Pilot Study by Liquid Biopsy in Gastrointestinal Stromal Tumors: Analysis of PDGFRA D842V Mutation and Hypermethylation of SEPT9 Presence by Digital Droplet PCR.

Tissue biopsy remains the standard for diagnosing gastrointestinal stromal tumors (GISTs), although liquid biopsy is emerging as a promising alternative in oncology. In this pilot study, we advocate for droplet digital PCR (ddPCR) to diagnose GIST in tissue samples and explore its potential for early diagnosis via liquid biopsy, focusing on the PDGFRA D842V mutation and SEPT9 hypermethylated gene. We utilized ddPCR to analyze the predominant PDGFRA mutation (D842V) in surgical tissue samples from 15 GIST patients, correlating with pathologists' diagnoses. We expanded our analysis to plasma samples to compare DNA alterations between tumor tissue and plasma, also investigating SEPT9 gene hypermethylation. We successfully detected the PDGFRA D842V mutation in GIST tissues by ddPCR. Despite various protocols to enhance mutation detection in early-stage disease, it remained challenging, likely due to the low concentration of DNA in plasma samples. Additionally, the results of Area Under the Curve (AUC) for the hypermethylated SEPT9 gene, analyzing concentration, ratio, and abundance were 0.74 (95% Confidence Interval (CI): 0.52 to 0.97), 0.77 (95% CI: 0.56 to 0.98), and 0.79 (95% CI: 0.59 to 0.99), respectively. As a rare disease, the early detection of GIST through such biomarkers is particularly crucial, offering significant potential to improve patient outcomes.

Therapeutic effect of adipose-derived mesenchymal stem cells in a porcine model of abdominal sepsis.

BACKGROUND: The term sepsis refers to a complex and heterogeneous syndrome. Although great progress has been made in improving the diagnosis and treatment of this condition, it continues to have a huge impact on morbidity and mortality worldwide. Mesenchymal stem cells are a population of multipotent cells that have immunomodulatory properties, anti-apoptotic effects, and antimicrobial activity. We studied these capacities in a porcine model of peritoneal sepsis. METHODS: We infused human adipose-derived mesenchymal stem cells (ADSCs) into a porcine model of peritoneal sepsis. Twenty piglets were treated with antibiotics alone (control group) or antibiotics plus peritoneal infusion of ADSCs at a concentration of 2 × 106 cells/kg or 4 × 106 cells/kg (low- and high-dose experimental groups, respectively). The animals were evaluated at different time points to determine their clinical status, biochemical and hematologic parameters, presence of inflammatory cytokines and chemokines in blood and peritoneal fluid, and finally by histologic analysis of the organs of the peritoneal cavity. RESULTS: One day after sepsis induction, all animals presented peritonitis with bacterial infection as well as elevated C-reactive protein, haptoglobin, IL-1Ra, IL-6, and IL-1b. Xenogeneic ADSC infusion did not elicit an immune response, and peritoneal administration of the treatment was safe and feasible. One day after infusion, the two experimental groups showed a superior physical condition (e.g., mobility, feeding) and a significant increase of IL-10 and TGF-ß in blood and a decrease of IL-1Ra, IL-1b, and IL-6. After 7 days, all animals treated with ADSCs had better results concerning blood biomarkers, and histopathological analysis revealed a lower degree of inflammatory cell infiltration of the organs of the peritoneal cavity. CONCLUSIONS: Intraperitoneal administration of ADSCs as an adjuvant therapy for sepsis improves the outcome and diminishes the effects of peritonitis and associated organ damage by regulating the immune system and reducing intra-abdominal adhesions in a clinically relevant porcine model of abdominal sepsis.

Prevalence of suicidality in children and adolescents with depressive disorders with and without epilepsy.

OBJECTIVE: Children with epilepsy (CWE) are at risk for a range of adverse emotional, behavioral, and social outcomes. Approximately one-third of CWE experience depressive disorders, and up to 20% of children and adolescents with epilepsy may experience suicidality, suggesting that epilepsy increases the risk for suicidality among children and adolescents with depressive disorders. Consequently, the goal of the present study is to compare rates of suicidality in children and adolescents diagnosed with depressive disorders with or without co-morbid epilepsy. PARTICIPANTS AND METHODS: A retrospective chart review was conducted for 100 pediatric patients with a history of both seizures and depressive disorders and 100 patients with a history of depressive disorders only. Cases were coded for depression diagnosis, suicidality, suicidal ideation, suicide attempts, psychiatric hospitalizations, and self-injury. The distributions of these variables for the two groups were compared. RESULTS: The age and sex distributions of the two groups were comparable. Patients with co-morbid depressive disorders and epilepsy found a high rate of suicidal ideation (69%) but did not differ from those with depressive disorders without epilepsy on any of the suicidality variables (all p > 0.20), with the exception of self-injury, which was higher in those without epilepsy. CONCLUSIONS: CWE and co-morbid depression are at significant risk for suicidality, including ideation, attempts, and hospitalizations, but at rates that are comparable to those with depressive disorders without seizures. However, patients with co-morbid epilepsy are less likely to engage in other self-injurious behaviors. These findings support the need for careful monitoring of the psychiatric status of children and adolescents with epilepsy.

Culturally Informed Neuropsychological Evaluations in Pediatric Epilepsy: Evidence-Based Practice Considerations.

OBJECTIVE: Epilepsy is one of the most common reasons for referral for a pediatric neuropsychological evaluation due its high prevalence in childhood and our well-established clinical role in tertiary care settings. Emerging evidence indicates that racial and ethnic minority populations experience increased epilepsy burden compared with White peers. Although there has been heightened recognition in our specialty regarding the dire need for culturally and linguistically responsive evaluations, the scientific evidence to support effective neuropsychological service delivery for bi/multilingual and bi/multicultural youth with epilepsy is comparatively scant and of poor quality. As a result, significant patient and clinical challenges exist, particularly in high stakes presurgical pediatric epilepsy evaluations of bi/multilingual and bi/multicultural children. METHOD: Given that Spanish is the most common language spoken in the United States after English, this paper will focus on Spanish and English measures, but will provide evidence-based practice considerations that can inform practices with other non-English speaking communities. Cultural and linguistic factors that affect clinical decision-making regarding test selection, test interpretation, and feedback with families are highlighted. RESULTS: We offer a review of neuropsychological profiles associated with pediatric epilepsy as well as a flexible, multimodal approach for the assessment of linguistically and culturally diverse children with epilepsy based on empirical evidence and the clinical experiences of pediatric neuropsychologists from diverse backgrounds who work with children with epilepsy. CONCLUSION: Limitations to this approach are discussed, including the lack of available measures and resources for culturally and linguistically diverse pediatric populations. A case illustration highlights a culturally informed assessment approach.

Long-term neuropsychological outcomes in children with febrile infection-related epilepsy syndrome (FIRES) treated with anakinra.

Background: Febrile-infection related epilepsy syndrome (FIRES) is a rare epilepsy syndrome in which a previously healthy individual develops refractory status epilepticus in the setting of a preceding febrile illness. There are limited data regarding detailed long-term outcomes. This study aims to describe the long-term neuropsychological outcomes in a series of pediatric patients with FIRES. Methods: This is a retrospective multi-center case series of pediatric patients with a diagnosis of FIRES treated acutely with anakinra who had neuropsychological testing at least 12 months after status epilepticus onset. Each patient underwent comprehensive neuropsychological evaluation as part of routine clinical care. Additional data collection included the acute seizure presentation, medication exposures, and outcomes. Results: There were six patients identified with a median age of 11.08 years (IQR: 8.19-11.23) at status epilepticus onset. Anakinra initiation was a median of 11 days (IQR: 9.25-13.50) after hospital admission. All patients had ongoing seizures and none of the patients returned to baseline cognitive function with a median follow-up of 40 months (IQR 35-51). Of the five patients with serial full-scale IQ testing, three demonstrated a decline in scores over time. Testing results revealed a diffuse pattern of deficits across domains and all patients required special education and/or accommodations for academic learning. Conclusions: Despite treatment with anakinra, neuropsychological outcomes in this series of pediatric patients with FIRES demonstrated ongoing diffuse neurocognitive impairment. Future research will need to explore the predictors of long-term neurocognitive outcomes in patients with FIRES and to evaluate if acute treatment interventions improve these outcomes.

Study of the Effect of Wild-Type and Transiently Expressing CXCR4 and IL-10 Mesenchymal Stromal Cells in a Mouse Model of Peritonitis.

Sepsis due to peritonitis is a process associated with an inflammatory state. Mesenchymal stromal cells (MSCs) modulate the immune system due to the paracrine factors released and may be a therapeutic alternative. Three treatment groups were developed in a murine model of peritonitis to verify the effect of human adipose mesenchymal stem cell (hASCs). Additionally, a temporary modification was carried out on them to improve their arrival in inflamed tissues (CXCR4), as well as their anti-inflammatory activity (IL-10). The capacity to reduce systemic inflammation was studied using a local application (peritoneal injection) as a treatment route. Comparisons involving the therapeutic effect of wild-type ASCs and ASCs transiently expressing CXCR4 and IL-10 were carried out with the aim of generating an improved anti-inflammatory response for sepsis in addition to standard antibiotic treatment. However, under the experimental conditions used in these studies, no differences were found between both groups with ASCs. The peritoneal administration of hASCs or genetically modified hASCs constitutes an efficient and safe therapy in our model of mouse peritonitis.

Discrepant expressive language lateralization in children and adolescents with epilepsy.

Neuronavigated transcranial magnetic stimulation (nTMS) has emerged as a presurgical language mapping tool distinct from the widely used functional magnetic resonance imaging (fMRI). We report fMRI and nTMS language-mapping results in 19 pediatric-epilepsy patients and compare those to definitive testing by electrical cortical stimulation, Wada test, and/or neuropsychological testing. Most discordant results occurred when fMRI found right-hemispheric language. In those cases, when nTMS showed left-hemispheric or bilateral language representation, left-hemispheric language was confirmed by definitive testing. Therefore, we propose nTMS should be considered for pediatric presurgical language-mapping when fMRI shows right-hemispheric language, with nTMS results superseding fMRI results in those scenarios.

Implication of stem cells from adipose tissue in wound healing in obese and cancer patients.

OBJECTIVE: Certain diseases such as obesity and cancer can cause impaired wound healing. Adipose tissue derived stem cells (ASCs) are a novel field of research. Many studies have evidenced their high degree of safety and potential for wound repair due to their immunomodulatory and tissue-regeneration properties. The purpose of this study is to determine the impact of obesity and cancer on the therapeutic potential of ASCs. MATERIALS AND METHODS: We isolated and characterized the phenotype, differentiation capacities, secretome, and in vitro migration capacities of ASCs. Furthermore, we analyze their capacity of in vitro migration associated with the plasma of the different patients. RESULTS: We observed that ASCs isolated from obese and cancer patients have the same phenotype, cell proliferation, and migration capacities as ASCs derived from healthy donors. However, they do not have the same differentiation potential and exhibit distinct profiles of both pro-inflammatory and regulatory secreted cytokines, which, together with the signals received from the bloodstream, could account for the impaired healing in patients with these diseases. CONCLUSIONS: We consider the ASCs from patients with either obesity or cancer are slightly altered, and this may be the cause of worse wound healing in these patients.

OBJETIVO: Enfermedades como la obesidad y el cáncer pueden alterar la cicatrización de las heridas. Las células madre derivadas del tejido adiposo (ASC) abren un nuevo campo de investigación ya que muchos estudios han demostrado su utilidad y alto grado de seguridad para la reparación de heridas debido a sus propiedades inmunomoduladoras y de regeneración tisular. El propósito de este estudio es determinar el impacto de la obesidad y el cáncer en el potencial terapéutico de las ASCs. MATERIAL Y MÉTODOS: Aislamos y caracterizamos el fenotipo, la capacidad de diferenciación, el secretoma y la capacidad de migración in vitro de las ASC. Asimismo, analizamos la capacidad de migración in vitro asociada al plasma de los diferentes pacientes. RESULTADOS: Observamos que las ASC aisladas de pacientes obesos y con cáncer tienen el mismo fenotipo, proliferación celular y capacidades de migración que las ASCaisladas de donantes sanos. Sin embargo, no tienen el mismo potencial de diferenciación y exhiben perfiles distintos de citoquinas secretadas tanto proinflamatorias como reguladoras. CONCLUSIONES: Consideramos que las ASC de pacientes con obesidad o cáncer están levemente alteradas. Esta puede ser la causa de una peor cicatrización de las heridas en este tipo de pacientes.

Oncological transformation in vitro of hepatic progenitor cell lines isolated from adult mice.

Colorectal cancer cells can transfer the oncogene KRAS to distant cells, predisposing them to malignant transformation (Genometastasis Theory). This process could contribute to liver metastasis; besides, hepatic progenitor cells (HPCs) have been found to be involved in liver malignant neoplasms. The objective of this study is to determine if mouse HPCs-Oval cells (OCs)-are susceptible to incorporate Kras GAT (G12D) mutation from mouse colorectal cancer cell line CT26.WT and if OCs with the incorporated mutation behave like malignant cells. To achieve this, three lines of OCs in different conditions were exposed to CT26.WT cells through transwell co-culture for a week. The presence of KrasG12D and capacity to form tumors were analyzed in treated samples by droplet digital PCR and colony-forming assays, respectively. The results showed that the KrasG12D mutation was detected in hepatic culture conditions of undifferentiated OCs and these cells were capable of forming tumors in vitro. Therefore, OCs are susceptible to malignant transformation by horizontal transfer of DNA with KrasG12D mutation in an undifferentiated condition associated with the liver microenvironment. This study contributes to a new step in the understanding of the colorectal metastatic process.

Toxicity study in a pig model of intraperitoneal collagenase as an "enzymatic scalpel" directed to break stroma in order to generate a new perspective for peritoneal carcinomatosis approach: an experimental research.

BACKGROUND: This study aimed to measure the toxicity resulting from collagenase administration to the peritoneal cavity in a pig model as a preliminary step to break down the stroma surrounding tumors. METHODS: Eight pigs were treated with 2 different collagenase concentrations previously tested in rats by our group. Time and temperature were controlled using a peritoneal lavage system (PRS System, Combat Medical Ltd.) identical to that used in human surgeries through hyperthermic intraperitoneal chemotherapy (HIPEC); 2 additional pigs were treated with peritoneal lavage only. Samples of blood and peritoneal fluid were collected pre-treatment, immediately after treatment, and 24 h postoperatively. In addition, histological studies and blood collagenase levels were measured. RESULTS: No complications were observed during the surgeries. Intraoperative images evidenced the release of peritoneal tissue during collagenase treatment. After surgery, the animals showed no signs of pain. Diet and mobility were normal at 4 h postoperatively, and there were no significant differences in hematologic or biochemical parameters. Quantification of MMP1 and MMP2 in all samples as measured by absorbance showed no differences in blood collagenase levels between pre-treatment, post-treatment, and 24 h postoperatively. None of the animals treated with collagenase showed peritoneal adhesions during the second surgery. Histologically, peritoneal organs and serous structures did not show any microscopic alterations associated with collagenase treatment in any group. CONCLUSION: Lavage of the peritoneal cavity with doses of up to 100,000 collagen digestion units/animal for 30 min is safe and removes connective tissue from the peritoneal cavity.

Comparative Analysis Between Mesenchymal Stem Cells From Subcutaneous Adipose Tissue and Omentum in Three Types of Patients: Cancer, Morbid Obese and Healthy Control.

Objective. The aims of this study are to compare 2 origins of adipose-derived mesenchymal stem cells (MSCs) (omentum and subcutaneous) from 2 pathologies (morbid obesity and cancer) vs healthy donors. Adipose tissue has revealed to be the ideal MSC source. However, in developing adipose-derived stem cells (ASCs) for clinical use, it is important to consider the effects of different fat depots and also the effect of donor variability. Methods. We isolated and characterized the membrane markers and differentiation capacities of ASCs obtained from patients with these diseases and different origin. During the culture period, we further analysed the cells' proliferation capacity in an in vitro assay as well as their secretome. Results. Adipose-derived stem cells isolated from obese and cancer patients have mesenchymal phenotype and similar cell proliferation as ASCs derived from healthy donors, some higher in cells derived from subcutaneous fat. However, cells from these 2 types of patients do not have the same differentiation potential, especially in cancer patients from omentum, and exhibit distinct secretion of both pro-inflammatory and regulatory cytokines, which could explain the differences in use due to origin as well as pathology associated with the donor. Conclusion. Subcutaneous and omentum ASCs are slightly different; omentum generates fewer cells but with greater anti-inflammatory capacity. Adipose-derived stem cells from patients with either obesity or cancer are slightly altered, which limits their therapeutic properties.

Intraperitoneal collagenase as a novel therapeutic approach in an experimental model of colorectal peritoneal carcinomatosis.

The usefulness of local collagenase in therapeutic approaches to solid tumors has been tested recently. In this study, we evaluate the safety and efficacy of intraperitoneal collagenase associated or not to mitomycin for treatment of colorectal peritoneal metastases in an experimental rat model. Using a fixed-dose procedure, we found that a dose of collagenase of 37 IU/mL administered for 15 min with a hyperthermia pump at 37.5 °C, both in isolation or associated to sequential treatment with intraperitoneal mitomycin, led to a macroscopic decrease in tumor volume as evaluated by the modified peritoneal cancer index (mPCI). Concerning the safety of the procedure, the animals showed no physiological or behavioral disorders during 8 weeks of follow-up. Local treatment for peritoneal metastases of colorectal origin with intraperitoneal collagenase has proved safe and effective in an experimental murine model. Therefore, the stroma-first approach by enzymatic breakdown of collagen from the tumor's extracellular matrix provides a new therapeutic target for colorectal peritoneal metastases.

Main histological parameters to be evaluated in an experimental model of myocardial infarct treated by stem cells on pigs.

Myocardial infarction has been carefully studied in numerous experimental models. Most of these models are based on electrophysiological and functional data, and pay less attention to histological discoveries. During the last decade, treatment using advanced therapies, mainly cell therapy, has prevailed from among all the options to be studied for treating myocardial infarction. In our study we wanted to show the fundamental histological parameters to be evaluated during the development of an infarction on an experimental model as well as treatment with mesenchymal stem cells derived from adipose tissue applied intra-lesionally. The fundamental parameters to study in infarcted tissue at the histological level are the cells involved in the inflammatory process (lymphocytes, macrophages and M2, neutrophils, mast cells and plasma cells), neovascularization processes (capillaries and arterioles) and cardiac cells (cardiomyocytes and Purkinje fibers). In our study, we used intramyocardial injection of mesenchymal stem cells into the myocardial infarction area 1 hour after arterial occlusion and allowed 1 month of evolution before analyzing the modifications on the normal tissue inflammatory infiltrate. Acute inflammation was shortened, leading to chronic inflammation with abundant plasma cells and mast cells and complete disappearance of neutrophils. Another benefit was an increase in the number of vessels formed. Cardiomyocytes and Purkinje fibers were better conserved, both from a structural and metabolic point of view, possibly leading to reduced morbidity in the long term. With this study we present the main histological aspects to be evaluated in future assays, complementing or explaining the electrophysiological and functional findings.

Rat model of anal sphincter injury and two approaches for stem cell administration.

AIM: To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area. METHODS: Adipose-derived stem cells (ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein (GFP) for cell tracking. Biosutures (sutures covered with ASCs) were prepared with 1.5 x 106 GFP-ASCs, and solutions of 106 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology (hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole (commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for: (1) Cell injection without repair; (2) biosuture repair; and (3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area. RESULTS: With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches (peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min (mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological assessment revealed that with the aforementioned surgical procedure, both sphincters were completely sectioned. In manometry, the described activity disappeared and was replaced by a gentle oscillation of basal line, without a recognizable pattern. Surprisingly, these findings appeared irrespective of injury repair or not. ASCs survived in this potentially septic area for 7 d, at least. We were able to identify them in 84% of animals, mainly in the muscular section area or in the tissue between the muscular endings. ASCs formed a kind of "conglomerate" in rats treated with injections, while in the biosuture group, they wrapped the suture. ASCs were also able to migrate to the damaged zone. No relevant adverse events or mortality could be related to the stem cells in our study. We also did not find unexpected tissue growths. CONCLUSION: The proposed procedure produces a consistent sphincter lesion. Biosutures and injections are suitable for cell delivery. ASCs survive and are completely safe in this clinical setting.

Response to clobazam in continuous spike-wave during sleep.

AIM: To evaluate the efficacy of clobazam treatment in reducing epileptiform discharges and modifying neuropsychological function in continuous spike-wave during slow wave sleep. METHOD: We performed a prospective clinical trial in patients with continuous spike-wave during sleep aged 4 to 10 years. Patients underwent neuropsychological assessment and overnight electroencephalographic monitoring before treatment, and subsequent repeat assessment and overnight electroencephalographic monitoring 3 months after treatment. Treatment consisted of 1mg/kg clobazam up to a maximum dose of 30mg during the first night, followed by 0.5mg/kg nightly for 3 months. RESULTS: Nine patients completed the study and had pre- and post-neuropsychological evaluation. There was a qualitative reduction in median (p25 -p75 ) spike percentage after 3 months (72.2 [68.0-75.8] vs 32.7 [4.7-81.7]). There were no marked changes in median (p25 -p75 ) IQ comparing pre- and post-clobazam treatment (80.0 [74.0-88.0] vs 80.0 [67.0-89.0]). There was a qualitative increase in Verbal IQ (83.0 [69.0-92.0] vs 95.0 [83.0-99.0]) and a qualitative decrease in Non-verbal IQ (84.0 [74.0-87.0] vs 71.0 [60.0-84.0]). INTERPRETATION: Qualitative improvements in epileptiform activity and cognition occurred in patients treated with clobazam for 3 months and the relationship between epileptiform activity and cognitive outcome should be studied in larger studies. WHAT THIS PAPER ADDS: Verbal IQ in patients with continuous spike-wave during sleep improved following short-term treatment with clobazam. Other neuropsychological improvements were observed, but varied by patient. Cognitive improvement was observed despite some worsening of epileptiform discharges.

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR.

AIM: To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. METHODS: In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well. RESULTS: Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION: ddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.

A Preliminary Study of the Action of Virtosomes from Non-dividing Cells on Tumour Cell Replication in vitro and in vivo.

BACKGROUND: A DNA-RNA-lipoprotein complex, termed as virtosome, is released spontaneously from healthy human, other mammalian, avian, amphibian and plant cells in a regulated and energy-dependent manner. Studies on human and mouse lymphocytes, hepatocytes, NIH 3T3 cells and mouse tumour cell lines have shown that virtosomes may be acting as inter-cellular messengers. In particular, virtosomes from non-dividing cells blocked 3H-thymidine incorporation into DNA in tumour cell lines. OBJECTIVE: Study of the effect of virtosomes on tumors "in vivo" and "in vitro". METHODS AND RESULTS: The present study shows in vitro effects of virtosomes isolated from rat liver, essentially nondividing cell populations, on cultures of healthy smooth muscle cells (SMC), human umbilical vein endothelial cells (HUVEC), human fibroblasts (h-fibroblasts) and mouse embryonic fibroblasts (NIH-3T3) together with two tumour cell lines, human Duke's type B colorectal adenocarcinoma cells (SW480) and human connective tissue fibrosarcoma cells (HT1080). Multiplication of all cell lines was inhibited by the liver virtosomal preparation even with various dilutions of the extract (100 - 0.5%). In an in vivo study, tumours were initiated by subcutaneous injection of 1.0 x 106 DHD/K12-PROb cells in 6 weeks old male BIDX rats. Visible tumours (<1cm) appeared after 3 weeks and lung metastases after 8 weeks (80%). Virtosomes introduced via a tail vein on tumour initiation resulted in a reduction in tumour size and number. CONCLUSION: Virtosomal preparation from a non-dividing cell population inhibited cell division, reduced tumour size and partially blocked metastasis.

Community-Based Needs Assessment of Neurodevelopment, Caregiver, and Home Environment Factors in Young Children Affected by HIV in Lima, Peru.

BACKGROUND: In many resource-poor settings such as Peru, children affected by HIV have a high prevalence of neurodevelopmental delays (NDDs) and remain excluded from adequate treatment. METHODS: Community health workers (CHWs) administered NDD screening instruments to assess child development and associated caregiver and household factors in 14 HIV-affected parent-child dyads. Focus group discussion with caregivers was conducted to explore their needs and behaviors around early child stimulation and to assess their perceptions of the screening experience. RESULTS: Over 70% of the children had abnormal classification in at least 1 (out of 5) developmental domains according to Ages and States Questionnaire-provided cutoff scores. Caregiver depression and stress were associated with abnormal development as were some parenting behavior factors. Knowledge about child development was low. Caregivers felt testing and discussing results with a CHW were very insightful. Reported caregiver behavior differed between caregivers with HIV-infected children and those with uninfected children. CONCLUSION: Taken together, these exploratory quantitative data suggest that parenting behaviors associated with low child development scores may be modifiable and that community-based testing is well received and informative to these HIV-infected caregivers.

Non-dividing Cell Virtosomes Affect In Vitro and In Vivo Tumour Cell Replication.

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

Potential of mesenchymal stem cell in stabilization of abdominal aortic aneurysm sac.

BACKGROUND: In their origin, abdominal aortic aneurysms (AAAs) are related to an inflammatory reaction within the aortic wall, which can lead to weakness and degeneration of this structure. One of the most widely accepted treatment modalities for AAAs is the placement of stent grafts. Nevertheless, in some patients blood re-enters the aneurysm sac, creating so-called leaks, which constitute a renewed risk of rupture and death.This study explores the possibility of filling aneurysm sacs treated by endovascular aneurysm repair with adipose tissue-derived mesenchymal stem cells (ASCs) in a porcine model. METHODS: We developed a porcine model using 22 animals by creating an artificial AAA made with a Dacron patch. AAAs were then treated with a coated stent that isolated the aneurysm sac, after which we introduced allogeneic ASC into the sac. Animals were followed-up for up to 3 mo. The experiment consisted of the aforementioned surgical procedure performed first, followed by computed tomography and echo-Doppler imaging during the follow-up, and finally, after sacrificing the animals, histologic analysis of tissue samples from the site of cell implantation by a blinded observer and the detection of implanted cells by immunofluorescence detection of the Y chromosome. RESULTS: Our findings demonstrate the survival of ASCs over the 3 mo after implantation and histologic changes associated with this treatment. Treated animals had less acute and chronic inflammation throughout the study period, and we observed increasing fibrosis of the aneurysm sac, no accumulation of calcium, and a regeneration of elastic fibers in the artery. CONCLUSIONS: The combination of endovascular aneurysm repair and cell therapy on AAAs has promising results for the stabilization of the sac, resulting in the generation of living tissue that can secure the stent graft and even showing some signs of wall regeneration. The therapeutic value of such cell-based therapy will require further investigation.