 350 developed hypertensive disorders of pregnancy (HDP)-of which 139 had PE, comprising 68 with severe PE and 47 with preterm PE-and were matched with two women with a normal pregnancy. METHODS: We selected a priori clinical characteristics and promising markers to create multivariable logistic regression models: body mass index (BMI), mean arterial pressure (MAP), placental growth factor, soluble Fms-like tyrosine kinase-1, pregnancy-associated plasma protein A and inhibin A. MAIN OUTCOME MEASURES: PE, severe PE, preterm PE, HDP. RESULTS: At false-positive rates of 5 and 10%, the estimated detection rates were between 15% (5-29%) and 32% (25-39%), and between 39% (19-59%) and 50% (34-66%), respectively. Considering the low prevalence of PE in this population, the positive predictive values were 7% (5-9%) to 10% (7-13%) for PE and 2% (1-4%) to 4% (3-6%) in the preterm and severe PE subgroups. The multivariable model yielded areas under the receiver operating characteristics curves (AUC) between 0.72 (0.61-0.81) and 0.78 (0.68-0.88). When only BMI and MAP were included in the model, the AUC were similar to those of the a priori model. CONCLUSIONS: In a population with a low prevalence of preterm PE, a multivariable risk algorithm using an a priori combination of clinical characteristics and biochemical markers did not reach a performance justifying clinical implementation as screening test early in pregnancy.

Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727.

Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser(727)) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser(727)-phosphorylated STAT3 molecule (pSTAT3Ser(727)) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer(727) modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser(727) overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.

OS090. Performance of candidate clinical and biochemical markers in screening early in pregnancy to detect women at high risk to develop preeclampsia.

INTRODUCTION: The advent of early preventive measures, such as low-dose aspirin targeting women at high risk of preeclampsia (PE), emphasizes the need for better detection. Despite the emergence of promising biochemical markers linked to the pathophysiological processes, systematic reviews have shown that, until now, no single tests fulfill the criteria set by WHO for biomarkers to screen for a disease. However, recent literature reveals that by combining various clinical, biophysical and biochemical markers into multivariate algorithms, one can envisage to estimate the risk of PE with a performance that would reach clinical utility and cost-effectiveness, but this remains to be demonstrated in various environments and health care settings. OBJECTIVES: To investigate, in a prospective study, the clinical utility of candidate biomarkers and clinical data to detect, early in pregnancy, women at risk to develop PE and to propose a multivariate prediction algorithm combining clinical parameters to biochemical markers. METHODS: 7929 pregnant women prospectively recruited at the first prenatal visit, provided blood samples, clinical and sociodemographic information. 214 pregnant women developed hypertensive disorders of pregnancy (HDP) of which 88 had PE (1.2%), including 44 with severe PE (0.6%). A nested case-control study was performed including for each case of HDP two normal pregnancies matched for maternal age, gestational age at recruitment, ethnicity, parity, and smoking status. Based on the literature we selected the most promising markers in a multivariate logistic regression model: mean arterial pressure (MAP), BMI, placental growth factor (PlGF), soluble Flt-1, inhibin A and PAPP-A. Biomarker results measured between 10-18 weeks gestation were expressed as multiples of the median. Medians were determined for each gestational week. RESULTS: When combined with MAP at the time of blood sampling and BMI at the beginning of pregnancy, the four biochemical markers discriminate normal pregnancies from those with HDP. At a 5% false positive rate, 37% of the affected pregnancies would have been detected. However, considering the prevalence of HDP in our population, the positive predictive value would have been only 15%. If all the predicted positive women would have been proposed a preventive intervention, only one out 6.7 women could have potentially benefited. In the case of severe PE, performance was not improved, sensitivity was the same, but the positive predictive value decreased to 3% (lower prevalence of severe PE). CONCLUSION: In our low-risk Caucasian population, neither individual candidate markers nor multivariate risk algorithm using an a priori combination of selected markers reached a performance justifying implementation. This also emphasizes the necessity to take into consideration characteristics of the population and environment influencing prevalence before promoting wide implementation of such screening strategies. In a perspective of personalized medicine, it appears more than ever mandatory to tailor recommendations for HDP screening according not only to individual but also to population characteristics.

PP054. Predicting preeclampsia at late mid-term pregnancy before occurrence of clinical symptoms: Clinical utility of biomarkers and clinical parameters in a low-risk population.

INTRODUCTION: Despite research efforts and healthcare improvement, preeclampsia (PE) continues to be a leading cause of maternal and fetal morbidity and mortality. Early identification of women at risk of developing PE is the most promising approach to implement preventive measures such as low-dose aspirin to reduce negative outcomes. However, it is still relevant to evaluate pregnant women to detect PE before the occurrence of clinical symptoms and/or to have better tools to assist in its differential diagnosis. Recently, measurements of biomarkers such as soluble fms-like tyrosine kinase-1 (SFLT-1) and placental growth factor (PlGF) have been proposed and some manufacturers are already marketing reagents for this purpose. OBJECTIVES: To examine in a prospective study the performance of selected clinical and biochemical markers for identifying late mid-term pregnancy women at risk of developing PE within a few weeks. METHODS: Seven thousand nine hundred and twenty nine pregnant women prospectively recruited at the first routine prenatal visit, provided blood samples, clinical and sociodemographic information. Two hundred and fourteen pregnant women developed hypertensive disorders of pregnancy (HDP) of which 88 had PE (1.2%), including 44 who presented with severe PE (0.6%). We performed a nested case-control study from the whole cohort including for each case of HDP two normal pregnancies after matching for maternal age, gestational age at recruitment, ethnicity, parity, and smoking status. Based on the literature, we selected the most promising clinical and biochemical markers to be included in a multivariate logistic regression model: mean arterial pressure and body mass index (BMI), PlGF, SFLT-1, inhibin A, and PAPP-A. All markers were measured between 20 and 32 weeks of gestation except for BMI (early pregnancy). All biological marker results were transformed in multiples of median. Medians were established for each gestational week. Multivariate logistic regression analyses were performed to develop prediction algorithm. RESULTS: The resulting regression model discriminated the affected from normal pregnancies as indicated by an area under the receiver operating characteristics (ROC) curve of 0.8. But at a 5% false positive rate, only 28% of the women who have developed HDP would have been detected. Even when the statistical analyses were limited to severe PE, the performance was poor: sensitivity 30%, positive predictive value 2.7%. CONCLUSION: In our low-risk Caucasian population, neither individual candidate markers nor multivariate risk algorithm using an a priori combination of selected clinical and biochemical markers reached a performance justifying implementation as a screening procedure. These results emphasize the necessity to take into consideration the environment, population and health care settings influencing prevalence and characteristics of HDP before promoting wide implementation of such screening strategies. It is imperative to tailor future recommendations for HDP screening not only according to the individual but also to the population characteristics if clinical utility has to be reached.

Lentivirus degradation and DC-SIGN expression by human platelets and megakaryocytes.

BACKGROUND AND AIM: As platelets are able to endocytose human immunodeficiency virus (HIV), we have investigated the fate of lentiviruses when endocytosed by human platelets and megakaryocytes (MK), and have characterized a specific receptor directly involved in this function. METHODS: Genetically modified (non-replicative) lentiviruses with an HIV envelope (HIV-e) or with a vesicular stomatitis virus protein G envelope (VSV-e) were alternatively used and their interaction with platelets and MK analyzed by electron microscopy (EM) and immunoEM. RESULTS: When incubated with platelets, HIV-e and VSV-e lentiviruses were internalized in specific endocytic vesicles and trafficked to the surface connected canalicular system (SCCS). Double immunolabeling for the viral P24 core protein and alpha-granule markers showed that lentiviruses were degraded in the SCCS after contact with alpha-granule proteins. In culture MK, lentiviruses were found in endocytic vesicles and accumulated in acid phosphatase-containing multivesicular bodies (MVB). The expression of the pathogen receptor dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN) was then demonstrated in platelets by flow cytometry, immunoEM and Western blot. Anti-DC-SIGN antibodies decreased HIV-e lentivirus internalization by platelets, showing that the receptor is functional. Specific signals for DC-SIGN protein and mRNA were also found in MK. CONCLUSION: This study indicates that platelets and MK can internalize lentiviruses in a pathway, which either provide a shelter to lentiviral particles or alternatively disrupts viral integrity. The receptor DC-SIGN is involved in this function.

Molecular study of the hematopoietic zinc finger gene in three unrelated families with gray platelet syndrome.

Hematopoietic zinc finger (HZF) null mice have features reminiscent of patients with gray platelet syndrome (GPS), a rare inherited bleeding disorder. This similarity has suggested that HZF deregulation might be involved in the human disease. The sequence of the eight exons of the HZF gene as well as the study of its expression in blood samples from five patients belonging to three different families did not reveal any modifications when compared with healthy donors. This study indicates that HZF is unlikely to be responsible for GPS.

The expression of prion protein (PrP(C)) in the megakaryocyte lineage.

BACKGROUND: Cellular prion protein (PrP(C)) is a naturally occurring protein in normal individuals which adopts an abnormal conformation, termed scrapie prion protein (PrP(Sc)) that is associated with disease. There is great concern that clinically asymptomatic variant Creutzfeldt-Jacob disease (vCJD) may transmit PrP(Sc) in blood transfusion products. PrP(C) is widely expressed and has been found in human blood. The majority of cellular borne PrP(C) is associated with platelets (84%). Although PrP(C) mRNA has been demonstrated in platelets, the quantity is unknown and may not reflect the total PrP(C) present. OBJECTIVE: To investigate the expression of PrP(C) in the megakaryocyte lineage. METHODS: The expression of PrP(C) was studied in CD34+ cells, cultured megakaryocytes and platelets using electron microscopy, flow cytometry, semi-quantitative RT-PCR and immunofluorescence confocal microscopy. RESULTS AND CONCLUSIONS: The expression of PrP(C) appeared to increase with differentiation and polyploidization in the megakaryocyte lineage. PrP(C) was located within platelet alpha-granules and its source is likely to be from megakaryocyte precursors. If PrP(Sc) has a similar distribution, these results have implications for the selection of blood donors and preparation of cell-depleted blood products.

The P-selectin cytoplasmic domain directs the cellular storage of a recombinant chimeric factor IX.

Hemophilia B was recognized as a good candidate for gene therapy. Several strategies have been attempted and gave promising results in hemophilic animals but failed to achieve corrective levels in humans. To overcome this inconvenience we aimed to generate intracellular pools of factor (F)IX in cells that are implicated in the hemostatic response, e.g. endothelial cells and platelets. Upon stimulation, these cells release their granule content, which in this case would result in an increase in local FIX concentration, and could locally produce an effective hemostasis. In an attempt to produce an intracellular pool of releasable coagulation FIX, the cytoplasmic domain of the P-selectin (pselCT) molecule was fused to the carboxy-terminal extremity of the human FIX protein. The properties of this chimeric molecule (FIX-pselCT) were studied in AtT20, a cell line which possesses storage granules. As previously shown for transmembrane molecules but not for a soluble protein such as FIX, the pselCT fragment induces the storage of FIX-pselCT. The coagulant activity of FIX-pselCT was not affected by the addition of the pselCT tail. The treatment of AtT20 cells with different inhibitors revealed that FIX-pselCT was not submitted to intracellular degradation and that the half-life of the chimeric molecule was at least two times longer than that of FIX-WT. An immunoelectron microscopic analysis demonstrated a specific localization of FIX-pselCT within the ACTH-containing granules. Cell stimulation using Phorbol Myristrate Acetate (PMA), ionophore A-23187 or 8-Br-cAMP induced efficient release of an active FIX-pselCT. These data demonstrate that the addition of the cytoplasmic domain of P-selectin to FIX modifies the cellular fate of the FIX molecule by directing the recombinant protein toward regulated-secretory granules without altering its coagulant activity.

Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion.

CD146 is a cell-surface molecule belonging to the immunoglobulin superfamily and expressed in all types of human endothelial cells. Confocal and electron microscopic analysis of confluent human umbilical vein endothelial cells (HUVECs) were used to demonstrate that CD146 is a component of the endothelial junction. Double immunolabeling with vascular endothelial cadherin showed that CD146 is localized outside the adherens junction. Moreover, CD146 expression is not restricted to the junction, since part of the labeling was detectable at the apical side of the HUVECs. Interestingly, cell-surface expression of CD146 increased when HUVECs reached confluence. In addition, the paracellular permeability of CD146-transfected fibroblast cells was decreased compared with that of control cells. Finally, CD146 colocalized with actin, was partly resistant to Triton X-100 extraction, and had its expression altered by actin-disrupting agents, indicating that CD146 is associated with the actin cytoskeleton. These results show the regulated expression of CD146 at areas of cell-cell junction and strongly suggest involvement of CD146 as a mediator of cell-cell interaction.

Of mice and men: comparison of the ultrastructure of megakaryocytes and platelets.

OBJECTIVE: Mice provide an excellent model for studying platelet and megakaryocyte (Mk) biology in vivo. Given the increasing use of transgenic and knockout mice, it is important that any similarities and differences between murine and human platelet/Mk biology be well defined. Therefore the objective of this study was to compare and contrast in detail any significant morphological differences between Mks, platelets, and mechanisms of thrombopoiesis in humans and mice. METHODS: The distinctive structural and ultrastructural features of murine and human platelets and Mks are reviewed. Several platelet and Mk glycoproteins were also localized in murine cells by immunoelectron microscopy using polyclonal antibodies directed against human platelet proteins and compared to existing human data. Finally, the ultrastructure of maturing murine and human Mks in culture and bone marrow were examined in detail to facilitate a comparison of either in vivo or in vitro platelet production. RESULTS: Human and murine platelets exhibit significant but well-established morphological differences. Murine platelets are smaller and more numerous and display much greater granule heterogeneity than their human counterparts. Immunoelectron microscopy also demonstrated that murine platelet alpha-granules are highly compartmentalized. In fact, they are remarkably similar to human alpha-granules, with asymmetrical distribution of von Willebrand factor (vWF), and labeling of alpha(IIb)beta(3) and P-selectin (CD62P) in the granule limiting membrane. In vivo, murine but not human Mks are also consistently localized within the spleen. Subcellular events accompanying platelet formation and release by murine Mks are presented for the first time, and compared to human. Consistent differences were found in the pathway of redistribution of demarcation membranes preceding platelet formation, which may be important for the clarification of the mechanism of platelet release. CONCLUSION: Human and murine platelets and Mks display several characteristic ultrastructural differences (size, number, histological distribution, platelet shedding) which have been emphasized and analyzed in this report. Nevertheless, since there are also many close similarities (organelle and glycoprotein subcellular distribution) mice offer an excellent in vivo model to study various aspects of human Mk and platelet biology.

Identification of PML oncogenic domains (PODs) in human megakaryocytes.

Megakaryocytes (Mks) are unique cells in the human body in that they carry a single and polyploid nucleus. It is therefore of interest to understand their nuclear ultrastructure. PML oncogenic domains (PODs) were described in several types of eukaryotic cells using human autoantibodies which recognize nuclear antigens with a specific speckled pattern (dots) in indirect immunofluorescence (IF). Two main antigens, PML and Sp 100, usually colocalize and concentrate in these nuclear subdomains. We investigated the presence of PODs using IF and immunoelectron microscopy (IEM) in cells from megakaryocytic lineage: the HEL cell line and human cultured Mks. Antibodies against PML, Sp100, and anti-nuclear dots were used in single and double labeling. PODs were identified in HEL cells and in human Mks, and their ultrastructure was characterized. We then used IF to quantify PODs within Mks and showed that their number increased proportionally to nuclear lobularity. In summary, we report the identification of PODs in human Mks at an ultrastructural level and an increase in PODs number in parallel with Mk ploidy. We show that endomitosis not only leads to DNA increase but also to the multiplication of at least one of the associated nuclear structures.

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques.

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.

Newly recognized cellular abnormalities in the gray platelet syndrome.

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased alpha-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34+ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in alpha-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of P-selectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal alpha-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS.

The storage defects in grey platelet syndrome and alphadelta-storage pool deficiency affect alpha-granule factor V and multimerin storage without altering their proteolytic processing.

Among proteins stored in alpha-granules, multimerin and factor V share unusual features: they bind to each other, are proteolysed to unique forms and are stored eccentrically in alpha-granules. These unique features of their processing led us to study these proteins in alpha delta storage pool deficiency (alphadelta-SPD) and grey platelet syndrome (GPS, alpha-SPD), two conditions known to impair alpha-granule protein storage. Platelet factor V and multimerin were severely reduced in GPS, whereas they ranged from reduced to normal in alphadelta-SPD. The platelet levels of factor V and multimerin in these disorders indicated multimerin deficiency was not predictive of platelet factor V deficiency, although it reduced the amount of multimerin associated with platelet factor V. In GPS only, the defect in storing proteins was associated with increased multimerin and multimerin-factor V complexes in plasma. Like normal platelets, GPS and alphadelta-SPD platelets contained factor V mainly in granules. Platelet factor V and multimerin were proteolysed to normal platelet forms in GPS and alphadelta-SPD platelets, indicating that these conditions preserve some aspects of normal alpha-granule protein processing. Although we found factor V can be stored in platelets deficient in multimerin, our data indicate that multimerin storage influences the point at which multimerin binds factor V.