Characterization of decellularized porcine oviduct- and uterine-derived scaffolds evaluated by spermatozoa-based biocompatibility and biotoxicity.

Decellularized extracellular matrix (dECM) are widely utilized in regenerative medicine and tissue engineering due to their ability to promote cell growth, proliferation, and differentiation. In reproduction, research is focused on the utilization of these scaffolds to treat pathologies causing reproductive dysfunction or to improve assisted reproduction technologies (ARTs). We developed an efficient protocol employing the immersion-agitation technique to decellularize porcine oviductal and uterine sections, comparing the efficacy of fresh versus frozen treatments. Both methods successfully generated acellular matrices with less than 3 % residual DNA, effectively preserving structural and protein integrity. Scanning and transmission electron microscopy confirmed the ultrastructural integrity, whereas Masson's Trichrome staining highlighted better collagen preservation in frozen treatments. Proteomic analysis of decellularized scaffolds revealed collagen and key macromolecules such as laminin, filamin, dermatopontin, and fibronectin, which are essential for extracellular matrix structure and cell functions such as adhesion and migration. Innovatively, we assessed the biocompatibility and cytotoxicity of the scaffolds using spermatozoa, demonstrating that thorough washing ensures the scaffold biocompatibility without compromising sperm viability or motility. Our findings not only contribute to the standardization of decellularization protocols for female reproductive organs but also emphasize the importance of evaluating sperm biocompatibility to ensure the safety of dECM scaffolds.

Development of decellularization protocols for female cat reproductive organs.

Decellularization is an innovative method to create natural scaffolds by removing all cellular materials while preserving the composition and three-dimensional ultrastructure of the extracellular matrix (ECM). The obtention of decellularized reproductive organs in cats might facilitate the development of assisted reproductive techniques not only in this species but also in other felids. The aim was to compare the efficiency of three decellularization protocols on reproductive organs (ovary, oviduct, and uterine horn) in domestic cats. The decellularization protocol involved 0.1% sodium dodecyl sulfate and 1%Triton X-100. Protocol 1 (P1) entailed 2-cycles of decellularization using these detergents. Protocol 2 (P2) was like P1 but included 3-cycles. Protocol 3 (P3) was similar to P2, with the addition of deoxyribonuclease incubation. Reproductive organs from nine cats were separated into two sides. One side served as the control (non-decellularized organ) while the contralateral side was the treated group (decellularized organ). The treated organs were subdivided into 3 groups (n = 3 per group) for each protocol. Both control and treated samples were analyzed for DNA content, histology (nuclear and ECM (collagen, elastin, and glycosaminoglycans (GAGs)) density), ultrastructure by electron microscopy, and cytotoxicity. The results of the study showed that P3 was the only protocol that displayed no nucleus residue and significantly reduced DNA content in decellularized samples (in all the studied organs) compared to the control (P < 0.05). The ECM content in the ovaries remained similar across all protocols compared with controls (P > 0.05). However, elastic fibers and GAGs decreased in decellularized oviducts (P < 0.05), while collagen levels remained unchanged (P > 0.05). Regarding the uterus, the ECM content decreased in decellularized uterine horns from P3 (P < 0.05). Electron microscopy revealed that the microarchitecture of the decellularized samples was maintained compared to controls. The decellularized tissues, upon being washed for 24 h, showed cytocompatibility following co-incubation with sperm. In conclusion, when comparing different decellularization methods, P3 proved to be the most efficient in removing nuclear material from reproductive organs compared to P1 and P2. P3 demonstrated its success in decellularizing ovarian samples by significantly decreasing DNA content while maintaining ECM components and tissue microarchitecture. However, P3 was less effective in maintaining ECM contents in decellularized oviducts and uterine horns.

Lipidomic Analysis Reveals Alterations in Hepatic FA Profile Associated With MASLD Stage in Patients With Obesity.

CONTEXT: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by the intracellular lipid accumulation in hepatocytes. Excess caloric intake and high-fat diets are considered to significantly contribute to MASLD development. OBJECTIVE: To evaluate the hepatic and serum fatty acid (FA) composition in patients with different stages of MASLD, and their relationship with FA dietary intake and MASLD-related risk factors. METHODS: This was a case-control study in patients with obesity undergoing bariatric surgery at a university hospital between January 2020 and December 2021. Participants were distributed in 3 groups: no MASLD (n = 26), steatotic liver disease (n = 33), and metabolic dysfunction-associated steatohepatitis (n = 32). Hepatic and serum FA levels were determined by gas chromatography-mass spectrometry. Nutritional status was evaluated using validated food frequency questionnaires. The hepatic expression of genes involved in FA metabolism was analyzed by reverse transcription quantitative polymerase chain reaction. RESULTS: The hepatic, but not serum, FA profiles were significantly altered in patients with MASLD compared with those without MASLD. No differences were observed in FA intake between the groups. Levels of C16:0, C18:1, and the C18:1/C18:0 ratio were higher, while C18:0 levels and C18:0/C16:0 ratio were lower in patients with MASLD, being significantly different between the 3 groups. Hepatic FA levels and ratios correlated with histopathological diagnosis and other MASLD-related parameters. The expression of genes involved in the FA metabolism was upregulated in patients with MASLD. CONCLUSION: Alterations in hepatic FA levels in MASLD patients were due to enhancement of de novo lipogenesis in the liver.

Liver Histology Predicts Liver Regeneration and Outcome in ALPPS: Novel Findings From A Multicenter Study.

BACKGROUND AND AIMS: Alterations in liver histology influence the liver's capacity to regenerate, but the relevance of each of the different changes in rapid liver growth induction is unknown. This study aimed to analyze the influence of the degree of histological alterations during the first and second stages on the ability of the liver to regenerate. METHODS: This cohort study included data obtained from the International ALPPS Registry between November 2011 and October 2020. Only patients with colorectal liver metastases were included in the study. We developed a histological risk score based on histological changes (stages 1 and 2) and a tumor pathology score based on the histological factors associated with poor tumor prognosis. RESULTS: In total, 395 patients were included. The time to reach stage 2 was shorter in patients with a low histological risk stage 1 (13 vs 17 days, P ˂0.01), low histological risk stage 2 (13 vs 15 days, P <0.01), and low pathological tumor risk (13 vs 15 days, P <0.01). Regarding interval stage, there was a higher inverse correlation in high histological risk stage 1 group compared to low histological risk 1 group in relation with future liver remnant body weight ( r =-0.1 and r =-0.08, respectively), and future liver remnant ( r =-0.15 and r =-0.06, respectively). CONCLUSIONS: ALPPS is associated with increased histological alterations in the liver parenchyma. It seems that the more histological alterations present and the higher the number of poor prognostic factors in the tumor histology, the longer the time to reach the second stage.

Immunolocalisation of aquaporins 3, 7, 9 and 10 in the epididymis of three wild ruminant species (Iberian ibex, mouflon and chamois) and sperm cryoresistance.

CONTEXT: In the epididymis, epithelial cells manage changes in the luminal environment for proper sperm maturation. Moreover, aquaglyceroporins, a subgroup of aquaporins (AQP), modulate the transport of water, glycerol and other small molecules in epithelial cells. AIMS: We aim to characterise the lining epithelium, quantify its cell composition and immunolocalise the aquaglyceroporins AQP3, AQP7, AQP9 and AQP10 alongside the epididymal ductus of three wild ruminant species, and to determine if species-specific differences could be associated with cauda sperm cryoresistance variations. METHODS: Epididymides from Iberian ibex (n =5), mouflon (n =5) and chamois (n =6) were obtained. Cauda spermatozoa were collected and sperm parameters were analysed before and after freezing. Histology and immunohistochemistry of AQP3, 7, 9, 10 and T-CD3 were performed in the caput, corpus and cauda epididymal regions. KEY RESULTS: This work first describes the lining epithelium in Iberian ibex, mouflon and chamois epididymis along the three anatomical regions, consisting of principal, basal, apical, clear and halo cells. However, the percentage of each cell type differed in ibex compared to mouflon and chamois. The positive T-CD3 immunolabeling of all the halo cells confirmed their T-lymphocyte nature. Aquaglyceroporin expression patterns were similar among species, except for differences in AQP7 and AQP10 immunolocalisation in ibex. Species-specific differences in epididymal sperm cryoresistance were confirmed. CONCLUSIONS: The epididymal epithelium of the three wild ruminants differ in their relative number of cell types and AQP immunolocalisation, which ultimately appears to affect cauda epidydimal spermatozoa cryoresistance. IMPLICATIONS: Our study provides information on the relevance of the quantitative composition and AQP pattern expression in epididymal lining epithelium on sperm cryoresistance.

Impact of rapid hypertrophy of tourniquet associating liver partition and portal vein ligation in the tumor progression pathways compared to two stage hepatectomy in patients with colorectal liver metastases.

BACKGROUND: It is not known if the inflammatory phenomena related to highly accelerated regeneration activate any signaling pathways that are associated with a major stimulus to colorectal liver metastases (CRLM) disease in tourniquet associating liver partition and portal vein ligation for staged hepatectomy (T-ALPPS) compared to two stage hepatectomy (TSH). METHODS: Between January 2012 and April 2018, we prospectively performed biopsies from future liver remnant and deportalized lobe in CRLM patients undergoing T-ALPPS in both stages. Immunohistopathological analysis was performed on the above tissue samples and compared to biopsy samples from patients who underwent TSH for CRLM at our center between September 2000 and August 2011. RESULTS: A total of 42 patients (20 TSH and 22 T-ALPPS) were included. There were no differences in the rates of recurrence, overall survival or any of the factors analyzed relating to tumor progression between stages 1 and 2. Regarding the anti-tumor effect, there was a significant reduction in the number of T-CD8 infiltrates in the second stage of TSH (12.5 vs. 5.5, p = 0.02). CONCLUSION: The results suggest that liver regeneration with T-ALPPS does not induce higher tumor progression or significant immunological changes in the tumor environment when compared to classical TSH.

Effectiveness of Intensively Applied Mirror Therapy in Older Patients with Post-Stroke Hemiplegia: A Preliminary Trial.

INTRODUCTION: The present work was carried out to determine the effectiveness of neuromuscular stimulation triggered by mirror therapy (MT) in older patients with post-stroke hemiplegia by two different intervention protocols, either intensively or spaced. METHODS: A preliminary trial conducted on Spanish rehabilitation centres was conducted. Forty older patients (>70 years) with diagnosed post-stroke hemiplegia were randomly distributed to intensive intervention group (5 times/week for 6 weeks) or to spaced intervention group (3 times/week for 10 weeks), which underwent a similar number of MT sessions (n = 30). Muscle strength and activity were measured at baseline and at the end of treatment. Functional ability was also evaluated. RESULTS: Although both interventions improved muscle activity parameters, intensive MT showed a significantly and statistically higher intervention effect on electromyographic activity (p < 0.001) and muscle strength (p < 0.001) than the spaced over time protocol. Attending to the Barthel Index scores, the effect on functionality was also greater in the intensive therapy group (p < 0.001), although the functional improvement measured by the Fugl-Meyer test was similar (p = 0.235). The effect of the interventions was independent of age and clinical antecedents. CONCLUSION: Intensive MT appears to be more effective than a more spaced over time therapy; therefore, at least in the older adults, this treatment protocol should be recommended in the post-stroke recovery of these patients. Further studies will confirm with certainty whether this treatment is the most suitable guideline for to treat these patients.

Biomarkers of Early Liver Graft Damage in Circulatory Death and Brain Death Donors: A Propensity Score Matching Analysis.

BACKGROUND: Donation after circulatory death (DCD) is related to a warm ischemia time and more complications compared with traditional donors (donation after brain death [DBD]). METHODS: This study included biopsy samples retrospectively collected from November 2014 to December 2018 to compare histologic and biological markers of DCD and DBD liver grafts. The analysis includes marker of early apoptosis (p21), senescence (telomerase reverse transcriptase [TERT]), cell damage (caspase-3 active), endothelial damage (vascular endothelial growth factor), stem cell (CD90), hypoxia (HIF1A), inflammatory activation (COX-2), and cross-organ allograft rejection (CD44). A propensity score matching (PSM) was used to match patients receiving DCD livers to those receiving DBD livers. We analyzed the immunohistochemical initial liver damage-related warm ischemia time. RESULTS: Positive staining expression of liver damage biomarkers (COX-2, CD44, TERT, HIF1A, and CD90) was found, but no significant differences were found between DCD and DBD and with ischemic cholangiopathy. After PSM, there was a significant relationship between CD90 and male donors (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.07-0.91), TERT with donor sodium (OR, 1.11; 95% CI, 1.02-1.2), HIF1A with steatosis (OR, 0.33; 95% CI, 0.13-0.83), and CD44 with donor vasoactive drugs (OR, 0.36; 95% CI, 0.13-1) and glutamic oxaloacetic transaminase 1 week increase (OR, 1.01; 95% CI, 1-1.03). CONCLUSIONS: DCD immunohistochemical initial liver damage was found to behave similarly to DBD. The increase in complications and cholangiopathy associated with warm ischemia could be related to a different later phenomenon.

Seasonal variation in sperm freezability associated with changes in testicular germinal epithelium in domestic (Ovis aries) and wild (Ovis musimon) sheep.

The aim of this study was to examine ovine sperm cryoresistance during the rutting season (RS) and its association with sperm head area and seminiferous epithelium proliferation. Small ruminants show fluctuating testosterone levels throughout the year, which could interfere with spermatogenesis and sperm cryopreservation. Ejaculates, testicular biopsies and blood were collected during the middle and at the end of the RS (Middle-RS vs End-RS) during periods of high and low testosterone levels in Merino and Mouflon rams. Fresh and frozen-thawed sperm quality, sperm morphometry, seminiferous tubule morphometry and testicular proliferation markers (proliferating cell nuclear antigen, proliferation marker protein Ki-67 and transcription factor GATA-4) were evaluated. Post-thaw sperm viability was higher in the End-RS group in both Merino (69.9±8.2 vs 41.6±7.3%; P=0.020) and Mouflon rams (40.9±3.3 vs 24.2±5.0%; P=0.008). Mouflons had larger sperm head area at the End-RS (38.3±0.2 vs 34.3±0.1µm2; P=0.029), whereas there was no difference between Merino groups (35.7±0.5 vs 34.8±1.0µm2). Seminiferous tubule morphometry and proliferation markers showed higher levels of germinal epithelium proliferation in the Middle-RS of both species. In conclusion, sperm freezability is affected during the RS in domestic and wild rams, which could be correlated with changes that occur during spermatogenesis, since there is an effect of season on cell proliferation in the testis.

Local Gastrointestinal Injury Exacerbates Inflammation and Dopaminergic Cell Death in Parkinsonian Mice.

The cause of progressive degeneration in Parkinson's disease is not clear, although, in the last years, different studies have suggested that both brain and peripheral inflammation could play a key role in the progression of this disorder. In our study, we aimed to analyze the effect of an acute inflammation confined to the colon on dopaminergic neuronal death and glial response in mice intoxicated with MPTP. The results obtained show a very significant decrease of dopaminergic neurons in the SNpc as well as a significant decrease of dopaminergic fibers in the striatum of the MPTP+DSS-treated group compared with the control animals. In addition, there was a significant exacerbation of microglial and astrocytes activation in MPTP+DSS animals compared with the control group. This data suggests that a specific gastrointestinal injury, which induces a systemic inflammatory response, is able to exacerbate cell death mechanisms of the remaining dopaminergic neurons and then contributes to the persistent progression of the disease. These results leave open new lines of research on the role of exclusive colonic inflammation and the progression of nigrostriatal dopaminergic degeneration.

Histopathological and Immunohistochemical Analysis of the Use of Collagen Dressing as a Reinforcement of Esophagic Anastomosis in a Rat Experimental Model. / Análisis histopatológico e inmunohistoquímico del uso del apósito de colágeno como refuerzo de anastomosis esofágica en un modelo experimental en rata.

INTRODUCTION: One of the most severe complications after esophaguectomy is anastomotic dehiscence. The use of collagen sponges could be an effective way to resolve this complication. Our objective was to perform an experimental model of esophageal anastomosis in rats to study these mechanisms. METHODS: A total of 50 Sprague-Dawley rats were used divided into 2 groups, Tachosil® group (n=25) and control group (n=25). After the section of the abdominal esophagus a single-layer esophago-gastric anastomosis was performed reinforced with 1cm of Tachosil® wrapping the anastomosis in group 1. A functional study was performed using manometry as well as histopathological and immunohistochemical studies for angiogenic, fibrogenic and growth factors. RESULTS: The mortality in our series was 8% in the collagen dressing group, compared to 36% in the control group. When esophageal manometry was performed, the dehiscence pressure was higher in the reinforced anastomosis, On microscopical analysis, in the collagen dressing group a profuse inflammatory reaction with abundant neutrophils and macrophages surrounded by a connective matrix with fibroblasts and blood vessels was observed, The expression of VEGF, FGF1 and FGF2 was noticeably higher in the collagen dressing group. CONCLUSIONS: These results show that the application of collagen dressing facilitates tissue reparation phenomena, and therefore could be very useful as a reinforcement of esophago-gastric anastomosis to prevent dehiscence.

Differential profile of activated regulatory T cell subsets and microRNAs in tolerant liver transplant recipients.

Regulatory T cells (Tregs) play a potential role in operational tolerance in liver transplantation (LT) patients, and microRNAs (miRNAs) are known to be involved in immunological responses and tolerance. Thus, we analyzed the implication of different peripheral blood Treg subsets and miRNAs on LT tolerance in 24 tolerant (Tol) and 23 non-tolerant (non-Tol) LT recipients by cellular, genetic, and epigenetic approximation. Non-Tol patients had a lower demethylation rate of the forkhead box P3 (FOXP3) regulatory T cell-specific demethylated region (TSDR) than Tol patients that correlated with the frequency of circulating Tregs. Tol patients presented a different signature of Treg subset markers compared with non-Tol patients with increased expression of HELIOS and FOXP3 and a higher proportion of latency-associated peptide (LAP)+ Tregs and CD45RA- human leukocyte antigen D related (HLA-DR)+ activated effector-memory Tregs. The expression of miR95, miR24, miR31, miR146a, and miR155 was higher in Tol than in non-Tol patients and was positively correlated with activated Treg markers. In conclusion, these data suggest that activated effector-memory Tregs and a TSDR-demethylation state of Tregs may play a role in the complex system of regulation of LT tolerance. In addition, we describe a set of miRNAs differentially expressed in human LT Tol patients providing suggestive evidence that miRNAs are implied in the preservation of self-tolerance as mediated by Tregs. Liver Transplantation 23 933-945 2017 AASLD.

SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer.

The development of metastases largely relies on the capacity of cancer cells to invade extracellular matrices (ECM) using two invasion modes termed 'mesenchymal' and 'amoeboid', with possible transitions between these modes. Here we show that the SCN4B gene, encoding for the ß4 protein, initially characterized as an auxiliary subunit of voltage-gated sodium channels (NaV) in excitable tissues, is expressed in normal epithelial cells and that reduced ß4 protein levels in breast cancer biopsies correlate with high-grade primary and metastatic tumours. In cancer cells, reducing ß4 expression increases RhoA activity, potentiates cell migration and invasiveness, primary tumour growth and metastatic spreading, by promoting the acquisition of an amoeboid-mesenchymal hybrid phenotype. This hyperactivated migration is independent of NaV and is prevented by overexpression of the intracellular C-terminus of ß4. Conversely, SCN4B overexpression reduces cancer cell invasiveness and tumour progression, indicating that SCN4B/ß4 represents a metastasis-suppressor gene.

Parp-1 genetic ablation in Ela-myc mice unveils novel roles for Parp-1 in pancreatic cancer.

Pancreatic cancer has a dismal prognosis and is currently the fourth leading cause of cancer-related death in developed countries. The inhibition of poly(ADP-ribose) polymerase-1 (Parp-1), the major protein responsible for poly(ADP-ribosy)lation in response to DNA damage, has emerged as a promising treatment for several tumour types. Here we aimed to elucidate the involvement of Parp-1 in pancreatic tumour progression. We assessed Parp-1 protein expression in normal, preneoplastic and pancreatic tumour samples from humans and from K-Ras- and c-myc-driven mouse models of pancreatic cancer. Parp-1 was highly expressed in acinar cells in normal and cancer tissues. In contrast, ductal cells expressed very low or undetectable levels of this protein, both in a normal and in a tumour context. The Parp-1 expression pattern was similar in human and mouse samples, thereby validating the use of animal models for further studies. To determine the in vivo effects of Parp-1 depletion on pancreatic cancer progression, Ela-myc-driven pancreatic tumour development was analysed in a Parp-1 knock-out background. Loss of Parp-1 resulted in increased tumour necrosis and decreased proliferation, apoptosis and angiogenesis. Interestingly, Ela-myc:Parp-1(-/-) mice displayed fewer ductal tumours than their Ela-myc:Parp-1(+/+) counterparts, suggesting that Parp-1 participates in promoting acinar-to-ductal metaplasia, a key event in pancreatic cancer initiation. Moreover, impaired macrophage recruitment can be responsible for the ADM blockade found in the Ela-myc:Parp-1(-/-) mice. Finally, molecular analysis revealed that Parp-1 modulates ADM downstream of the Stat3-MMP7 axis and is also involved in transcriptional up-regulation of the MDM2, VEGFR1 and MMP28 cancer-related genes. In conclusion, the expression pattern of Parp-1 in normal and cancer tissue and the in vivo functional effects of Parp-1 depletion point to a novel role for this protein in pancreatic carcinogenesis and shed light into the clinical use of Parp-1 inhibitors.