[Study of - maximum systolic velocities and mean maximum velocities of skull base arteries recorded with transcranial Doppler in adult sickle cell patients without neurovascular complication]. / Étude des vitesses systoliques maximales et des moyennes des vitesses - maximales des artères de la base du crâne enregistrées au Doppler transcrânien chez l'adulte drépanocytaire sans complication neurovasculaire.

INTRODUCTION: Sickle cell disease is the leading genetic disease in Île-de-France. Stroke is one of its most severe complications. In SS sickle cell children, transcranial Doppler (TDC) is, through the study of average speeds of the skull base arteries, the gold standard for screening and diagnosis of vasculopathy. To our knowledge, in adults with sickle cell disease, no standards have been established for the speed of the arteries at the base of the skull. It therefore seemed useful to us to establish an approach to brain speeds recorded in adults with sickle cell disorders without neurovascular complications. MATERIAL AND METHODS: This was an observational, prospective, monocentric study conducted between February 2017 and June 2017. The main objective of the study was to determine the mean and standard deviation of maximum systolic velocities (MSS) and mean maximum velocities for all arteries recorded during the transcranial Doppler echo. The secondary objectives were to compare the mean maximum systolic velocities in sickle cell adults with those of healthy adults, to compare the mean maximum systolic velocities in sickle cell adults with those of sickle cell children, and to determine whether parameters could influence the speeds recorded at TCD. RESULTS: Forty patients were included between February 1, 2017 and June 30, 2017, with an average age of 39.3 years. The mean maximum velocities recorded were: 78cm/s for the middle cerebral arteries; 59.6cm/s for the internal carotid arteries; 61cm/s for the anterior cerebral arteries; 44cm/s for the posterior cerebral arteries and 55cm/s for the basilar trunk. DISCUSSION: The highest circulatory velocities are found in the middle cerebral arteries. The speeds found in the internal carotid arteries and anterior cerebral arteries are faster than in the vertebrobasilar system. Speeds in sickle cell adults are slower than those described in sickle cell children SS but significantly faster than those found in healthy adults. CONCLUSION: To our knowledge, this study is the first to evaluate transcranial Doppler circulatory velocities in adult sickle cell patients. This work has limitations due to its small sample size, however, it provides a basis for further studies on transcranial Doppler in sickle cell adults.

Risk factors and impact of orthopaedic monitoring on the outcome of avascular necrosis of the femoral head in adults with sickle cell disease: 215 patients case study with control group.

INTRODUCTION: Sickle cell disease is a public health problem. The WHO has recommended that global management be implemented to reduce mortality and morbidity. Since no comprehensive care programme for bone and joint complications exists, the Caribbean Sickle Cell Disease Center added orthopaedic consultation to screen for and monitor these complications in 1992. HYPOTHESIS: Comprehensive medical and surgical care of patients with sickle cell disease will reduce the complications and disability associated with this disease. POPULATIONS AND METHODS: Two populations were compared to evaluate the impact of comprehensive disease management on the occurrence of avascular necrosis (AVN) of the femoral head (femoral head AVN). The case-control series, [E-1994], included 115 patients (58 SS and 57 S) without orthopaedic monitoring and was evaluated retrospectively. The other patient series, [E-2008], included 215 patients (94 SS and 121 SC) with systematic orthopaedic care and was followed prospectively. Age, gender, duration of follow-up, haemoglobin levels, genotype, pain before treatment, associated humerus AVN and leg ulcers were analysed. RESULTS: Femoral head AVN occurred in young adult patients (35.3 ± 4 years for [E-1994] and 29 ± 3.4 years for [E-2008]). Only elevated haemoglobin levels were associated with the occurrence of femoral head AVN, which suggests that increased blood viscosity contributes to the condition ([E-1994], P<0.0001; [E-2008], P=0.001). Treatment in [E-2008] patients reduced the number of femoral head AVN cases from 36.5% in [E-1994] to 14.4% in [E-2008] (P<0.0001). DISCUSSION: The prevention and management of femoral head AVN must include medical treatment of the disease to reduce the occurrence of painful vaso-occlusive crises, which are known to trigger femoral head AVN. The effectiveness of this programme hinged on identifying risk factors and using simple approaches (hydration, pain medication, rest and crutches) to manage painful joint crises before femoral head AVN appeared. These approaches could be implemented in disadvantaged countries where sickle cell disease is prevalent. CONCLUSION: By knowing the risk factors, symptomatic patients who are at risk for femoral head AVN can be identified and additional evaluations can be performed early on in cases of hip pain.

Ventilatory and lactic thresholds in subjects with sickle cell trait.

This study investigated 1) whether ventilatory and lactic thresholds (VT and LT, respectively) are different in sickle cell trait carriers (SCTc) and subjects with normal hemoglobin (control group), and 2) whether the first LT and VT and the second LT and VT are respectively coincident in the two populations. Seven SCTc and 8 control subjects performed an incremental exercise test (IET). Blood lactate concentration and cardioventilatory variables were analyzed at rest and during IET. No significant difference in the ventilatory parameters (notably, maximal oxygen uptake [VO (2max)] and the ventilatory thresholds) or the lactic thresholds was observed between the two groups. In both SCTc and control subjects, the LTs and VTs did not occur at the same exercise intensity. The first VT did not coincide with the first LT, in contrast with the second VT and the second LT, which coincided in both groups. In conclusion, SCTc exhibited normal ventilatory and lactic responses during a progressive and maximal exercise test assessing aerobic physical fitness.

Sickle cell trait in French West Indian elite sprint athletes.

The aim of this study was to establish the percentage of sickle cell trait (SCT) carriers among French West Indian sprinters selected for the French National Team in 2000. The investigation determined the number of SCT carriers and the number of national records they had established. Sixteen athletes were indexed (6 males and 10 females). The athletes were within the ranges of 20-33 years, 161-186 cm and 60-80 kg. The results showed the presence of SCT carriers in this population among whom three were SCT carriers (2 males and 1 female) (18.75%). Moreover, there is a significantly higher percentage of titles and records held by the SCT carriers (38.6% and 50%, respectively). In conclusion, this study shows that sickle cell trait carriers are able to perform sprint exercises at the highest levels, and it further indicates that brief and explosive exercise involving mainly the alactic anaerobic metabolism may be enhanced by HbS.

[Sickle cell anemia and pregnancy: review of 68 cases in Guadeloupe]. / Drépanocytose et grossesse: revue de 68 observations en Guadeloupe.

Pregnancy in women with major sickle cell syndromes is a high risk maternofetal situation. This descriptive study presents the features and the clinical course of 68 pregnancies in sickle cell women who were delivered in Guadeloupe from January 1(st) 1993 to December 31(st) 1997. Specific complications were observed in all hemoglobin types, but with a severer course in SS women. Painful vaso-occlusive crises were the main causes of hospitalisation (88% of SS pregnancies and 27% of SC pregnancies) associated most often with worsening anemia and / or infection. Acute chest syndrome was observed in all genotypes at any time throughout pregnancy and during the post partum period. One death occurred (a 16 years old SBeta(+)thal woman). Fetal mortality and morbidity were also high, intrauterine growth retardation and fetal death being the most frequent fetal complications. The rates of prematurity (21%) and caesarean section (48%) were higher than in the whole population. Three (3) neonatal deaths occurred. A multidisciplinary and specific approach, vigilance of health care providers and patient compliance are required to manage efficiently pregnancy, delivery and post partum in sickle cell women.

[Analysis of hospitalization of adult sickle-cell patients in Guadeloupe]. / Analyse des hospitalisations chez les patients drépanocytaires adultes en Guadeloupe.

PURPOSE: To determine the characteristics of acute hospitalizations in adult patient with sickle-cell disease in Guadeloupe. METHODS: We retrospectively studied clinical features of adult patients followed up by the "Centre Caribeen de la Drépanocytose" (CCD) in 1996. Data were collected from the medical records of the hospitalized patients and the longitudinal records of the CCD. RESULTS: Sixty-three (25%) of the 251 patients who were followed up by the CCD required hospitalization in 87 cases (1.38 hospitalizations/patient). Mean age of the hospitalized patients was 27.5 years (range 17 to 71 years). Most hospitalizations involved men (29 [31%] vs 34 [22%] for women, P < 0.05), and most were for homozygous patients with sickle-cell anemia: 39 (31%) SS, 19 (18.55%) SC and five (21.75%) S beta thal. A painful vaso-occlusive crisis was noted in 67 episodes. There were nine acute chest syndromes (ACS), six of them occurred following a vaso-occlusive crisis. We noted 39 infectious episodes. The increase in C-reactive protein (> 100 mg/L) was associated with ACS or urinary infection. A patient with renal failure died during septicemia. CONCLUSION: This study confirms the need for prevention of painful crises and other severe complications in patients with sickle-cell disease.

[Arterial blood pressure in homozygote patients with drepanocytosis]. / Pression artérielle chez les patients drépanocytaires homozygotes.

BACKGROUND: Relative hypotension has been reported in sickle cell patients. The aim of this study was to compare blood pressure in patients with SS disease and subjects with normal hemoglobin genotype AA and to assess whether the same clinical, biological and socio-demographic variables are associated to the mean arterial pressure in patients with sickle cell disease and normal subjects. METHOD: Blood pressure was measured with a standardized automated oscillometric method in 88 SS patients et 88 AA control subjects seen in the University Hospital of Pointe-à-Pitre (Guadeloupe). A multiple linear regression analysis for mean arterial pressure was done including type of hemoglobin (forced variable), age, sex, body mass index, pulse rate, hemoglobin concentration and interaction terms between type of hemoglobin and other variables. A regression was also fitted separately for each population. A downward stepwise strategy was used to simplify the models. RESULTS: The two groups were similar for age, height and gender ratio and pulse rate. Mean arterial pressure was significantly lower in sickle cell patients (81.6 mmHg in SS patients vs 89.9 mmHg in AA subjects, p < 10(-4)). The final model included type of hemoglobin, age, sex, body mass index, pulse rate and an interaction between type of hemoglobin and age (global F = 22.04, adjusted R2 = 42%). The separate models indicated that sex was associated with mean arterial pressure only in patients with sickle cell disease and that age and hemoglobin concentration was associated with mean arterial pressure only in normal subjects. CONCLUSION: Blood pressure determinants are not similar in the two populations. The effect of age, especially, is not the same in patients with sickle cell disease and in normal subjects. These results confirm that specific patho-physiological models should be defined in sickle cell disease.

A randomized trial of captopril for microalbuminuria in normotensive adults with sickle cell anemia.

PURPOSE: Nephropathy is a common complication of sickle cell anemia and is often preceded by proteinurea. Our aim was to evaluate the effect of angiotensin-converting enzyme inhibition on microalbuminuria in sickle cell patients. PATIENTS AND METHODS: We performed a randomized, double-blind, placebo-controlled trial in 22 normotensive patients with sickle cell anemia and persistent microalbuminuria. Patients received captopril (25 mg/day) or placebo and were followed up for 6 months. Albuminuria, blood pressure, and serum creatinine and hemoglobin concentrations were measured at baseline and at 1, 3, and 6 months. The primary outcome variable was the 6-month change in albuminuria between the two groups. RESULTS: Baseline albuminuria was 121 (SD 66) mg per 24 hours in the captopril group and 107 (SD 86) mg per 24 hours in the placebo group. Microalbuminuria decreased from baseline in the captopril group but increased in the placebo group. The mean absolute change and the mean percentage change in microalbuminuria were significantly different between the two groups at 6 months (absolute change -45 mg per 24 hours in the captopril group versus +18 mg per 24 hours in the placebo group, P <0.01; and percentage change -37% in the captopril group versus +17% in the placebo group, P <0.01). The 95% confidence intervals (CI) for the difference in albuminuria between the two groups were 63 (CI 40 to 86) mg per 24 hours for the mean absolute change and 54% (CI 22% to 85%) for the mean percentage change. Blood pressure decreased slightly from baseline in captopril-treated patients and did not change in the placebo group. The change was significantly different between the two groups only for diastolic blood pressure at 6 months (P <0.01). CONCLUSION: Captopril reduces albuminuria and slightly decreases blood pressure in patients with sickle cell anemia. More studies are required to demonstrate the sustained benefit on protein excretion.

Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells.

Flt3/flk-2 ligand (Flt3-L) co-stimulates and synergizes with cytokines such as granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin in the proliferation of bone marrow and cord blood hematopoietic stem and progenitor cells. To study the biological effects of Flt3-L on the Flt3-L responsive AML5 cell line, the retroviral vector L(Flt3-L)SN was constructed based on the vector LXSN, but containing the human Flt3-L cDNA transcriptionally regulated by the Mo-MLV LTR. High-titer amphotropic producer cells that generated 10(6) cfu/mL after shuttle packaging through ecotropic packaging cells were isolated. AML5 cells were cultured overnight with L(Flt3-L)SN retroviral supernatant, 8 micrograms/mL polybrene, and 100 U/mL G-CSF, and expanded 1 week in medium with G-CSF. Transduced cells were selected in medium containing 0.4 mg/mL G418 and then in medium with 1.0 mg/mL G418. Retroviral mediated gene transfer in G418-resistant cells was confirmed after amplification by PCR of neo-specific sequences in genomic DNA. Northern blot analysis demonstrated L(Flt3-L)SN mRNA expression. Soluble Flt3-L was undetectable (< 100 pg/mL) by ELISA assay of conditioned medium from transduced cells, but Flt3-L was detected on the surface of AML5 cells by FACS analysis. Cells were plated in colony assay with and without 100 ng/mL Flt3-L, 100 U/mL G-CSF, and the combination. Gene transfer or growth factor treatment increased somewhat the clonogenicity of the nontransduced AML5 cells. More strikingly, L(Flt3-L)SN and each growth factor combination greatly increased the size of the resultant colonies such that the size of colonies from AML5/Flt3-L cells without added growth factor approximated that of the AML5 cells stimulated by exogenous soluble Flt3-L. Moreover, MAP kinase activity in L(Flt3-L)SN-transduced cells cultured without soluble Flt3-L was increased to the level induced in control cells by soluble Flt3-L. These results indicate that retroviral mediated gene transfer and autologous expression of the Flt3-L enhances growth and intracellular signaling of AML5 cells, information that should be of value for studying the effects of Flt3-L on immature subsets of primary hematopoietic stem and progenitor cells.

Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins.

As an approach to cell targeting by retroviruses, the lack of which constitutes one major limitation of retroviral vector technology, we engineered the Moloney murine leukemia virus ecotropic envelope glycoprotein. When inserted between amino acids 6 and 7 of the latter, a single-chain antibody fragment (ScFv) specific for human major histocompatibility complex class I molecules was shown to be able to redefine the tropism of ecotropic Moloney murine leukemia virus-derived retroviral particles by allowing infection of major histocompatibility complex class I-positive human cells. At variance with other recently described experimental systems, the type of modification adopted here allowed targeted infection in the absence of coexpressed wild-type env-encoded protein molecules. Interestingly, the chimeric ScFv-env protein also retained the ability to recognize the ecotropic receptor and allowed infection of murine cells, albeit at a reduced efficiency.

Cord blood transplantation and the potential for gene therapy. Gene transduction using a recombinant adeno-associated viral vector.

Cord blood, which contains a high frequency of immature stem/progenitor cells with extensive proliferative and replating capacity in vitro was used as a clinical source of transplantable stem and progenitor cells. These cells can be efficiently transduced with new genetic material by using AAV or retroviral vectors. Using a recombinant AAV vector, high level expression of the lacZ gene under a CMV promoter was demonstrated in immature subsets of cord blood progenitor cells.

Cloning and expression of a single-chain antibody fragment specific for a monomorphic determinant of class I molecules of the human major histocompatibility complex.

B9.12.1 is a monoclonal antibody specific for a monomorphic determinant of human MHC class I molecules. It is currently used for cell typing and is useful for targeting infection of human cells by murine ecotropic retroviruses. We have cloned and expressed it in the form of a single-chain variable fragment (ScFv) that recognizes the same epitope as the parental antibody. Through genetic engineering, this ScFv may be used for developing new cell-typing probes and new retroviral targeting approaches.

Differential sensitivity of FOS and JUN family members to calpains.

Degradation of c-fos protein (c-FOS) in the cytoplasm is very rapid in vivo and constitutes a crucial regulation of the nuclear steady-state level through the control of the amount of full-length molecules available for nuclear transport. Using cytoplasmic extracts from various origins, we report herein that c-FOS degradation can be initiated in a calcium-dependent manner which involves cysteine proteases called milli- and micro-calpain. Interestingly, FOS-B, a member of the fos multigene family, as well as all members of the jun family (JUN-B, c-JUN and JUN-D) are also sensitive to calpains albeit to different extents. FRA-2, which is a c-FOS-related protein, is resistant to micro- but not to milli-calpain whereas FRA-1, another member of the fos family, is resistant to both proteases. Given the fact that a work by others (Hiraï et al., 1991b) suggests that calpains can be involved in c-FOS and c-JUN degradation in vivo, our observations raises the possibility of a novel contribution to the regulation of AP-1 transcription complex activity through a differential control of the steady-state level of some of its components that involves calpains.

Concerted evolution in the GAPDH family of retrotransposed pseudogenes.

In murine rodents the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) multigene family includes more than 300 retroprocessed pseudogenes. Its single functional gene encodes GAPDH, an enzyme of glycolysis. Because of its manageable size, this family is a good model for the study of genome cohesion and evolution. By sequence comparison of several GAPDH pseudogenes in Rattus norvegicus and Mus musculus, we have obtained evidence that (i) the GAPDH family still generates new pseudogenes; we note in each species the beginning of a process of species-specific evolution since the pseudogenes of one genus on average cluster more with one another than they do with those of the other genus, and (ii) the GAPDH family contains diversified subfamilies. These findings suggest a certain level of transcription and transposition of the pseudogenes independent of the functional gene which may result from various mechanisms. The homogenization we observe may be due to the pseudogenes themselves (concerted evolution in a strict sense), which explains the occurrence of long-term homogenization of old sequences and subfamily groupings.

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker.

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

Cell targeting by murine recombinant retroviruses.

Cell targeting by murine retroviruses carrying recombinant genes would have numerous applications such as immortalization of under-represented cell types from complex cellular mixtures, increasing therapeutical yields in the case of gene therapy and delivery of specific genes at any location and at any moment of the life-time of living animals. To this aim, we are currently developing techniques that allow binding of viral particles to specific cell membrane markers different from the natural receptors. We have shown that biochemical bi-specific molecular adaptors able to bind both viral particles and cell surface molecules may reveal appropriate for piloting viruses toward specific cell types. More recently, we have undertaken the genetic engineering of the retroviral envelope glycoprotein in order to modify its natural tropism. This approach is discussed below.

Identification and tissue localization of an eosinophil 17 kDa protein accumulating in rat uterus upon estradiol treatment.

In a previous paper (J. Steroid Biochem. 29 (1988) 475-480), the isolation of a 17 kDa protein that was dramatically induced in the uterus of estrogen-treated spayed rats was presented. We now describe a new purification procedure that is compatible with microsequencing of the 17 kDa protein. The protein partial N-terminal amino acid sequence analysis gave 28 residues that revealed a strong homology to the human major basic protein (MBP) of eosinophils described by Wasmoen et al. (J. Biol. Chem. 263 (1988) 12559-12563). Polyclonal rabbit antibodies were raised against this protein and used for tissue or blood cell analysis after electrophoresis and Western blotting. The 17 kDa protein was found to be constitutively present in the stomach and small intestine of the rat and guinea-pig. Estrogen treatment had a clearcut effect in guinea-pig uterus, but not as drastic as that observed in rat uterus. The protein was abundant in purified rat eosinophils. The antibodies cross-reacted with human MBP and an equivalent molecular weight human polymorphonuclear leukocyte protein. Immunohistochemical staining of rat uterus sections showed that the protein was first only associated with eosinophils that emigrate upon estrogen treatment; it then spread throughout the stroma and the deep glandular epithelium. It was not found in the myometrium. In conclusion, the appearance of a 17 kDa protein that is presumably the rat MBP is clearly regulated in the rat uterus.