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This research aims to create a three-dimensional (3D) organoid model of the spinal cord and skeletal muscle from stem cells for the investigation and potential treatment of neuromuscular diseases, specifically spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The focus is on understanding comparative and different pathological features and disease mechanisms, ultimately to evaluate therapeutic strategies. SMA and ALS are severe neurological disorders marked by the gradual deterioration of motor neurons. Establishing robust human models is crucial for enhancing our knowledge of and identifying therapeutic approaches for these neuromuscular disorders, which are known for causing paralysis and are attributed to disruptions in the complex network that controls voluntary movement. We created organoids representing the human spinal cord and muscle tissue using induced pluripotent stem cells (iPSCs) taken from patients with SMA, ALS C9ORF72 (C9) mutations, and non-affected individuals. Our methods of analysis included comprehensive transcriptomics, single-cell RNA sequencing, multielectrode array assessments, and immunophenotyping. As regards pharmacological intervention, SMA organoids were treated with a Risdiplam-like compound while C9 organoids with an antisense oligonucleotide with morpholino chemistry with proved efficacy in the murine counterpart of the C9 mouse model as C9-derived dipeptide modulator. So far, our organoid models for SMA and C9-ALS displayed significant differences in cellular and molecular development compared to controls, involving various cell types and extending beyond motor neurons, indicating a developmental aspect to both diseases. Specifically, for SMA organoids, treatment with the Risdiplam-like compound resulted an efficient correction of the proportion of full-length to truncated SMN2 protein accompanied by a modulation of about 15% of the genes, showcasing significant in vitro durability. Importantly, this treatment corrected and reversed multiple pathological indicators within the SMA organoids. The organoids proved effective for studying drug interactions and the impact of therapies, highlighting the profound and early developmental influence of SMA and C9ALS. Our results promote the potential of Risdiplam-like therapies in treating extensive facets of SMA pathology and emphasize the need of deeply understanding the pharmacodynamics involved. This research advances the prospect of refining therapeutic approaches and suggests avenues for developing treatments for patients with neuromuscular conditions. [ABSTRACT FROM AUTHOR]

Background: Although invasiveness is one of the major determinants of the poor glioblastoma (GBM) outcome, the mechanisms of GBM invasion are only partially understood. Among the intrinsic and environmental processes promoting cell-to-cell interaction processes, eventually driving GBM invasion, we focused on the pro-invasive role played by Extracellular Vesicles (EVs), a heterogeneous group of cell-released membranous structures containing various bioactive cargoes, which can be transferred from donor to recipient cells., Methods: EVs isolated from patient-derived GBM cell lines and surgical aspirates were assessed for their pro-migratory competence by spheroid migration assays, calcium imaging, and PYK-2/FAK phosphorylation. Brain invasiveness was investigated in human cortical organoids-based assembloids and in vivo orthotopic xenografts. EV molecular features were specified by multiplex bead-based flow cytometry., Results: Results unveil a self-sustaining mechanism triggering migration through autocrine release and engagement of a specific population of EVs of large size (L-EVs), isolated from either patient-derived cell lines or surgical aspirates. L-EVs act through modulation of calcium transients via Connexin 43-Gap Junctions (Cx43-GJ) and phospho-activation of PYK2. Pre-incubation with blocking antibodies targeting Cx43 hemichannels demonstrated a dose-dependent inhibition of the L-EV-mediated GBM migration. By exploiting patients' surgical aspirates, we show that only L-EVs deriving from tumoral cells, and not those with immune origin, promote tumor migration, impacting more prominently the tumoral cells with mesenchymal subtype., Conclusions: We demonstrate that L-EVs released by GBM cells, but not by the immune cells of the tumor microenvironment, represent a relevant and unique autocrine pro-migratory input for the tumor., (© The Author(s) 2025. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Triggering receptor expressed on myeloid cells 2 (Trem2) is a myeloid cell-specific gene expressed in brain microglia, with variants that are associated with neurodegenerative diseases, including Alzheimer's disease. Trem2 is essential for microglia-mediated synaptic refinement, but whether Trem2 contributes to shaping neuronal development remains unclear. Here, we demonstrate that Trem2 plays a key role in controlling the bioenergetic profile of pyramidal neurons during development. In the absence of Trem2, developing neurons in the hippocampal cornus ammonis (CA)1 but not in CA3 subfield displayed compromised energetic metabolism, accompanied by reduced mitochondrial mass and abnormal organelle ultrastructure. This was paralleled by the transcriptional rearrangement of hippocampal pyramidal neurons at birth, with a pervasive alteration of metabolic, oxidative phosphorylation, and mitochondrial gene signatures, accompanied by a delay in the maturation of CA1 neurons. Our results unveil a role of Trem2 in controlling neuronal development by regulating the metabolic fitness of neurons in a region-specific manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations., (© 2023. The Author(s).)

Mass spectrometry imaging (MSI) is commonly used to map the spatial distribution of small molecules within complex biological matrices. One of the major challenges in imaging MS-based spatial metabolomics is molecular identification and metabolite annotation, to address this limitation, annotation is often complemented with parallel bulk LC-MS2-based metabolomics to confirm and validate identifications. Here we applied MSI method, utilizing data-dependent acquisition, to visualize and identify unknown molecules in a single instrument run. To reach this aim we developed MSIpixel, a fully automated pipeline for compound annotation and quantitation in MSI experiments. It overcomes challenges in molecular identification, and improving reliability and comprehensiveness in MSI-based spatial metabolomics., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mammalian neocortical neurons span one of the most diverse cell type spectra of any tissue. Cortical neurons are born during embryonic development, and their maturation extends into postnatal life. The regulatory strategies underlying progressive neuronal development and maturation remain unclear. Here we present an integrated single-cell epigenomic and transcriptional analysis of individual mouse and marmoset cortical neuron classes, spanning both early postmitotic stages of identity acquisition and later stages of neuronal plasticity and circuit integration. We found that, in both species, the regulatory strategies controlling early and late stages of pan-neuronal development diverge. Early postmitotic neurons use more widely shared and evolutionarily conserved molecular regulatory programs. In contrast, programs active during later neuronal maturation are more brain- and neuron-specific and more evolutionarily divergent. Our work uncovers a temporal shift in regulatory choices during neuronal diversification and maturation in both mice and marmosets, which likely reflects unique evolutionary constraints on distinct events of neuronal development in the neocortex., (© 2022. The Author(s).)

Decoding the complexity of the brain requires an understanding of the architecture, function, and development of its neuronal circuits. Neuronal classifications that group neurons based on specific features/behaviors have become essential to further analyze the different subtypes in a systematic and reproducible way. A comprehensive taxonomic framework, accounting for multiple defining and quantitative features, will provide the reference to infer generalized rules for cells ascribed to the same neuronal type, and eventually predict cellular behaviors, even in the absence of experimental measures. Technologies that enable cell-type classification in the nervous system are rapidly evolving in scalability and resolution. While these approaches depict astonishing diversity in neuronal morphology, electrophysiology, and gene expression, a robust metric of the coherence between different profiling modalities leading to a unified classification is still largely missing. Focusing on GABAergic neurons of the cerebral cortex, Gouwens et al . 1 pioneered the first integrated cell-type classification based on the simultaneous analysis of the transcriptional networks, the recording of intrinsic electrophysiological properties, and the reconstruction of 3D morphologies of the same cell. Their comprehensive and high-quality data provide a new framework to shed light on what may be considered a "neuronal cell type.", Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2022 Faculty Opinions Ltd.)

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Up to 40% of patients with inflammatory bowel disease present with psychosocial disturbances. We previously identified a gut vascular barrier that controls the dissemination of bacteria from the intestine to the liver. Here, we describe a vascular barrier in the brain choroid plexus (PVB) that is modulated in response to intestinal inflammation through bacteria-derived lipopolysaccharide. The inflammatory response induces PVB closure after gut vascular barrier opening by the up-regulation of the wingless-type, catenin-beta 1 (Wnt/β-catenin) signaling pathway, rendering it inaccessible to large molecules. In a model of genetically driven closure of choroid plexus endothelial cells, we observed a deficit in short-term memory and anxiety-like behavior, suggesting that PVB closure may correlate with mental deficits. Inflammatory bowel disease–related mental symptoms may thus be the consequence of a deregulated gut–brain vascular axis.

Background: Preterm birth affects almost 9-11% of newborns and is one of the leading causes of childhood neurodevelopmental disabilities; the underlying molecular networks are poorly defined. In neurons, retrotransposons LINE-1 (L1) are an active source of genomic mosaicism that is deregulated in several neurological disorders; early life experience has been shown to regulate L1 activity in mice., Methods: Very preterm infants were randomized to receive standard care or early intervention. L1 methylation was measured at birth and at hospital discharge. At 12 and 36 months, infants' neurodevelopment was evaluated with the Griffiths Scales. L1 methylation and CNVs were measured in mouse brain areas at embryonic and postnatal stages., Results: Here we report that L1 promoter is hypomethylated in preterm infants at birth and that an early intervention program, based on enhanced maternal care and positive multisensory stimulation, restores L1 methylation levels comparable to healthy newborns and ameliorates neurodevelopment in childhood. We further show that L1 activity is fine-tuned in the perinatal mouse brain, suggesting a sensitive and vulnerable window for the L1 epigenetic setting., Conclusions: Our results open the field on the inspection of L1 activity as a novel molecular and predictive approach to infants' prematurity-related neurodevelopmental outcomes., Trial Registration: ClinicalTrial.gov ( NCT02983513 ). Registered on 6 December 2016, retrospectively registered.

The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism., (Copyright © 2020, American Association for the Advancement of Science.)

The neocortex is by far one of the most complex regions of the mammalian brain, characterized by an extraordinary diversity of neuronal and non-neuronal cell types, whose coordinated development and function guarantee the execution of high-order cognitive, sensory, and motor behaviours. Decoding its heterogeneity and understanding the molecular strategies upon which the cerebral cortex is built during development have been at the core of neuroscientists' work for decades. Here, we will focus on the current classification of neuronal types (both excitatory and inhibitory) of the neocortex in light of the insights provided by recent single-cell omic technologies, which have offered - with unprecedented resolution - an extended framework to interpret cortical diversity and its developmental origin. We will cover the impact of neuronal subtype identity on generating specific neuronal networks (neuron-to-neuron interaction), as well as their effect on the development of the non-neuronal populations in the cerebral cortex., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: Terra dei Fuochi (TdF), the so-called 'Land of Fires' in Southern Italy, is an agricultural territory characterized by illegal dumping of toxic waste known to occur since the 1980s. It is unknown whether prognosis of patients developing cancer and living in that area may differ compared to those living in areas not exposed to this specific type of pollution. We retrospectively analyzed the 5-year survival rates of patients originating from the TdF diagnosed with lung cancer compared to patients from other areas., Materials and Methods: Patients consecutively operated on for non-small cell lung cancer (NSCLC) between November 2004 and April 2013 at the Division of Thoracic Surgery of the National Cancer Institute of Naples were eligible. The study outcome was overall survival (OS). In addition, the TdF and non-TdF groups were compared through propensity score matching (PSM)., Results: Overall, 439 patients with resectable NSCLC were operated on, 123 (28%) from the TdF and 316 (72%) from other referral centers of our catchment area. There were 301 males and 138 females; the median age of the entire surgical population was 65 years (range=25-83) years. Apart from a different prevalence of hypertension and underweight patients, preoperative factors were evenly distributed between the two groups. At univariate analysis, OS was not different between the TdF and non TdF group (median 72 and 68 months, respectively; p=0.75 log-rank test). Multivariable analysis confirmed that living in the TdF area had no prognostic impact (hazard ratio=1.05; 95% confidence interval=0.70-1.57; p=0.78) on OS. PSM confirmed no statistically significant difference of OS (hazard ratio=1.01, 95% confidence interval=0.67-1.52; p=0.93)., Conclusion: Following surgery for lung cancer, TdF and non-TdF surgical candidates had similar long-term survival. Originating from the TdF does not seem to be associated with worse outcomes after surgical treatment of patients with lung cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The mammalian cerebral cortex is responsible for the highest levels of associative, cognitive and motor functions. In the central nervous system (CNS) the cortex stands as a prime example of extreme neuronal diversity, broadly classified into excitatory projection neurons (PNs) and inhibitory interneurons (INs). We review here recent progress made in understanding the strategies and mechanisms that shape PN diversity during embryogenesis, and discuss how PN classes may be maintained, postnatally, for the life of the organism. In addition, we consider the intriguing possibility that PNs may be amenable to directed reprogramming of their class-specific features to allow enhanced cortical plasticity in the adult., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The neocortex is the part of the brain responsible for execution of higher-order brain functions, including cognition, sensory perception, and sophisticated motor control. During evolution, the neocortex has developed an unparalleled neuronal diversity, which still remains partly unclassified and unmapped at the functional level. Here, we broadly review the structural blueprint of the neocortex and discuss the current classification of its neuronal diversity. We then cover the principles and mechanisms that build neuronal diversity during cortical development and consider the impact of neuronal class-specific identity in shaping cortical connectivity and function.

The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype-specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.

Background: Metabolic Syndrome (MS) has been correlated to breast carcinogenesis. MS is common in the general population (34%) and increases with age and body mass index. Although the link between obesity, MS and hormone related cancer incidence is now widely recognized, the molecular mechanisms at the basis of such increase are still poorly characterized. A crucial role is supposed to be played by the altered insulin signalling, occurring in obese patients, which fuels cancer cell growth, proliferation and survival. Therefore we focused specifically on insulin resistance to investigate clinically the potential role of insulin in breast carcinogenesis., Methods: 975 patients were enrolled and the association between MS, insulin resistance, and breast cancer was evaluated. Women were stratified by age and menopausal status. Insulin resistance was measured through the Homeostasis Model Assessment score (HOMA-IR). The cut off value to define insulin resistance was HOMA-IR ≥ 2.50., Results: Higher prevalence of MS (35%) was found among postmenopausal women with breast cancer compared to postmenopausal healthy women (19%) [OR 2.16]. A broad range of BMI spanning 19-48 Kg/m2 was calculated. Both cases and controls were characterized by BMI ≥ 25 Kg/m2 (58% of cases compared to 61% of controls). Waist circumference >88 cm was measured in 53% of cases - OR 1.58- (95% CI 0.8-2.8) and in 46% of controls. Hyperinsulinemia was detected in 7% of cases - OR 2.14 (95% CI 1.78-2.99) and only in 3% of controls. HOMA-IR score was elevated in 49% of cases compared to 34% of controls [OR 1.86], suggesting that insulin resistance can nearly double the risk of breast cancer development. Interestingly 61% of women operated for breast cancer (cases) with HOMA-IR ≥ 2.5 presented subclinical insulin resistance with fasting plasma glucose levels and fasting plasma insulin levels in the normal range. Both android fat distribution and insulin resistance correlated to MS in the subgroup of postmenopausal women affected by breast cancer., Conclusions: Our results further support the hypothesis that MS, in particular insulin resistance and abdominal fat, can be considered as risk factors for developing breast cancer after menopause. We suggest that HOMA-IR, rather than fasting plasma glucose and fasting plasma insulin levels alone, could be a valuable tool to identify patients with subclinical insulin resistance, which could be relevant for primary prevention and for high risk patient screening.

Cortical and striatal interneurons are both generated within the ventral telencephalon, but their migratory journey takes them to very different destinations. Two articles in this issue (van den Berge et al., 2013; McKinsey et al., 2013) add an important molecular component to our understanding of how, during development, interneurons reach the cerebral cortex., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In the mammalian cerebral cortex, the developmental events governing the integration of excitatory projection neurons and inhibitory interneurons into balanced local circuitry are poorly understood. We report that different subtypes of projection neurons uniquely and differentially determine the laminar distribution of cortical interneurons. We find that in Fezf2⁻/⁻ cortex, the exclusive absence of subcerebral projection neurons and their replacement by callosal projection neurons cause distinctly abnormal lamination of interneurons and altered GABAergic inhibition. In addition, experimental generation of either corticofugal neurons or callosal neurons below the cortex is sufficient to recruit cortical interneurons to these ectopic locations. Strikingly, the identity of the projection neurons generated, rather than strictly their birthdate, determines the specific types of interneurons recruited. These data demonstrate that in the neocortex individual populations of projection neurons cell-extrinsically control the laminar fate of interneurons and the assembly of local inhibitory circuitry., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator-like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells.

Transcription factors with gradients of expression in neocortical progenitors give rise to distinct motor and sensory cortical areas by controlling the area-specific differentiation of distinct neuronal subtypes. However, the molecular mechanisms underlying this area-restricted control are still unclear. Here, we show that COUP-TFI controls the timing of birth and specification of corticospinal motor neurons (CSMN) in somatosensory cortex via repression of a CSMN differentiation program. Loss of COUP-TFI function causes an area-specific premature generation of neurons with cardinal features of CSMN, which project to subcerebral structures, including the spinal cord. Concurrently, genuine CSMN differentiate imprecisely and do not project beyond the pons, together resulting in impaired skilled motor function in adult mice with cortical COUP-TFI loss-of-function. Our findings indicate that COUP-TFI exerts critical areal and temporal control over the precise differentiation of CSMN during corticogenesis, thereby enabling the area-specific functional features of motor and sensory areas to arise.

The idea of repairing damaged neuronal circuitry in the mammalian central nervous system (CNS) has challenged neuroscientists for centuries. This is mainly due to the notorious inability of neurons to regenerate and the unparalleled cellular diversity of the nervous system. In the mammalian cerebral cortex, one of the most complex areas of the CNS, multipotent neural stem and progenitor cells undergo progressive specification during development to generate the staggering variety of projection neuron subtypes that are found in the adult. How is this process orchestrated in the embryo? And, can developmental signals be used to regenerate projection neuron subtypes in the adult or in the dish? Here, we first provide an overview of the diversity and fate potential of neural progenitors of the cerebral cortex during development. Further, we discuss the plasticity of neural progenitors and the roles of intrinsic and extrinsic signals over progenitor fate. Finally, we discuss the relevance of developmental signals for efforts to direct the differentiation of pluripotent stem cells into specific types of cortical projection neurons for therapeutic benefit.

Background and Aim: To assess the validity of hospital discharge diagnoses (HDDs) as a means for the surveillance of diabetes-related lower extremity amputations on a population basis, and to compare the demographic and clinical characteristics of diabetic and non-diabetic amputees., Methods and Results: All of the 1996 hospital discharge diagnoses reporting ICD-9-CM codes 84.10-84.19 were reviewed in the Campania Region, an area in Southern Italy with 5.7 million inhabitants. Diabetes was defined as any concomitant 250 ICD-9 code. The completeness of ascertainment and accuracy in reporting diabetes were validated in a sub-sample of cases by matching their HDDs with the register of buried limbs and the clinical records of the hospital. During the study period, 701 individuals underwent 738 amputations, 330 (47.1%) of whom had diabetes mentioned in their discharge diagnosis. All of the amputations reported in the buried limbs register were identified by the HDDs, whereas some minor amputations recorded in the HDDs were not reported in the registers. On the basis of the clinical charts, the diagnosis of diabetes was underreported by 10%. The proportion of females was significantly higher among the diabetic amputees (41% vs 35%), whereas the proportion of people aged less than 50 years was significantly lower (5% vs 13%) (p < 0.002). The duration of hospitalisation was similar for the diabetics and non-diabetics (23 +/- 20 vs 22 +/- 18 days). Minor amputations were significantly more frequent in the individuals with diabetes (38.8% vs 29.1%; p < 0.001), as was reamputation (7.2% vs 2.9%; p < 0.01)., Conclusion: These findings confirm the markedly increased risk of amputation among diabetics and demonstrate that HDDs represent a suitable procedure for the surveillance of lower extremity amputations.