Induction of cartilage integration by a chondrocyte/collagen-scaffold implant

The integration of implanted cartilage is a major challenge for the success of tissue engineering protocols. We hypothesize that in order for effective cartilage integration to take place, matrix-free chondrocytes must be induced to migrate between the two tissue surfaces. A chondrocyte/collagen-scaffold implant system was developed as a method of delivering dividing cells at the interface between two cartilage surfaces. Chondrocytes were isolated from bovine nasal septum and seeded onto both surfaces of a collagen membrane to create the chondrocyte/collagen-scaffold implant. A model of two cartilage discs and the chondrocyte/collagen-scaffold sandwiched in between was used to effect integration in vitro. The resulting tissue was analysed histologically and biomechanically. The cartilage–implant–cartilage sandwich appeared macroscopically as one continuous piece of tissue at the end of 40 day cultures. Histological analysis showed tissue continuum across the cartilage–scaffold interface. The integration was dependent on both cells and scaffold. Fluorescent labeling of implanted chondrocytes demonstrated that these cells invade the surrounding mature tissue and drive a remodelling of the extracellular matrix. Using cell-free scaffolds we also demonstrated that some chondrocytes migrated from the natural cartilage into the collagen scaffold. Quantification of integration levels using a histomorphometric repair index showed that the chondrocyte/collagen-scaffold implant achieved the highest repair index compared to controls, reflected functionally through increased tensile strength. In conclusion, cartilage integration can be achieved using a chondrocyte/collagen-scaffold implant that permits controlled delivery of chondrocytes to both host and graft mature cartilage tissues. This approach has the potential to be used therapeutically for implantation of engineered tissue.     

Genetic basis of neurodevelopmental disorders in 103 Jordanian families

We recruited 103 families from Jordan with neurodevelopmental disorders (NDD) and patterns of inheritance mostly suggestive of autosomal recessive inheritance. In each family, we investigated at least one affected individual using exome sequencing and an in-house diagnostic variant interpretation pipeline including a search for copy number variation. This approach led us to identify the likely molecular defect in established disease genes in 37 families. We could identify 25 pathogenic nonsense and 11 missense variants as well as 3 pathogenic copy number variants and 1 repeat expansion. Notably, 11 of the disease-causal variants occurred de novo. In addition, we prioritized a homozygous frameshift variant in PUS3 in two sisters with intellectual disability. To our knowledge, PUS3 has been postulated only recently as a candidate disease gene for intellectual disability in a single family with three affected siblings. Our findings provide additional evidence to establish loss of PUS3 function as a cause of intellectual disability.     

Somatic FGF9 mutations in colorectal and endometrial carcinomas associated with membranous beta-catenin

We previously described striking molecular features including high frequency of membranous beta-catenin in subsets of familial colon cancers with as yet unknown predisposition. We hypothesized that such tumors might carry mutations in Wnt/beta-catenin target genes. Fibroblast growth factor 9 (FGF9) was an attractive target, as it maps to a common area of loss of heterozygosity (LOH) in colorectal carcinomas on 13q12.11. Here, we report, for the first time, the occurrence of FGF9 mutations in human cancers. We found a total of six distinct FGF9 mutations including one frameshift, four missense, and one nonsense, in 10 (six colorectal and four endometrial) out of 203 tumors and cell lines. The frameshift mutation was detected in five different tumors. Mapping of these mutations onto the crystal structure of FGF9 predicted that they should all lead to loss of function albeit through variable mechanisms. The p.R173K mutation should diminish ligand affinity for heparin/heparan sulfate, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations should negatively impact ligand's interaction with receptor, while p.G84E and p.E142X (FGF9(Delta142-208)) mutations should interfere with ligand folding. Consistent with these structural predictions, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations impaired the ability of ligand to activate mitogen-activated protein kinase (MAPK) cascade in cultured cells expressing FGF receptors. LOH was observed in seven out of nine FGF9 mutant tumors, supporting the predicted loss of function. Interestingly, eight out of 10 (80%) of the FGF9 mutant tumors showed normal membranous beta-catenin expression and the absence of mutation in the beta-catenin gene (CTNNB1). These data suggest that FGF9 plays a role in colorectal and endometrial carcinogenesis.     

Successful pregnancy in an ovarian agenesis patient after modified natural cycle IVF oocyte donation

The recovery of a mature oocyte from a modified natural cycle followed by in-vitro fertilization (nIVF) is an attractive alternative to conventional IVF, involving ovarian stimulation, in the treatment of female infertility. Ovarian agenesis is a rare disorder resulting in primary amenorrhoea and infertility in affected females. A couple sought help for infertility due to ovarian agenesis of the female partner and decided to pursue treatment utilizing oocyte donation. Modified natural-cycle egg retrieval was carried out on the donor; one mature oocyte was retrieved and underwent IVF using a sperm sample from the male partner. A good-quality embryo was transferred. A viable pregnancy was confirmed by ultrasound scan and resulted in the delivery of a healthy baby boy at 36 weeks' gestation. This is the second published report of an ongoing clinical pregnancy and subsequent birth resulting from oocyte donation recovered during a modified natural cycle. The use of less invasive assisted reproduction techniques such as nIVF can be used in oocyte donation cycles successfully.     

Optical coherence tomography and vessel diameter changes after intravitreal bevacizumab in diabetic macular oedema

Purpose: To assess the effect of intravitreal bevacizumab on diabetic macular oedema (DMO) and retinal vessel calibres. Methods: We performed a consecutive case series study in which 10 consecutive eyes with diffuse DMO, two of which had not previously been treated, received an intravitreal injection of bevacizumab 1 mg, which was followed by two more injections at 6‐week intervals. Fundus photography and optical coherence tomography (OCT) were carried out at baseline immediately before injection and at 1, 2.5 and 4 months after the first injection. Outcome measures were best corrected visual acuity (BCVA) in Early Treatment Diabetic Retinopathy Study letters, macular volume, foveal subfield thickness and vessel diameter measurement. Results: Intravitreal administration of bevacizumab was followed by a mean increase in BCVA of 7.3 ± 17 (mean ± standard deviation) letters between baseline and month 4, which was 1 month after the last injection (p < 0.0001). This was accompanied by a reduction in mean macular volume from 9.90 ± 1.9 mm³ to 8.96 ± 2.4 mm³ (p = 0.002) and in foveal subfield thickness from 447 ± 117 μm to 388 ± 117 μm (p = 0.03). Two eyes with early proliferative diabetic retinopathy lost all signs of proliferation without any evidence of fibrosis. Although there was a trend towards vasoconstriction, the changes in vessel diameters (arteries and veins) after 4 months of intravitreal Avastin injection were not statistically significant (p = 0.9 and p = 0.17, respectively). Foveal thickness in non‐injected fellow eyes with DMO changed from 428 ± 153 μm at baseline to 383 ± 151 μm at 4 months (p = 0.1), which did not reach statistical significance. Conclusions: Intravitreal bevacizumab 1 mg every 6 weeks was followed by a moderate reduction in DMO without normalization of foveal and macular thickness. Our observations suggest that a larger study where patients are examined sooner after injection is needed to elucidate the potential relationship between changes in retinal vessel diameters and thickness changes in DMO.     

Both lipid and protein intakes stimulate increased generation of reactive oxygen species by polymorphonuclear leukocytes and mononuclear cells

BACKGROUND: It was recently shown that glucose challenge leads to increased generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNCs). OBJECTIVE: To further elucidate the relation between nutrition and ROS generation, we investigated the effect of lipid and protein challenges on ROS generation by leukocytes. DESIGN: After having fasted overnight, one group of healthy subjects consumed a carbohydrate- and protein-free cream preparation (1257 kJ) and another group of healthy subjects consumed an equienergetic pure preparation of casein. Sequential blood samples were obtained after the intake of cream and casein. ROS were measured by chemiluminescence after stimulation by N-formyl-methionyl-leucinyl-phenylalanine. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) and alpha-tocopherol was measured by HPLC. RESULTS: ROS generation by MNCs and PMNs increased significantly 1, 2, and 3 h after cream intake and 1 h after protein intake. Cholesterol concentrations did not change significantly, whereas triacylglycerol concentrations increased significantly 2 h after cream intake. Total TBARS concentrations increased 1 h after cream intake and remained elevated 3 h after intake, but the increase was not significant when corrected for changes in triacylglycerol. After casein intake, total cholesterol, triacylglycerol, and TBARS concentrations did not change significantly. alpha-Tocopherol concentrations did not change significantly after either cream or casein intake. CONCLUSIONS: Both fat and protein intakes stimulate ROS generation. The increase in ROS generation lasted 3 h after cream intake and 1 h after protein intake. Cream intake also caused a significant and prolonged increase in lipid peroxidation. These data are important because increased ROS generation and lipid peroxidation are key events in atherogenesis.     

Restoring mismatch repair does not stop the formation of reciprocal translocations in the colon cancer cell line HCA7 but further destabilizes chromosome number

An important anticarcinogenic function of the mismatch repair (MMR) system is its role in preventing recombination between similar, but nonidentical (homeologous) sequences, thus preventing chromosomal rearrangements. We recently identified a novel chromosomal instability (CIN) phenotype in an MMR defective colon cancer cell line (HCA7) characterized by an ongoing tendency to multiple reciprocal chromosomal translocations. To analyse the relation between MMR and chromosomal changes more closely, the HCA7 stem clone was divided into three stocks. The first was stably transfected with MLH1 expression plasmid, the second was regularly exposed to the demethylating agent 5-azacytidin to re-express the hypermethylated MLH1 gene, and the third was an unmanipulated control stock. All stocks were propagated in vitro for 55-80 passages and, furthermore, some of the early passages were irradiated to induce DNA double-strand breaks. Multiplex-fluorescent in situ hybridization (M-FISH) analysis showed that all three stocks acquired varying numbers of reciprocal translocations and other structural changes at some point. Interestingly, the control stock, which is MMR defective, maintained its numerical chromosomal stability, while some of the MMR-proficient clones showed additional numerical instability. Although the control stock was less sensitive to irradiation, its surviving clones showed marked stability of chromosome structure and number compared to the MMR-competent stocks. These results show that restoring MMR does not prevent the development of reciprocal translocations but rather predisposes cells to numerical CIN after irradiation. Thus, the accumulating data suggest that MMR defect may not be necessary for the development of reciprocal chromosomal translocations but might be permissive.