Deciphering the role of FUS::DDIT3 expression and tumor microenvironment in myxoid liposarcoma development.

BACKGROUND: Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. METHODS: To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. RESULTS: Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. CONCLUSIONS: Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Native Spleen Preservation During Visceral Transplantation Inhibits Graft-Versus-Host-Disease Development: Clinical and Experimental Study.

OBJECTIVE: We aimed to assess whether native spleen preservation during visceral transplantation (VT) affects graft-versus-host-disease (GVHD) incidence. SUMMARY BACKGROUND DATA: GVHD is one of the most severe and frequently lethal hematological complications after VT procedures. Because there is no specific treatment for GVHD, it is imperative to develop a strategy to reduce donor lymphocyte engraftment and proliferation. METHODS: Our study included both clinical and experimental data. A total of 108 patients were divided into 3 groups: a native spleen preservation group, a native spleen removal with no donor spleen group, and a donor spleen included (allogeneic spleen) group. We also used an allogeneic VT rat model, in which recipients were divided into 2 groups: a native spleen preservation (+SP) group and a native spleen removal (-S) group. Skin rash appearance, histopathological changes, chimerism, and spleen effects on circulating allogeneic T-cells were assessed. RESULTS: The patients with native spleen preservation showed a lower rate of GVHD ( P <.001) and better survival ( P <.05) than those in the other groups. Skin and histological signs of GVHD were lower in the rats in the +SP group ( P <.05). The donor T-cell frequency in the bloodstream and skin was also significantly reduced when the native spleen was preserved ( P <.01 and P <.0001, respectively). CONCLUSIONS: The clinical and experimental data indicate that recipient spleen preservation protects against GVHD after VT, and donor cell clearance from the bloodstream by spleen macrophages could be the underlying mechanism. Therefore, spleen preservation should be considered in VT procedures, whenever possible.

European Liver Transplant Registry: Donor and transplant surgery aspects of 16,641 liver transplantations in children.

BACKGROUND AND AIMS: The European Liver Transplant Registry (ELTR) has collected data on liver transplant procedures performed in Europe since 1968. APPROACH AND RESULTS: Over a 50-year period (1968-2017), clinical and laboratory data were collected from 133 transplant centers and analyzed retrospectively (16,641 liver transplants in 14,515 children). Data were analyzed according to three successive periods (A, before 2000; B, 2000-2009; and C, since 2010), studying donor and graft characteristics and graft outcome. The use of living donors steadily increased from A to C (A, n = 296 [7%]; B, n = 1131 [23%]; and C, n = 1985 [39%]; p = 0.0001). Overall, the 5-year graft survival rate has improved from 65% in group A to 75% in group B (p < 0.0001) and to 79% in group C (B versus C, p < 0.0001). Graft half-life was 31 years, overall; it was 41 years for children who survived the first year after transplant. The late annual graft loss rate in teenagers is higher than that in children aged <12 years and similar to that of young adults. No evidence for accelerated graft loss after age 18 years was found. CONCLUSIONS: Pediatric liver transplantation has reached a high efficacy as a cure or treatment for severe liver disease in infants and children. Grafts that survived the first year had a half-life similar to standard human half-life. Transplantation before or after puberty may be the pivot-point for lower long-term outcome in children. Further studies are necessary to revisit some old concepts regarding transplant benefit (survival time) for small children, the role of recipient pathophysiology versus graft aging, and risk at transition to adult age.

Preclinical Study of DCD and Normothermic Perfusion for Visceral Transplantation.

Considering recent clinical and experimental evidence, expectations for using DCD-derived intestines have increased considerably. However, more knowledge about DCD procedure and long-term results after intestinal transplantation (ITx) is needed. We aimed to describe in detail a DCD procedure for ITx using normothermic regional perfusion (NRP) in a preclinical model. Small bowel was obtained from pigs donors after 1 h of NRP and transplanted to the recipients. Graft Intestinal samples were obtained during the procedure and after transplantation. Ischemia-reperfusion injury (Park-Chiu score), graft rejection and transplanted intestines absorptive function were evaluated. Seven of 8 DCD procedures with NRP and ITx were successful (87.5%), with a good graft reperfusion and an excellent recovery of the recipient. The architecture of grafts was well conserved during NRP. After an initial damage of Park-chiu score of 4, all grafts recovered from ischemia-reperfusion, with no or very subtle alterations 2 days after ITx. Most recipients (71.5%) did not show signs of rejection. Only two cases demonstrated histologic signs of mild rejection 7 days after ITx. Interestingly intestinal grafts showed good absorptive capacity. The study's results support the viability of intestinal grafts from DCD using NRP, contributing more evidence for the use of DCD for ITx.

A Multi­Center, Open­Label, Single­Arm Trial to Evaluate the Efficacy, Pharmacokinetics, and Safety and Tolerability of IGSC 20% in Subjects with Primary Immunodeficiency.

PURPOSE: The purpose of this phase 3 study was to evaluate the efficacy, pharmacokinetics (PK), and safety of Immune Globulin Subcutaneous (Human), 20% Caprylate/Chromatography Purified (IGSC 20%) in patients with primary immunodeficiency (PI). METHODS: Immunoglobulin treatment-experienced subjects with PI received 52 weeks of IGSC 20% given weekly at the same dose as the subject's previous IgG regimen (DAF 1:1); the minimum dose was 100 mg/kg/week. The primary endpoint was serious bacterial infections (SBIs [null vs alternative hypothesis: SBI rate per person per year ≥ 1 vs < 1]). IgG subclasses and specific pathogen antibody levels were also measured. RESULTS: Sixty-one subjects (19 children [≤ 12 years], 10 adolescents [> 12-16 years], and 32 adults) were enrolled. The rate of SBIs per person per year was 0.017. The 1-sided 99% upper confidence limit was 0.036 (< 1), and the null hypothesis was rejected. The rate of hospitalization due to infection per person per year was 0.017 (2-sided 95% confidence interval: 0.008-0.033) overall. The mean trough total IgG concentrations were comparable to the previous IgG replacement regimen. The average of the individual mean trough ratios (IGSC 20%:previous regimen) was 1.078 (range: 0.83-1.54). The average steady-state mean trough IgG concentrations were 947.64 and 891.37 mg/dL, respectively. Seven subjects had serious treatment-emergent adverse events (TEAEs); none was drug-related. The rate of all TEAEs, including local infusion site reactions, during 3045 IGSC 20% infusions was 0.135. Most TEAEs were mild or moderate. CONCLUSIONS: IGSC 20% demonstrated efficacy and good safety and tolerability in subjects with PI.

First multivisceral transplantation in Mitchell-Riley/Martinez-Frias syndrome.

BACKGROUND: MRS/MFS is a rare multisystem disorder with a poor prognosis. The high mortality rate of this syndrome is related to the severity of the associated gastrointestinal, pancreatic, and hepatobiliary conditions, as most of them are not amenable to conventional medical and surgical treatments. METHODS: We report the case of a Romani girl with all the key clinical features of MRS/MFS, and a review of cases reported in the literature. Our patient is a newborn from consanguineous parents who presented duodenal atresia, hypoplastic pancreas, gallbladder agenesis, and neonatal diabetes. Given the clinical suspicion of MRS/MFS, a genetic analysis was performed which revealed the presence of a homozygous variant in the RFX6 gene. During the course of the disease, the patient presented intractable secretory diarrhea and severe intestinal failure. RESULTS: At 2 years of age, she underwent MVT of the stomach, duodenum, small intestine, colon, liver, and pancreas. There were no surgical complications. Histologic evaluation of the small bowel showed extensive patches of gastric heterotopia. After more than 10 years of follow-up, she had presented with normal gastrointestinal, hepatic, and pancreatic function. She has one of the longest survival periods in the literature. CONCLUSIONS: Our experience suggests that multivisceral transplantation may be a promising option in select cases of MRS/MFS.

Graft infusion of adipose-derived mesenchymal stromal cells to prevent rejection in experimental intestinal transplantation: A feasibility study.

BACKGROUND: Mesenchymal stromal cells (MSC) have been proposed as a promising complement to standard immunosuppression in solid organ transplantation because of their immunomodulatory properties. The present work addresses the role of adipose-derived MSC (Ad-MSC) in an experimental model of acute rejection in small bowel transplantation (SBT). MATERIAL/METHODS: Heterotopic allogeneic SBT was performed. A single dose of 1.5x106 Ad-MSC was intra-arterially delivered just before graft reperfusion. Animals were divided into CONTROL (CTRL), CONTROL+Ad-MSC (CTRL_MSC), tacrolimus (TAC), and TAC+Ad-MSC (TAC_MSC) groups. Each Ad-MSC groups was subdivided in autologous and allogeneic third-party groups. RESULTS: Rejection rate and severity were similar in MSC-treated and untreated animals. CTRL_MSC animals showed a decrease in macrophages, T-cell (CD4, CD8, and Foxp3 subsets) and B-cell counts in the graft compared with CTRL, this decrease was attenuated in TAC_MSC animals. Pro- and anti-inflammatory cytokines and some chemokines and growth factors increased in CTRL_MSC animals, especially in the allogeneic group, whereas milder changes were seen in the TAC groups. CONCLUSION: Ad-MSC did not prevent rejection when administered just before reperfusion. However, they showed immunomodulatory effects that could be relevant for a longer-term outcome. Interference between tacrolimus and the MSC effects should be addressed in further studies.

Delayed introduction of sirolimus in paediatric intestinal transplant recipients: indications and long-term benefits.

To review our experience using sirolimus in a single centre paediatric intestinal transplantation cohort. Intestinal transplant patients with more than 3 months follow-up were divided into two groups according to their immunosuppression regimen: tacrolimus, (TAC group, n = 45 grafts) or sirolimus (SRL group, n = 38 grafts), which included those partially or completely converted from tacrolimus to sirolimus. The indications to switch were tacrolimus side effects and immunological complications. Survival and complications were retrospectively analysed comparing both groups. SRL was introduced 9 months (0 months-16.9 years) after transplant. The main cause for conversion was worsening renal function (45%), followed by haemolytic anaemia (21%) and graft-versus-host-disease (16%). Both groups showed a similar overall patient/graft survival (P = 0.76/0.08) and occurrence of rejection (24%/17%, P = 0.36). Immunological complications did not recur after conversion. Renal function significantly improved in most SRL patients. After a median follow-up of 65.17 months, 28/46 survivors were on SRL, 26 with monotherapy, with good graft function. Over one-third of our patients eventually required SRL conversion that allowed to improve their kidney function and immunological events, without entailing additional complications or survival impairment. Further trials are warranted to clarify the potential improvement of the standard tacrolimus maintenance by sirolimus conversion or addition.

Epigenetic footprint enables molecular risk stratification of hepatoblastoma with clinical implications.

BACKGROUND & AIMS: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. METHODS: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. RESULTS: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. CONCLUSIONS: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. LAY SUMMARY: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer.

Donor-Specific Antibodies in Pediatric Intestinal and Multivisceral Transplantation: The Role of Liver and Human Leukocyte Antigen Mismatching.

Rejection is one of the most important drawbacks for graft and patient survival in intestinal and multivisceral transplantation. However, there is no consensus on the diagnostic criteria for humoral rejection, and the literature about the role of donor-specific antibodies (DSA) on allograft outcome and the risk factors that contribute to their development is scant with contradictory results. The present study analyzes the role of DSA exclusively in a pediatric cohort of 43 transplants. Among our patients, 11.6% showed preformed DSA, but they did not correlate with more rejection or less allograft survival. Having previous transplants was the main sensitization factor with an odds ratio (OR) = 44.85 (P = 0.001). In total, 16.3% of recipients developed de novo donor-specific antibodies (dnDSA), mostly directed against human leukocyte antigen (HLA) class II, polyspecific and complement fixing. Additionally, the presence of dnDSA had a deleterious effect on graft rejection (hazard ratio [HR] = 11.00; P = 0.01) and survival (HR = 66.52; P < 0.001) in an observational period of 5 years after transplantation. The inclusion of the liver emerged as the main protective factor against dnDSA development with an OR = 0.07 (P = 0.007). The analysis of HLA compatibility at the serological and epitope level with the computational tools HLAMatchmaker and PIRCHE revealed no association between HLA mismatching and dnDSA. In conclusion, this study performed in pediatric recipients shows the deleterious effect of dnDSA on intestinal transplantation supported by the complement-fixing activity observed. Additionally, the liver inclusion in the allografts showed to be a protective factor against dnDSA generation.

Long-term results of surgery for bowel lengthening: how many transplants are avoided, for which patients?

PURPOSE OF REVIEW: One of the biggest successes of intestinal rehabilitation programs is that more patients achieve enteral autonomy without transplantation. Many factors are responsible of this accomplishment including new parenteral formulas, better catheter management, surgical management, and the experience of the teams. The purpose of this review is to analyze recent published papers regarding intestinal lengthening procedures trying to find out how many transplantations are avoided and for which patients. RECENT FINDINGS: A trend towards performing less intestinal transplants has been identified in the last years. The general improvement of intestinal rehabilitation accounts for this step forward. However, the role of intestinal lengthening has not been clarified. SUMMARY: Surgical techniques for autologous reconstructive surgery are not limited to bowel lengthening. Longitudinal intestinal lengthening and tailoring and serial transverse enteroplasty offered good results in terms of intestinal adaptation, long-term survival, and subsequent need of intestinal transplantation. In recent series, less than one quarter of patients who underwent intestinal lengthening required salvage intestinal transplantation.

Biochemical and nutritional factors associated with blood viscosity in adults living in a mountain chain, (Imbabura), Ecuador. / Factores bioquímicos y nutricionales asociados a la viscosidad sanguínea en adultos de la sierra urbana (Imbabura), Ecuador.

The association of blood viscosity (BV) with hypertension and diabetes mellitus type 2 indicates that it should be considered, especially in populations living in mountain chains where the hemoglobin and hematocrit values are higher. In fact, this work analyzed the association of BV with nutritional and biochemical risk factors in the development of cardio metabolic diseases in healthy adults (20-60 years old; 46.39% female and 53.61% male) living in the urban Ecuadorian sierra. The body mass index (BMI) was calculated. The body fat percentage, body water percentage, waist circumference (WC) and blood pressure were measured. Determinations of biochemical and hematological parameters were performed using established methodologies and a validated formula was used to determine the BV. Hyperviscosity (HV) was present in 14.76% of the population: 10.46% men, 4.3% women. In this group, 42.8% showed metabolic syndrome (MS) against 33% in the normoviscosity group (NV). Uric acid (UA) was observed statistically higher in HV and NV with MS groups. A simple positive linear correlation was found between glucose (GLU) and triglycerides (TRI) concentrations and BV in HV without MS group. The multiple linear regression analysis indicated that GLU, UA and systolic pressure influence to increase BV in the HV without MS group. Glucose concentration and percentage of water, independently influence the BV in the NV without MS group. The increase in GLU and UA concentrations are the most influencing factors on BV in this population.

Effect of consumption of tree tomato juice (Cyphomandra betacea) on lipid profile and glucose con centrations in adults with hyperlipidemia, Ecuador.

In this work the effect of consumption of tree tomato juice (Cypho-mandra betacea) was evaluated on nutritional and biochemical parameters in 54 volunteers (44 women and 10 men) aged 45 ±8 years-Ecuador. A nutritional and biochemical evaluation was performed in volunteers; then they were invited to drink tree tomato juice (IOOg of fruit in 150 ml of water) daily for 6 weeks. Finished these 6 weeks, volunteers were nutritional and biochemical evaluated again. 67% abdominal obesity was found, decreasing at 53% after drinking the established dosage of juice. 87% of the volunteers before treatment, showed -hypercholesterolemia, 40.7% and 46.3% had hypertriglyceridemia and elevated LDL, respectively and 18.5% glucose concentrations between 98-130 mg/dL. These values decrease significantly in this group after drinking tomato juice. In ge- neral, Total Cholesterol, LDL and glucose concentrations decrease significantly after drinking tree tomato juice in all the voluntaries with or without overweight. There is no change in HDL concentrations. The consumption of tree tomato juice did not affect the activity of alanine aminotransferase or aspartate aminotransferase enzymes either creatinine, urea and uric acid concentrations, neither blood pressure suggesting that does not affect renal or liver function. These results indicate that consumption of tree tomato juice for six weeks appears to have a lipid-lowering and modulating effect on glucose metabolism, suggesting C betacea as one of the high Andean fruits nutraceutical potential. However, this issue should be investigated in more detail.

Antiadenohypophysis autoantibodies in patients with nongluten-related gastroenteropathies.

AIM: To investigate the presence of antipituitary antibodies (APA) in the serum of patients undergoing gastroenteropathies (GEP) other than celiac disease (CD). METHODS: APA were analyzed in GEP patients (n = 103), CD patients (n = 94), idiopathic growth hormone (GH) deficiency patients (n = 21), and 98 age- and sex-matched controls. Indirect immunofluorescence was performed in cryostat sections of baboon pituitary gland. RESULTS: APA were detected in 30% of GEP patients, 38% of them showed failure to thrive. No significant differences were found when we compared thrive impairment in negative and positive APA GEP patients. Indeed, normal values of insulin-like growth factor 1 were found in 93% of positive APA GEP patients. APA were detected in 52% of the CD patients. Ninety-one percent of them, in contrast to GEP group, showed significant growth impairment (P < 0.05) when compared to APA negative CD individuals. GH-deficient non-CD patients did not show APA. CONCLUSIONS: We have shown the presence of APA in patients with nongluten-related enteropathies. The presence of antipituitary autoantibodies in GEP patients does not seem to be associated with failure to thrive as it occurs in CD.

[Relationships between biomarkers of oxidative stress and nutritional status in adults, Ecuador]. / Marcadores de estrés oxidativo y su relación con el estado nutricional en adultos, Ecuador.

In this work it was evaluated the relationship between oxidative stress biomarkers (uric acid, bilirubin and C-reactive protein) with nutritional status in 321 adults of Ecuador, belonging to administrative staff of of the Universidad Tècnica del Norte, aged 43 ± 10 years old (46 30% female and 53.61% male). Socio demographic and epidemiological information and lifestyle were obtained through a survey; The Body Mass Index (BMI) and body fat and body water percentages were calculated; waist circumference (WC) and blood pressure was measured. Determinations of uric acid, bilirubin, and serum C-reactive protein (PCR) were performed. 17.9% of the populations were obese and 51.72% overweight. The highest values of uric acid were found in obese, hypertensive and physical activity groups. The total direct and indirect bilirubin were found in upper limits in abdominal obesity and physical activity groups. The CRP level was influenced by % fat and % water in the low body fat group and in females. In male, BMI and WC were associated with CRP. Uric acid showed relationship with % fat and WC in overweight, high body fat and PHT groups, uric acid was associated with the % water and BMI in obese. Finally, uric acid was associated with % water and the WC in the abdominal obesity, and HT groups'. The body water percentage is an important indicator to development of oxidative stress in this population.

Digital RNA sequencing using unique molecular identifiers enables ultrasensitive RNA mutation analysis.

Mutation analysis is typically performed at the DNA level since most technical approaches are developed for DNA analysis. However, some applications, like transcriptional mutagenesis, RNA editing and gene expression analysis, require RNA analysis. Here, we combine reverse transcription and digital DNA sequencing to enable low error digital RNA sequencing. We evaluate yield, reproducibility, dynamic range and error correction rate for seven different reverse transcription conditions using multiplexed assays. The yield, reproducibility and error rate vary substantially between the specific conditions, where the yield differs 9.9-fold between the best and worst performing condition. Next, we show that error rates similar to DNA sequencing can be achieved for RNA using appropriate reverse transcription conditions, enabling detection of mutant allele frequencies <0.1% at RNA level. We also detect mutations at both DNA and RNA levels in tumor tissue using a breast cancer panel. Finally, we demonstrate that digital RNA sequencing can be applied to liquid biopsies, analyzing cell-free gene transcripts. In conclusion, we demonstrate that digital RNA sequencing is suitable for ultrasensitive RNA mutation analysis, enabling several basic research and clinical applications.

A novel phenotype variant of severe congenital neutropenia caused by G6PC3 deficiency.

Severe congenital neutropenia type 4 (SCN4) is associated with mutations in the G6PC3 gene. To date, all patients bearing the p.Gly260Arg variant of the G6PC3 gene show heart defects. Here, we present a case of the p.Gly260Arg variant in a patient who did not have structural or functional heart anomalies. Treatment with granulocyte colony-stimulating factor recovered the absolute neutrophil count and neutrophil functional competence.