Interaction between clinicians and artificial intelligence to detect fetal atrioventricular septal defects on ultrasound: how can we optimize collaborative performance?

OBJECTIVES: Artificial intelligence (AI) has shown promise in improving the performance of fetal ultrasound screening in detecting congenital heart disease (CHD). The effect of giving AI advice to human operators has not been studied in this context. Giving additional information about AI model workings, such as confidence scores for AI predictions, may be a way of further improving performance. Our aims were to investigate whether AI advice improved overall diagnostic accuracy (using a single CHD lesion as an exemplar), and to determine what, if any, additional information given to clinicians optimized the overall performance of the clinician-AI team. METHODS: An AI model was trained to classify a single fetal CHD lesion (atrioventricular septal defect (AVSD)), using a retrospective cohort of 121 130 cardiac four-chamber images extracted from 173 ultrasound scan videos (98 with normal hearts, 75 with AVSD); a ResNet50 model architecture was used. Temperature scaling of model prediction probability was performed on a validation set, and gradient-weighted class activation maps (grad-CAMs) produced. Ten clinicians (two consultant fetal cardiologists, three trainees in pediatric cardiology and five fetal cardiac sonographers) were recruited from a center of fetal cardiology to participate. Each participant was shown 2000 fetal four-chamber images in a random order (1000 normal and 1000 AVSD). The dataset comprised 500 images, each shown in four conditions: (1) image alone without AI output; (2) image with binary AI classification; (3) image with AI model confidence; and (4) image with grad-CAM image overlays. The clinicians were asked to classify each image as normal or AVSD. RESULTS: A total of 20 000 image classifications were recorded from 10 clinicians. The AI model alone achieved an accuracy of 0.798 (95% CI, 0.760-0.832), a sensitivity of 0.868 (95% CI, 0.834-0.902) and a specificity of 0.728 (95% CI, 0.702-0.754), and the clinicians without AI achieved an accuracy of 0.844 (95% CI, 0.834-0.854), a sensitivity of 0.827 (95% CI, 0.795-0.858) and a specificity of 0.861 (95% CI, 0.828-0.895). Showing a binary (normal or AVSD) AI model output resulted in significant improvement in accuracy to 0.865 (P < 0.001). This effect was seen in both experienced and less-experienced participants. Giving incorrect AI advice resulted in a significant deterioration in overall accuracy, from 0.761 to 0.693 (P < 0.001), which was driven by an increase in both Type-I and Type-II errors by the clinicians. This effect was worsened by showing model confidence (accuracy, 0.649; P < 0.001) or grad-CAM (accuracy, 0.644; P < 0.001). CONCLUSIONS: AI has the potential to improve performance when used in collaboration with clinicians, even if the model performance does not reach expert level. Giving additional information about model workings such as model confidence and class activation map image overlays did not improve overall performance, and actually worsened performance for images for which the AI model was incorrect. © 2024 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Genetic evaluation of AMPD1, CPT2, and PGYM metabolic enzymes in patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a disease that can seriously impair one's quality of life; patients complain of excessive fatigue and myalgia following physical exertion. This disease may be associated with abnormalities in genes affecting exercise tolerance and physical performance. Adenosine monophosphate deaminase (AMPD1), carnitine palmitoyltransferase II (CPT2), and the muscle isoform of glycogen phosphorylase (PYGM) genes provide instructions for producing enzymes that play major roles in energy production during work. The aim of this study was to look for evidence of genotype-associated excessive muscle fatigue. Three metabolic genes (AMPD1, CPT2, and PYGM) were therefore fully sequenced in 17 Italian patients with CFS. We examined polymorphisms known to alter the function of these metabolic genes, and compared their genotypic distributions in CFS patients and 50 healthy controls using chi-square tests and odds ratios. One-way analysis of variance with F-ratio was carried out to determine the associations between genotypes and disease severity using CF scores. No major genetic variations between patients and controls were found in the three genes studied, and we did not find any association between these genes and CFS. In conclusion, variations in AMPD1, CPT2, and PGYM genes are not associated with the onset, susceptibility, or severity of CFS.

Chronic fatigue syndrome: Features of a population of patients from northern Italy.

In this study we analyzed the clinical features of a population of Italian patients with chronic fatigue syndrome (CFS) diagnosed according to the CDC-1994 criteria. The aim was to investigate CFS patients and their relatives, in order to search for events related to the onset of the disease and to identify correlations with other diseases. The analysis was carried out by examining medical records belonging to 82 patients suffering from the syndrome. The documentation was collected between 2008 and 2011 and provided by the non-profit Italian organization AMCFS (Associazione Malati di CFS). The influence of gender on the age of onset and association with potential risk factors were investigated in patients and in their relatives. From the results a significant correlation between the age of onset and autoimmunity was observed.

Mitochondrial alterations, oxidative stress and neuroinflammation in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The primary cause and sequence of its progression are only partially understood but abnormalities in folding and accumulation of insoluble proteins such as beta-amyloid and Tau-protein are both associated with the pathogenesis of AD. Mitochondria play a crucial role in cell survival and death, and changes in mitochondrial structure and/or function are related to many human diseases. Increasing evidence suggests that compromised mitochondrial function contributes to the aging process and thus may increase the risk of AD. Dysfunctional mitochondria contribute to reactive oxygen species which can lead to extensive macromolecule oxidative damage and the progression of amyloid pathology. Oxidative stress and amyloid toxicity leave neurons chemically vulnerable. The mitochondrial toxicity induced by beta-amyloid is still not clear but may include numerous mechanisms, such as the increased permeability of mitochondrial membranes, the disruption of calcium homeostasis, the alteration of oxidative phosphorylation with a consequent overproduction of reactive oxygen species. Other mechanisms have been associated with the pathophysiology of AD. Inflammatory changes are observed in AD brain overall, particularly at the amyloid deposits, which are rich in activated microglia. Once stimulated, the microglia release a wide variety of pro-inflammatory mediators including cytokines, complement components and free radicals, all of which potentially contribute to further neuronal dysfunction and eventually death. Clinically, novel approaches to visualize early neuroinflammation in the human brain are needed to improve the monitoring and control of therapeutic strategies that target inflammatory and other pathological mechanisms. Similarly, there is growing interest in developing agents that modulate mitochondrial function.

Chronic fatigue syndrome/myalgic encephalomyelitis: an update.

Chronic Fatigue Syndrome (CFS), also referred to as Myalgic Encephalomyelitis (ME), is a disease of unknown origin. It is classified as Post Viral Fatigue Syndrome (PVFS) in the WHO International Classification of Diseases (ICD) and listed as sub-category at G93.3 under chapter G93, other disorders of the brain. ME/CFS is primarily an endemic disorder but occurs in both epidemic and sporadic forms. It affects all racial-ethnic groups and is seen in all socioeconomic strata. A diagnosis of CFS is a diagnosis of exclusion, meaning other medical conditions, including psychiatric disorders, must be first ruled out. CFS is diagnosed if there is no other explanation for the fatigue and if the other symptoms did not develop before the fatigue. The estimated worldwide prevalence of CFS is 0.4?1 percent. The disease predominantly affects young adults, with a peak age of onset of between 20 and 40 years, and women, with a female to male ratio of 6:1. Mean illness duration ranges from 3 to 9 years. The patho-physiological mechanism of CFS is unclear but the immunological pattern of CFS patients gleaned from various studies indicates that the immune system is chronically activated. Besides the role of environmental insults (xenobiotics, infectious agents, stress) the genetic features of patients are studied to evaluate their role in triggering the pathology. At present there are no specific pharmacological therapies to treat the disease but a variety of therapeutic approaches have been described as benefiting patients. Treatment programs are directed at relief of symptoms, with the goal of the patient regaining some level of preexisting function and well-being.

[Acute abdomen in renal transplant recipients. Epidemiology and treatment in not referral transplantation centers]. / L'addome acuto nel paziente trapiantato renale. Considerazioni epidemiologiche, diagnostiche e terapeutiche in DEA non dedicati.

The incidence of gastrointestinal complications in renal transplant recipients is relatively high while about 10% is related to acute abdomen. Data concerning gastrointestinal (GI) complications were reported in literature mainly from referral center studies. A multicenter retrospectively survey was performed in Lazio, Italy, in order to evaluate the incidence of acute abdomen in renal transplant recipients observed to the emergency departments of not referral transplantation centers. Clinical and demographic findings regarding 14 patients who experienced acute abdomen between February 2005 and Dicember 2008 have been collected. The following data was investigated: etiology, diagnostic workup, duration of symptoms, elapsed time between admission and emergency operation if performed, morbility and mortality. The severity of disease at presentation was assessed by mean of the Acute Physiology and Chronic Health Evaluation score (APACHE II). Acute abdomen was due to pancreatitis in three patients (23.1%); to cholecystitis in three (23.1%); to acute diverticolitis with colon perforation in two patients (15.4%); to acute appendicitis in two (15.4%) and to intestinal obstruction in 2 patients (15.4%). Small bowel perforation was observed in two patients (15.4%) which one case, upon pathological examination, showed malignant lymphoma. The mean APACHE II score was 14.0 ± 5.9. Ten patients (71.4%) were submitted to surgery. Overall mortality and morbidity were 35% and 42% respectively. Statistical analysis showed admission APACHE II score (p<0.01), duration of symptoms (p<0.05), and total time elapsed between the onset of symptoms and treatment (p<0.04) as factors significantly related to mortality.

Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle.

Sarcocystis cruzi and Neospora caninum infections in cattle are common throughout the world, and cause important economical losses. N. caninum can be transmitted horizontally by ingestion of oocysts or vertically from the infected dam to the fetus via the placenta. Vertical transmission for S. cruzi is infrequent and horizontal transmission is considered the most important route of infection. The objectives of this study were to evaluate the frequency of horizontal and vertical transmission for S. cruzi and N. caninum in a dairy cattle herd and to analyze IFAT titers as predictors of vertical transmission. Serum samples (n = 173) were collected from dairy calves at birth prior to colostrum ingestion and from their dams. In addition, 12 calves were also sampled after ingestion of colostrum, 25 female calves were sampled at 7 months, and 81 of the dams were also sampled at breeding. Sera were evaluated for S. cruzi and N. caninum antibodies by IFAT starting at 1:25 dilution. For S. cruzi, vertical transmission frequency was 1.7%, and all female calves evaluated at 7 months and cows were seropositive. Seroprevalence for N. caninum was 80.9% in cows and 30% in precolostrum calves. Vertical transmission frequency was 37.1%. Cows with high antibody titers (> or = 400) showed higher vertical transmission frequency (94.8%) than cows with low antibody titers (between 25 and 200) (14.8%). Negative precolostrum calves (7/12) had postcolostrum N. caninum titers 2-8 times higher than their dams. Estimated horizontal transmission frequency was 51 and 47%, based on differences of seroprevalences in calves and dams, and on the seroconversion of 9/19 negative precolostrum female calves when retested at 7 months, respectively. Average N. caninum titers of cows at breeding and calving were 120.6 and 320.9, respectively. Cows with a high titer at breeding had a high titer at calving. Therefore, N. caninum IFAT titers at breeding and calving could potentially be used as predictors of vertical transmission.

Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats (Suricata suricatta) with fatal toxoplasmosis in Argentina.

In this study, the diagnosis of fatal disseminated toxoplasmosis in three captive slender-tailed meerkats (Suricata suricatta) in the zoo of La Plata, Argentina and the invitro isolation and molecular characterization of Toxoplasma gondii are reported. The animals showed depression, dyspnea and hypothermia, and also ataxia in one case, and died within 1-5 days. The main histopathological lesions included interstitial pneumonia, non-suppurative inflammatory changes and focal necrosis in liver, spleen, kidney and brain. Tachyzoites or tissue cysts were present in lung, liver, spleen, brain, striated muscle, kidney, intestine and mesenteric lymph node sections, and stained strongly with T. gondii antiserum in immunohistochemical analysis. T. gondii was isolated in Swiss mice and in bovine monocytes cultures from tissues of one of the meerkats. The isolate was cryopreserved and it was named TG-Suricata-1. T. gondii DNA was demonstrated in tissues of all three animals and in tachyzoites isolated in cell cultures. The PCR-RFLP analysis of markers based in the loci 3'-SAG2, 5'-SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico of T. gondii produced patterns corresponding to the clonal type III. Type III strains of T. gondii possess no or only little virulence in the mouse model, however their association with virulence in other animal species is uncertain. In the present case, T. gondii of the clonal lineage III was responsible for fatal cases in S. suricatta. To our knowledge, this is the first report of isolation and genotyping of T. gondii from S. suricatta.

Seroprevalence of Neospora caninum, Toxoplasma gondii and Sarcocystis sp. in llamas (Lama glama) from Jujuy, Argentina.

Llamas (Lama glama) are South American camelids described as intermediate hosts of Neospora caninum, Toxoplasma gondii and Sarcocystis aucheniae. Due to the potential role of these protozoan infections as a cause of economic losses, the aim of this study was to determine the seroprevalence for T. gondii, N. caninum and Sarcocystis sp. in llamas from Argentina. Serum samples from 308 llamas (>2 years old) were collected between 2005 and 2007. A total of 55 farms located in six departments of Jujuy province, Argentina were sampled. Presence of antibodies to N. caninum, T. gondii and Sarcocystis sp. was determined by the indirect fluorescent antibody test (IFAT). For Sarcocystis, 2 different bradyzoites-based antigens were prepared using S. aucheniae and S. cruzi. Sera were tested at dilutions 1:25 and 1:50. Antibodies to N. caninum were found in 4.6% serum samples. Fifty percent of departments and 14.5% of farms had positive animals. Antibodies to T. gondii were found in 30% of samples, distributed in 66% of departments and 43.6% of farms. Antibodies to Sarcocystis sp. were detected in 96% of samples and all departments and farms had positive animals, suggesting frequent contact between llamas and canids. Co-infection with N. caninum, T. gondii and Sarcocystis sp. was also recorded. Low seroprevalence of N. caninum in llamas detected in this study could be related to climatic and geographical conditions that limit cattle breeding activity, reducing the source of infection for definitive hosts. Seroprevalence of T. gondii and the positive animal distribution suggest frequent contamination of grass with felid faeces. In conclusion, this is the first report of combined seroprevalence for N. caninum, T. gondii and Sarcocystis sp. in llamas. Further studies are needed to determine the potential role of these protozoan infections as cause of abortion in Argentina as well as presence of these protozoans in llama meat used for human consumption.

Multiparametric ultrasound evaluation with CEUS and shear wave elastography for carotid plaque risk stratification.

PURPOSE: To assess the diagnostic effectiveness of Multiparametric ultrasound (MPUS), which includes color Doppler ultrasound (CDUS), CEUS and Shear wave elastography (SWE), for evaluating carotid plaque as compared with CT-angiography (CTA) and histology. MATERIALS AND METHODS: Forty-three consecutive patients scheduled to undergo carotid endarterectomy underwent MPUS. Then, after periods ranging from 2 days to 2 weeks, all underwent CTA. Each plaque was classified by means of dedicated scores for CEUS and SWE as compared with CTA features. At surgery, each plaque was removed in a single fragment to facilitate histological analysis, which evaluated 4 features: extension of the lipid core, thickness of the fibrous cap, inflammatory infiltrate (CD68 + and CD3 + markers) and the presence of intraplaque microvessels. For the CEUS, SWE and CTA, the following values for identifying plaque vulnerability were evaluated: sensitivity, specificity, accuracy, negative predictive value (NPV), positive predictive value (PPV) and Area under the curve (AUC). Cohen's kappa was used to evaluate the concordance between measurements in the different imaging methods. A p < 0.05 was considered statistically significant. RESULTS: At histology, 31 out of 43 plaques were identified as vulnerable because of the presence of at least one of the following criteria: fibrous cap < 200 µm, lipid core, intraplaque hemorrhage, inflammatory infiltrate or intraplaque neovascularization. CTA showed a sensitivity of 87.1%, a specificity of 100%, a PPV of 100%, an NPV of 75% and an AUC of 93.5%. SWE showed a sensitivity of 87.1%, a specificity of 66.7%, a PPV of 87.1%, an NPV of 66.7% and an AUC of 76.9%. CEUS showed a sensitivity of 87.1%, a specificity of 58.3%, a PPV of 84.4%, an NPV of 63.6% and an AUC of 72.7%. CONCLUSIONS: Multiparametric ultrasound is an effective modality to obtain comprehensive information on carotid plaques. Further studies are needed to determine whether it can be considered a diagnostic standard.

The transcriptional landscape of polyploid wheat.

The coordinated expression of highly related homoeologous genes in polyploid species underlies the phenotypes of many of the world's major crops. Here we combine extensive gene expression datasets to produce a comprehensive, genome-wide analysis of homoeolog expression patterns in hexaploid bread wheat. Bias in homoeolog expression varies between tissues, with ~30% of wheat homoeologs showing nonbalanced expression. We found expression asymmetries along wheat chromosomes, with homoeologs showing the largest inter-tissue, inter-cultivar, and coding sequence variation, most often located in high-recombination distal ends of chromosomes. These transcriptionally dynamic genes potentially represent the first steps toward neo- or subfunctionalization of wheat homoeologs. Coexpression networks reveal extensive coordination of homoeologs throughout development and, alongside a detailed expression atlas, provide a framework to target candidate genes underpinning agronomic traits in wheat.

Toxoplasmosis in captive Bennett's wallabies (Macropus rufogriseus) in Argentina.

Wallabies and other Australian marsupials are among the most susceptible species to Toxoplasma gondii. Fatal generalized toxoplasmosis was diagnosed in two captive 3 year-old female Bennett's wallabies (Macropus rufogriseus) from Argentina (w 1 and w 2) with a history of sudden death. Both animals had internal joeys which died 2 days after their mothers. Serologically, both females and one adult male without clinical signs from the same enclosure (w 3) had antibody titers for T. gondii>or=800 by the modified agglutination test (MAT); another adult male (w 4) was negative (MAT titer<25). Microscopically, tachyzoites were observed associated to non-suppurative meningoencephalitis, hepatitis, myositis, myocarditis and severe enteritis in hematoxylin and eosin stained sections from both w 1 and w 2. Immunohistochemically, parasites in heart, brain and liver sections of both female wallabies reacted with T. gondii antiserum. T. gondii was isolated from brain tissues of w 1 and w 2 by bioassay in mice and by culture in bovine monocytes and both isolates were cryopreserved. Genomic DNA was isolated from tachyzoites grown in cultures derived from both animals. The primer pair B22/B23 specific for T. gondii produced 115bp amplicons on poliacrylamide electrophoretic gels. Stray cats were suspected as the possible source of infection. Not all infected macropods were ill, showing that the infection may be asymptomatic and is not always fatal. A vertical infection could not be proved in the joey from w 2. As far as we know, this is the first confirmed report of toxoplasmosis in Bennet's wallabies in Argentina.

What is the role of contrast-enhanced ultrasound in the evaluation of the endoleak of aortic endoprostheses? A comparison between CEUS and CT on a widespread scale.

OBJECTIVE: To evaluate the accuracy of contrast-enhanced ultrasound (CEUS) in comparison to CT angiography (CTA) to identify and classify endoleaks following abdominal aortic aneurism repair with endoprosthesis. MATERIALS AND METHODS: A retrospective analysis of 181 patients treated with EVAR, from September 2009 to September 2014, was performed. Patients were evaluated with CEUS, CTA and angiography in the cases requiring treatment. Sac diameter, sac integrity, identification and classification of endoleaks were taken into consideration. Sensitivity, specificity, accuracy and negative predictive values were considered for each modality of endoleak identification. RESULTS: Forty-two endoleaks (23.2%; type II: 39 cases, type III: 3 cases) were documented. Sensitivity and specificity of CEUS and CT were, respectively, 97.6 and 90.5, 100 and 100%. In two cases, CEUS was able to better classify endoleaks compared to CT. CONCLUSIONS: CEUS accuracy to identify endoleaks following EVAR is similar to CT. CEUS should be considered as an effective modality for the long-term surveillance of EVAR because of its capability to correctly classify endoleaks with no ionizing radiation exposure.

Transcriptional induction of the PML growth suppressor gene by interferons is mediated through an ISRE and a GAS element.

PML is a nuclear matrix protein with growth suppressing properties, whose expression is deregulated during oncogenesis. Moreover, in the t(15;17) translocation of acute promyelocytic leukaemia (APL), PML fusion to the retinoic acid receptor alpha (RAR alpha) is the likely molecular basis of leukaemogenesis. Here we show that interferons (IFNs) alpha, beta, and gamma upregulate PML mRNA expression. Analysis of 5' genomic sequences of the PML gene revealed an IFN-alpha/-beta stimulated response element (ISRE) and an IFN-gamma activation site (GAS) in the untranslated first exon. Binding of IFN signal transducers and activators of transcription (STATs) was demonstrated to be weak for the PML GAS, but strong for the PML ISRE which also seemed to contribute substantially to the IFN-gamma response. Thus, PML is a primary target gene of IFNs and would appear as a suitable candidate for mediating some of their antiproliferative effects. Abnormalities of PML structure, localisation or expression in human malignancy, constitute examples of how an IFN target gene may be altered in oncogenesis.

Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms.

The acute promyelocytic leukaemia (APL)-specific chromosome 15;17 translocation leads to the fusion of a newly identified putative transcription factor, PML, and the retinoic acid receptor alpha. We have characterized the structure of the PML genomic locus and preliminarily characterized its expression pattern. The PML locus spans a minimum of 35 kb and is subdivided into nine exons. The putative PML DNA binding site is encoded by exons 2 and 3. We isolated a large number of alternatively spliced PML transcripts that encode numerous PML isoforms. Two groups of isoforms were identified that differed either in their C-terminal region or in the length of their central region, but retained the putative DNA-binding and dimerization domains. RNAase protection experiments revealed that the different PML isoforms are equally expressed in established cell lines of different histological origin.

TIF1gamma, a novel member of the transcriptional intermediary factor 1 family.

We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1gamma. Similar to TIF1alpha and TIF1beta, the structure of TIF1beta is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1alpha and TIF1beta, TIF1gamma presents several functional differences. In contrast to TIF1alpha, but like TIF1beta, TIF1 does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1alpha and TIF1beta were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1alpha, MODI (HP1beta) and MOD2 (HP1gamma) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1gamma does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1gamma, like TIF1alpha and TIF1beta, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1gamma, this motif likely participates in TIF1 dependent repression.

In vitro treatment with retinoids or the topoisomerase inhibitor, VP-16, evidences different functional apoptotic pathways in acute promyelocytic leukemic cells.

Understanding the mechanisms inherent to malignant cell eradication is a major determinant for cancer therapy. Recent data have demonstrated that apoptosis may be one of the mechanisms through which both cytotoxic and differentiating drugs may eliminate malignant cells. Treatment of acute promyelocytic leukemia (APL) by all-trans retinoic acid (ATRA) is the first model of differentiation therapy allowing achievement of more than 90% complete remission (CR). However, disease-free survival (DFS) is short if patients are not subsequently treated with chemotherapy. In order to address the question of APL cells' elimination during ATRA therapy, we studied phenotypic and molecular features of 14 APL cases relative to cell survival in primary culture in the presence or absence of ATRA. Compared to other acute myeloid leukemia (AML) subtypes, APL cells in short-term suspension culture present a better survival rate (P < 0.001). After incubation with ATRA, cell survival was not altered and was correlated with a concomitant absence of apoptosis, despite a significant decrease of the BcL-2 protein in APL differentiated cells. Indeed, after 6 days of culture, only 3 +/- 0.5% of APL cells exhibit morphological features of apoptosis after ATRA treatment compared to 30 +/- 5% in HL-60-treated cells. Treatment of APL cells with 9-cis RA, 13-cis RA or analogs of RAR alpha or RXR alpha also failed to induce apoptosis. Treatment of either APL or ATRA-differentiated APL cells with 40 microM etoposide resulted in DNA fragmentation and morphological changes characteristic of apoptosis in 23 +/- 5% cells after only 20 h of treatment and 68 +2- 3% after 48 h suggesting that other pathways of apoptosis are still functional in APL cells. Though these in vitro data cannot fully represent the mechanism of cell death and cell elimination in vivo, they clearly indicate that ATRA alone may not induce leukemic clone eradication by apoptosis correlating with the persistence of minimal residual disease and constant relapse after CR obtained with ATRA alone.

Induction of the PML protein by interferons in normal and APL cells.

PML has been identified through its fusion to the RAR alpha gene in acute promyelocytic leukemia (APL). The PML protein is specifically associated to nuclear bodies (NBs) whose alterations in APL were proposed to contribute to leukemogenesis. The role of this nuclear domain (which also harbors the Sp100 autoantigen and the NDP52 protein) is unknown. Here, we show that the PML protein, like Sp100 and NDP52, is induced by interferons (IFNs alpha, beta and gamma) in a large variety of human cells. Interestingly, the NBs that contain the three IFN-induced proteins appear to be associated to speckles labelled by the IFN-mediator Mx1. These observations link NBs to IFN response pathways, which may contribute to the elucidation of the biological role of these structures. In APL cells, IFNs induced both PML and PML/RAR alpha expression, resulting in an increased sequestration of PML and RXRs in the microspeckles induced by the fusion protein. As PML has growth suppressing properties, it may mediate some of the antiproliferative effects of IFN. In APL, inactivation of PML may result in disruption of growth control.