Intention to leave, depersonalisation and job satisfaction in physicians and nurses: a cross-sectional study in Europe.

The European healthcare sector faces a significant shortage of healthcare workers. Assessing the prevalence of this issue and understanding its direct and indirect determinants are essential for formulating effective recruitment programs and enhancing job retention strategies for physicians and nurses. A multicentric cross-sectional study was conducted, involving 381 physicians and 1351 nurses recruited from eight European hospitals in Belgium, the Netherlands, Italy, and Poland. The study focused on assessing turnover intentions among healthcare workers based on the Job Demands-Resources model, using an online questionnaire. Structural equation models were employed to test the data collection questionnaires' construct validity and internal consistency. The turnover intention was assessed by agreement with the intention to leave either the hospital or the profession. Among physicians, 17% expressed an intention to leave the hospital, while 9% intended to leave the profession. For nurses, the figures were 8.9% and 13.6%, respectively. The internal consistency of the questionnaires exceeded 0.90 for both categories of health workers. Depersonalization and job dissatisfaction were identified as direct determinants of turnover intention, with work engagement being particularly relevant for nurses. We found a higher intention to leave the hospital among physicians, while nurses were more prone to leave their profession. To mitigate turnover intentions, it is recommended to focus on improving job satisfaction, work engagement and fostering a positive working climate, thereby addressing depersonalisation and promoting job retention.

Nonalcoholic fatty liver disease is associated with a decreased myocardial mechano-energetic efficiency.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is linked to a raised risk of cardiovascular diseases (CVD), although the underlying mechanisms are not completely known. A reduced myocardial mechano-energetic efficiency (MEE) has been found to be an independent predictor of CVD. OBJECTIVE: To evaluate the association between NAFLD and a compromised MEE. METHODS: Myocardial MEE was assessed by a validated echocardiography-derived measure in 699 nondiabetic individuals subdivided into two groups according to ultrasonography defined presence of NAFLD. RESULTS: Subjects with NAFLD displayed higher levels of systolic (SBP) and diastolic blood pressure (DBP), triglycerides, fasting and postload glucose, high-sensitivity C-reactive protein (hsCRP), insulin resistance (IR) estimated by HOMA-IR and liver IR index, and lower values of high-density lipoprotein (HDL) in comparison with those without NAFLD. Presence of NAFLD was associated with increased levels of myocardial oxygen demand and reduced values of MEE. MEE was negatively correlated with male sex, age, BMI, waist circumference, SBP, DBP, total cholesterol, triglycerides, fasting and postload glucose, HOMA-IR and liver IR index, hsCRP and positively with HDL levels. In a multivariable regression analysis, presence of NAFLD was associated with MEE regardless of several cardio-metabolic risk factors such as age, gender, waist circumference, SBP, DBP, total and HDL cholesterol, triglycerides, glucose tolerance and hsCRP (ß = -0.09, P = 0.04), but not independently of IR estimates. CONCLUSION: Ultrasound-defined presence of NAFLD is associated with a decreased MEE, a predictor of adverse cardiovascular events. The relationship between NAFLD and a compromised MEE is dependent of IR.

Liraglutide improves cardiac function in patients with type 2 diabetes and chronic heart failure.

PURPOSE: To compare the effect of liraglutide, sitagliptin and insulin glargine added to standard therapy on left ventricular function in post-ischemic type-2 diabetes mellitus patients. METHODS: We evaluated 32 type-2 diabetes mellitus Caucasians with history of post-ischemic chronic heart failure NYHA class II/III and/or left ventricular ejection fraction ≤45 %. Participants underwent laboratory determinations, electrocardiogram, echocardiogram, Minnesota Living with Heart Failure questionnaire and 6 min walking test at baseline and following 52 weeks treatment. Patients were treated with standard therapy for chronic heart failure and were randomized to receive liraglutide, sitagliptin and glargine in addition to metformin and/or sulfonylurea. RESULTS: Liraglutide treatment induced an improvement in left ventricular ejection fraction from 41.5 ± 2.2 to 46.3 ± 3 %; P = 0.001). On the contrary, treatment with sitagliptin and glargine induced no changes in left ventricular ejection fraction (41.8 ± 2.6 vs. 42.5 ± 2.5 % and 42 ± 1.5 vs. 42 ± 1.6 %, respectively; P = NS). Indexed end-systolic LV volume was reduced only in liraglutide-treated patients (51 ± 9 vs. 43 ± 8 ml/m2; P < 0.05). Liraglutide treatment induced also a significant increase in the anterograde stroke volume (39 ± 9 vs. 49 ± 11 ml; P < 0.05), whereas no differences were observed in the other two groups. Cardiac output and cardiac index showed a significant increase only in liraglutide-treated patients (4.4 ± 0.5 vs. 5.0 ± 0.6 L/min; P < 0.05 and 1.23 ± 0.26 vs. 1.62 ± 0.29 L/m2; P = 0.005, respectively). Liraglutide treatment was also associated with an improvement of functional capacity and an improvement of quality of life. CONCLUSIONS: These data provide evidence that treatment with liraglutide is associated with improvement of cardiac function and functional capacity in failing post-ischemic type-2 diabetes mellitus patients.

Aerobic physical activity based on fast walking does not alter blood pressure values in non-dipper essential hypertensives.

AIM: Walking is a very acceptable form of aerobic exercise. Several trials have demonstrated significant benefits of fast walking on the risk factors of cardiovascular disease, particularly for hypertension. Aim of our study was to assess whether physical activity obtained through fast walking might lead to a different reduction of blood pressure levels in hypertensive patients in relation to different circadian profile of blood pressure. METHODS: We have enrolled 84 hypertensive patients, with evidence of stage I hypertension and non-dipper nocturnal profile. All subjects underwent a six weeks physical intervention based on fast walking, three sessions a week. Main outcome measurements were diurnal, nocturnal and 24-h blood pressure levels. RESULTS: After the sixth week of physical exercise there was not any significant change in 24-hour mean systolic blood pressure and diastolic blood pressure ABPM values when compared to baseline (respectively 143.2±5.2 vs. 141±4.4 and 91.4±4.8 vs. 90.1±2.5); also no differences in heart rate have been found. CONCLUSION: In non-dipper hypertensives a light aerobic program of physical activity based on fast walking seems to be less effective to reduce blood pressure values, contrary to what has been observed in dipper ones.

Effects of ACE-inhibitors and angiotensin receptor blockers on inflammation.

The role of inflammation in cardiovascular disease and in hypertensive disease above all, is complex. Several studies confirm that activation of renin-angiotensin-aldosterone system (RAAS), through increase in the production of angiotensin II (Ang II), is closely related to local vascular inflammation. Over the BP lowering effects of anti-hypertensive treatments, several ancillary effects for every class may be found, distinguishing the various drugs from one another. Given the pro-inflammatory effects of Ang II and aldosterone, agents that interfere with the components of RAAS, such as ACE inhibitors, Angiotensin Receptor Blockers (ARBs), and mineralocorticoid receptor antagonists (spironolactone or the more selective eplerenone), represent logical therapeutic tools to reduce vascular inflammation and cardiovascular risk, as suggested in large clinical trials in patients with hypertension and diabetes. Regarding ACE inhibitors, actually there is no convincing evidence indicating that ACEi's reduce plasma levels of major inflammatory markers in hypertension models. Lack of evidence concerns especially these inflammation markers, such as fibrinogen of CRP, which are less closely related to atherosclerotic disease and vascular damage and conversely are affected by several more aspecific factors. Results obtained by trials accomplished using ARBs seem to be more univocal to confirm, although to great extent, these is an anti-inflammatory effect of drugs blocking AT1 receptor. In order to strictly study the effects of blockage of RAAS on inflammation, future studies may explore different strategies by, for example, simultaneously acting on the ACE and the AT1 angiotensin receptors.

Análisis de los registros de presión arterial en recién nacidos pretérminos en consultorio de control ambulatorio / Analysis of records of blood pressure in preterm infants in an outpatient clinic control

El registro de PA en controles ambulatorios de los niños nacidos pretérminos no es una práctica habitual. Los objetivos de este estudio fueron: Evaluar PA y prevalencia de HTA de RNPT en control ambulatorio. Asociar HTA a factores de riesgo y patologías perinatales. POBLACIÓN. RNPT < 36 semanas (sem) dados de alta de UTIN y atendidos en consultorio ambulatorio. Período agosto 2008 a febrero 2009. Las variables estudiadas fueron edad gestacional, edad actual corregida, medidas antropométricas al nacimiento y al momento del registro: peso, talla. Patologías en UTIN. Se midió PA con tensiómetro oscilométrico manual con auscultación y palpación del pulso. Se siguió técnica y definición de PA, pre HTA e HTA 1 Y 2 de la 4a Task Force. Diseño descriptivo de corte transversal. Análisis estadístico simple y de asociación. RESULTADOS: 36 RNPT<36 s fueron estudiados, 57 por ciento de sexo femenino. Los promedios observados al nacer fueron; EG de 31.3 s IC 95 por ciento (30;32), PN 1533 gr IC95 por ciento (1397;1668), TN 40 cm (38.8; 41.3). Al momento del registro, EAC 11.8 m IC 95 por ciento (8.9;14.7), PA 8594 gr IC95 por ciento (7763; 9424). 19 por ciento con BPEG; 16 por ciento con TBEG; 3 po ciento TAEG. Se registró PAS a 32 niños (86,5 por ciento) , 6 por ciento fueron Pre-HTAS (P90-95); 6 por ciento HTAS grado I y 6 por ciento HTAS grado 11. En 50 por ciento se registró PAD, 6 por ciento pre HTAD; 25 por ciento con HTAD 1; 6% con HTAD 2. 27 niños tuvieron algún antecedente patológico perinatal, CAU 19 por ciento, 57 por ciento EMH, 51 por ciento sepsis, 41 por ciento ARM, 14 por ciento HIV, esta última se asoció significativamente con HTA (p=0.01). DISCUSIÓN Y CONCLUSIONES: En los más pequeños fue difícil registrar PA con este método. Encontramos mayor prevalencia de HTA en este grupo y asociación de HTA con HIV. El seguimiento será fundamental...

The registration of BP (Blood Pressure) in ambulatory monitoring of children born pre-term is not common practice. The objectives of this study were: to evaluate BP and Hypertension of newborns in outpatient control, associate Hypertension risk factors and perinatal pathology. POPULATION: pre term newborns of 36 weeks discharged from NICU and treated at an outpatient clinic. PERIOD: August 2008 to February 2009. The variables studied were: gestational age, corrected current age, anthropometric measurements at birth and the time of recording: weight, height. Pathologies in NICU. BP (Blood Pressure) was measured with an oscillometric blood pressure monitor with manual auscultation and pulse palpation. It was followed by technique and definition of BP, pre Hipertension, e Hipertension 1 y 2 of the fourth Task Force. Cross sectional descriptive design. Simple and association statistical analysis. RESULTS: 36 newbons 36 weeks were studied, 57 per cent female. The averages observed at birth were: GA (Gestational Age) 31.3 CI (Confidence Interval) 95 per cent (30:32), WB (Weight at Birth) 1533 gr 95 per cent (1397, 1668), HB (Height at Birth) 40 cm (38.8; 41.3). At the time of registration: CCA (Corrected Current Age) 118 m 95 per cent (8.9; 14.7), 8594 BP (Blood Pressure) 95 per cent gr (7763, 9424). 19 per cent in LWGA (Low Weight for Gestational Age), LHGA (Low Height for Gestational Age) 16 per cent, RHGA (Right Height for Gestational Age) 3 per cent. SBP (Systolic Blood Pressure) was recorded in 32 children (86,5 per cent), 6 per cent were pre-HBPs (P 90-95), 6 per cent HBP grade 1, and 6 per cent were HBP grade II...

Endothelial dysfunction and non-alcoholic liver steatosis in hypertensive patients.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease, characterized by insulin resistance, has been correlated with several clinical and pathological manifestations, such as intima-media thickness. At present, no data are available regarding endothelial dysfunction, the first step in atherosclerosis, and non-alcoholic fatty liver disease. The aim of this study was to test a possible association between non-alcoholic fatty liver disease and endothelium-dependent vasodilation in a group of hypertensive patients. METHODS AND RESULTS: A total of 40 never-treated uncomplicated hypertensive outpatients were enrolled. Patients underwent a complete clinical and biochemical work-up including ultrasonographic scanning to detect liver steatosis. Insulin sensitivity was estimated by using the homeostasis model assessment (HOMA) index. Endothelial function was assessed by strain-gauge plethysmography during intra-arterial infusion of increasing doses of acetylcholine and sodium nitroprusside. Endothelium-dependent vasodilation was significantly reduced in hypertensive patients with liver steatosis in comparison with those without. Statistical analysis demonstrated that the HOMA index was the strongest predictor of both endothelium-dependent vasodilation and liver steatosis. In particular, one point of HOMA accounts for 37.9% of forearm blood flow variation, and increases the risk of liver steatosis by 86.4%. CONCLUSION: Our data demonstrate that hypertensive patients with liver steatosis have a reduced endothelium-dependent vasodilation and highest insulin resistance. In keeping with this, it is possible to hypothesize that liver steatosis may be considered a marker of vascular damage in essential hypertension.

[Thrombocytopenia associated with brucellosis: a case report]. / Trombocitopenia in corso di brucellosi: descrizione di un caso.

Brucellosis, a common disease in some areas of the world, beside its typical signs and symptoms, as fever, arthropathy, hepatomegaly and splenomegaly, sometimes can complicate into thrombocytopenia, even in severe forms. The pathogenesis of thrombocytopenia in course of brucellosis is variable, and a main role is played by immunological reactions. Authors describe a case report of an eight years child who presented a severe thrombocytopenia in course of acute brucellosis. The patient responded efficaciously to the antibiotic therapy combined with immunoglobulin intravenous therapy.

Recurrent venous thromboembolism complicated by heparin-induced thrombocytopenia as a first manifestation of an occult cancer: a case report.

Heparin-Induced Thrombocytopenia (HIT) is a serious and potentially fatal complication of patients on heparins. Its management is difficult and it can be more complicated in patients with cancer because of the hemorrhagic risk carried out by direct inhibitor of thrombin, the currently approved drug for HIT. At present, it is not clear whether cancer patients also have an increased risk of HIT. We describe the case of a patient with occult cancer at the moment of the index venous thrombosis, who developed Deep Vein Thrombosis (DVT) and concomitant HIT with thrombotic complications (recurrent contra-lateral venous thrombosis). The management of HIT was efficaciously based on the combined use of alternative antithrombotic regimens (Dermatan-Sulphate and Defibrotide), without an increased risk of bleeding. This case highlights the potential relationship between DVT, as first episode of an occult cancer, and the risk of developing HIT. The use of alternative antithrombotic therapy seems to be efficacious even in this high-risk cancer patient.

[Angiosarcoma of the nasal cavity. Report of a case and review of the literature]. / Angiosarcoma della cavità nasale. Descrizione di un caso e revisione della letteratura.

We report an unusual case of primary angiosarcoma of the nasal cavity (AS-nc). Clinical--monolateral epistaxis in a young person--, radiological--polypoid hemorrhagic tumor arising within the nasal cavity and expanding into paranasal sinuses--, pathological--a network of anastomosing channels and solid areas immunoreactive for CD31 and CD34--and prognostic features--patient alive and well 36 months after the original diagnosis--are superimposable to those of previously reported AS-nc, suggesting that this lesion should be considered as a peculiar variant of classical AS.

Monocyte and lymphocyte apoptosis resistance in acute and chronic brucellosis and its possible implications in clinical management.

This study evaluated the level of susceptibility of monocytes and lymphocytes to spontaneously induced and CH11-induced apoptosis in 16 patients with Brucella infection. The expression of some immunological and apoptotic markers was evaluated. Before therapy, monocytes showed a high level of resistance to spontaneously induced or CH11-induced apoptosis in all patients. In patients with acute infection, this resistance persisted for 10-20 days after treatment was initiated, then decreased; in chronically infected patients, it persisted after 45 days of treatment. Lymphocytes were also more resistant to CH11-induced apoptosis. The level of activated CD8(+) T lymphocytes was high in patients with acute infection. The data indicate that the CD95-mediated apoptotic pathway is not involved in CH11 resistance. Lymphocytes are not infected by Brucella, so their resistance to apoptosis may be due to a soluble factor released by infected monocytes. The evaluation of levels of susceptibility to CH11-induced apoptosis in monocytes may be used to test the effectiveness of the therapy.

A clinical and bacteriological study of children suffering from haemolytic uraemic syndrome in Tucuman, Argentina.

Haemolytic uraemic syndrome (HUS) is a disease with serious consequences for children, such as terminal chronic renal failure. During the last few years there have been numerous studies undertaken to determine whether there is a relationship between this disease and the presence of Shiga toxin-producing bacteria. Escherichia coli (E. coli) O157:H7 is one of the most frequent etiologic agents of HUS. It acts through cytotoxins called Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) and carries a 90-Kb plasmid codified for an adhesion fimbria which is part of its pathogenicity. The objectives of this study were to: 1). confirm whether there exists a relationship between severity and clinical presentation of HUS; 2). prove the existence of Stx1 and/or Stx2 in the faeces of HUS patients; and 3). detect the presence of Stx1- and/or Stx2-producing E. coli. Our results did not show any difference in the average age, sex or clinical behavior between children with diarrhea positive (D+) HUS and diarrhea negative (D-) HUS. Male patients were predominant, as was incidence during summer, considering all cases. Nor could we find any relationship between severity and HUS type. E. coli O157:H7 was isolated in 40% of the patients with (D+) HUS and in 50% of patients with (D-) HUS. Another serotype, O55:K59, was also isolated (7%). Stx1 and/or Stx2 were found in all HUS cases. The following virulence factors of E. coli strains isolated from 12 patients were found: Adhesion fimbria (100%), Stx1 (16%), Stx2 (32%), and Stx1 + Stx2 (50%). None of these factors was found in control patients. Sixty-three percent of the HUS cases showed seroconversion for lipopolysaccharides of E. coli O157. We drew the following conclusions: 1). there is no significant relationship between seriousness of HUS and type of disease; 2). an association exists between HUS and the production of Stx1 and Stx2; 3). the incidence of E. coli O157:H7 was high in Tucuman, Argentina; and 4). Stx2 alone or in association with Stx1 was the predominant toxin.

Association between hemolytic uremic syndrome and verotoxin-producing strains of E. coli.

One of the most frequent etiologic agents of Hemolytic Uremic Syndrome (HUS) is Escherichia coli O157H7, a microorganism that possesses virulence factors (Shiga-like Toxins I and II and adhesion fimbriae). The present study was set up to determine the relationship between HUS and the presence of Verotoxin in patients of "Niño Jesús" Children's Hospital. Tucumán, Argentina. 19 Children between 0 and 4 years old suffering from HUS (typical and atypical symptoms) and 15 control children of similar sex and age were selected. Presence of enterohemorrhagic E. coli was studied in both groups using molecular hybridization techniques. Free Verotoxin and Verotoxin-producing E. coli were analyzed in Vero cells. The following results were obtained: 1) The cytotoxic effect on Vero cells from fecal filtrates was observed in all children suffering from HUS 2) Verotoxin-producing E. coli was detected in only 12 of them 3) None of the filtrates of feces from control children presented a cytotoxic effect on Vero cells 4) In 8 of the patients suffering from HUS serotype O157H7 was isolated, in one O55K59 and in 3 typification of E. coli was not possible with the serums assayed 5) 77.5% of the strains isolated from HUS patients gave a positive molecular hybridization reaction, showing the following: Adhesion Fimbriae (AF) (25%); AF + Shiga-like Toxin I (13.75%); AF + Shiga-like Toxin II (20%); AF + Shiga-like Toxins I and II (41.25%). In patients suffering from atypical HUS a combination of AF + Shiga-like Toxins I and II was found. The 15 control children did not hybridize to the probes assayed. From the results obtained we may conclude that there exists a relationship between HUS and the presence of Verotoxin in the children suffering from HUS studied. The predominant serotype in our cases was O157H7 and Shiga-like Toxin II was found with highest frequency.

Granulomatous slack skin: report of a case associated with Hodgkin's disease and a review of the literature.

We report a case of granulomatous slack skin (GSS) associated with Hodgkin's disease, and review the literature on this entity. GSS, a variant of cutaneous T-cell lymphoma, clinically presents with erythematous patches in the flexures, which gradually transform into bulky, pendulous areas of skin. Histology shows an elastolytic granulomatous infiltrate, with atypical lymphoid cells, and occasional epidermotropism. As far as we are aware, 10 cases of GSS, including our patient, have been reported in detail. The male:female ratio of these cases is 9:1, and the age range 15-51 years. Five cases were associated with Hodgkin's disease, one with small lymphocytic lymphoma, and one developed cutaneous T-cell lymphoma. The axillae, abdomen and groins were the most frequently affected areas. No definitive management for GSS has been established. Surgery has been performed in localized forms, and systemic treatments have included corticosteroids, dapsone, chlorambucil, nitrogen mustard, and radiotherapy. Our patient was treated with chemotherapy for his Hodgkin's disease, and this resulted in complete remission of the lymphoma. Subsequent maintenance therapy with interferon-alpha produced good control of the cutaneous lesions.

Tratamiento profiláctico de la infección urinaria recidivante con cefalexina oral / Profilactic treatment to recidivant urinary infection with oral cephalexine

Bajo la hipótesis que los daños renales están relacionados con la frecuencia e intensidad de las infecciones urinarias, se implementó el tratamiento profilático de éstas con quimioterápicos urinarios. Debido a problemas de resistencia, intolerancia y limitaciones en su uso durante los primeros meses de vida instituimos el uso de la Cefalexina. En este trabajo mostramos nuestra experiencia en cuanto a su efectividad en este tipo de tratamiento. Se estudió una muestra retrospectiva de 43 pacientes (p) que recibieron tratamiento profiláctico con Cefalexina a 30 mg/Kg/día en dos dosis en niños menores de 2 años y dosis única nocturna en mayores, con diferentes diagnósticos causales de sus cuadros de infección urinaria recidivante. La duración media del tratamiento fue de 13,6 meses (m) (Rango 3 m a 40 m), de los cuales 34 p (79 por ciento) se mantuvieron sin recidiva por un lapso promedio de 12,9 m (3 m - 40 m). De los 43 p, 26 habían recibido tratamiento rotatorio previo a la Cefalexina, con quimioterápicos por uden tiempo medio de 12,7 m (3 m - 31 m), con registro de recidivas en 17 de ellos (65 por ciento). Proponemos la Cefalexina como tratamiento profiláctico de elección en las I.U.recidivantes, ya que la recurrencia de las mismas fue menor que con el uso de otros quimioterápicos

Tratamiento profiláctico de la infección urinaria recidivante con cefalexina oral / Profilactic treatment to recidivant urinary infection with oral cephalexine

Bajo la hipótesis que los daños renales están relacionados con la frecuencia e intensidad de las infecciones urinarias, se implementó el tratamiento profilático de éstas con quimioterápicos urinarios. Debido a problemas de resistencia, intolerancia y limitaciones en su uso durante los primeros meses de vida instituimos el uso de la Cefalexina. En este trabajo mostramos nuestra experiencia en cuanto a su efectividad en este tipo de tratamiento. Se estudió una muestra retrospectiva de 43 pacientes (p) que recibieron tratamiento profiláctico con Cefalexina a 30 mg/Kg/día en dos dosis en niños menores de 2 años y dosis única nocturna en mayores, con diferentes diagnósticos causales de sus cuadros de infección urinaria recidivante. La duración media del tratamiento fue de 13,6 meses (m) (Rango 3 m a 40 m), de los cuales 34 p (79 por ciento) se mantuvieron sin recidiva por un lapso promedio de 12,9 m (3 m - 40 m). De los 43 p, 26 habían recibido tratamiento rotatorio previo a la Cefalexina, con quimioterápicos por uden tiempo medio de 12,7 m (3 m - 31 m), con registro de recidivas en 17 de ellos (65 por ciento). Proponemos la Cefalexina como tratamiento profiláctico de elección en las I.U.recidivantes, ya que la recurrencia de las mismas fue menor que con el uso de otros quimioterápicos

Long-term marrow culture in patients with aplastic anemia compared with marrow transplant recipients and normal controls.

UNLABELLED: Patients with severe aplastic anemia (SAA; n = 46) were studied in long-term bone marrow culture (LTBMC) systems and compared with allogeneic marrow transplant (BMT) recipients (n = 16) (within 30 days following BMT) and normal control patients (n = 12). SAA patients were divided in two groups: transfusion-dependent (Tx-D) SAA patients (group A; n = 15) and transfusion-independent (Tx-I) patients after treatment with antilymphocyte globulin (group B; n = 31). Cultures were analyzed at three levels: stromal layer (SL) formation (score: 0, no SL; 1, half confluent SL; and 2, confluent SL), number of nucleated cells in suspension, and growth of CFU-GM colonies. SL formation was rapid and complete in SAA patients, groups A and B (mean score on day 14: 1.3 and 1.4), similar to controls (mean score on day 14: 1.3), whereas an impairment of SL formation was seen in BMT recipients (mean score on day 14: 1.0). The number of nucleated cells in suspension increased significantly on day 7 of culture in controls (7.6-fold), significantly more than in BMT and SAA patients, and declined thereafter. Colony formation was also significantly increased on day 7 in Tx-I SAA patients, BMT recipients, and normal controls (4-, 5-, and 16-fold, respectively), lasting respectively 2, 3, and 4 weeks. Increments of colony formation were also obtained in Tx-D SAA patients, but in the first week of culture only. IN CONCLUSION: 1) a significant impairment of SL formation was seen in BMT recipients, but not in SAA patients; 2) a significant increment of granulocyte-macrophage colony-forming units (CFU-GM) growth can be obtained in patients with marrow failure early after starting long-term culture; 3) the number of CFU-GM grown in these culture conditions from Tx-I SAA patients parallels the number of progenitors from early post-BMT recipients; and 4) progenitor cells from Tx-D SAA patients are not only reduced in numbers, but also exhibit a poor ability to survive in LTBMC.

Sequence specificity of the human mRNA N6-adenosine methylase in vitro.

N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency.