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Data on the different HLA-antibody (Ab) categories in pediatric kidney recipients developing de novo donor-specific Abs (DSA) after transplantation are scarce. We retrospectively evaluated 82 consecutive nonsensitized pediatric recipients of a first kidney graft for de novo HLA Ab occurrence and antigen specificity. At a median follow-up of 6 years, 29% of patients developed de novo DSA, while 45% had de novo non-DSA. DSA appeared at 25-month median time post-transplant and were mostly directed toward HLA-DQ antigens. Considering each HLA antigen, the estimated rate of DQ DSA (7.55 per 100 person-years) was much higher than the rates observed for non-DQ DSA. The HLA-DQ Ab recognized determinants of the DQβ chain in 70% of cases, α chain in 25% of cases, and both chains in one patient. Non-DSA peaked earlier than DSA, and were largely directed against HLA class I specificities that belonged to HLA-A- and HLA-B-related cross-reacting epitope groups (CREG) in 56% of cases. Our results indicate a need for evaluating HLA-DQ compatibilities in kidney allocation, in order to minimize post-transplant development of de novo DSA, known to be responsible for antibody-mediated rejection and graft loss., (© 2014 Steunstichting ESOT.)

The Italian Space Agency, in line with its scientific strategies and the National Utilization Plan for the International Space Station (ISS), contracted Thales Alenia Space Italia to design and build a spaceflight payload for rodent research on ISS: the Mice Drawer System (MDS). The payload, to be integrated inside the Space Shuttle middeck during transportation and inside the Express Rack in the ISS during experiment execution, was designed to function autonomously for more than 3 months and to involve crew only for maintenance activities. In its first mission, three wild type (Wt) and three transgenic male mice over-expressing pleiotrophin under the control of a bone-specific promoter (PTN-Tg) were housed in the MDS. At the time of launch, animals were 2-months old. MDS reached the ISS on board of Shuttle Discovery Flight 17A/STS-128 on August 28(th), 2009. MDS returned to Earth on November 27(th), 2009 with Shuttle Atlantis Flight ULF3/STS-129 after 91 days, performing the longest permanence of mice in space. Unfortunately, during the MDS mission, one PTN-Tg and two Wt mice died due to health status or payload-related reasons. The remaining mice showed a normal behavior throughout the experiment and appeared in excellent health conditions at landing. During the experiment, the mice health conditions and their water and food consumption were daily checked. Upon landing mice were sacrificed, blood parameters measured and tissues dissected for subsequent analysis. To obtain as much information as possible on microgravity-induced tissue modifications, we organized a Tissue Sharing Program: 20 research groups from 6 countries participated. In order to distinguish between possible effects of the MDS housing conditions and effects due to the near-zero gravity environment, a ground replica of the flight experiment was performed at the University of Genova. Control tissues were collected also from mice maintained on Earth in standard vivarium cages.

Dufd1 (DUF729 domain containing 1) is related to Mtfr1 (mitochondrial fission regulator 1), a gene involved in the regulation of antioxidant activity in the mouse testis. The present study was undertaken to better understand their role in regulating mitochondrial architecture and function in the mouse. We show that Dufd1 is expressed as a 2 kb mRNA and has a more specific tissue pattern compared to Mtfr1, with highest level of expression in testes, lower level in spleen, and negligible levels in other organs and/or tissues. In the male gonad, Dufd1 mRNA expression increases during postnatal development, similarly to Mtfr1. In situ hybridization and real-time PCR analyses show that Dufd1 is expressed in the seminiferous tubules by middle-late pachytene spermatocytes and spermatids. In transfected cells, the Dufd1-tagged protein is located in mitochondria, associated with the tips of mitochondrial tubules and to tubules constrictions, and induces mitochondrial fission although with a lesser efficiency than Mtfr1. We also found that both endogenous Dufd1 and Mtfr1 proteins are associated with membrane-enriched subcellular fractions, including mitochondria. Inhibition of Mtfr1 and/or Dufd1 expression, in a testicular germ cells line, severely impairs O(2) consumption and indicates that both genes are required for mitochondrial respiration. Accordingly, analysis of testes mitochondria from Mtfr1-deficient mice reveals severely reduced O(2) consumption and ATP synthesis compared to wt animals. These data show that, in murine testis, Dufd1 and Mtfr1 have redundant functions related to mitochondrial physiology and represent genes with a potential role in testicular function., (© 2010 Wiley-Liss, Inc.)

Lipocalin-2 (LCN2) is a member of the lipocalin family, small secreted proteins functioning as modulators of many different physiological processes including cell differentiation, proliferation and apoptosis. LCN2 expression is also up-regulated in several pathological conditions, including inflammation and cancer. LCN2 synthesis has been described in epithelia, bone and cells of the immune system. Despite its wide expression the role of LCN2 remains to be fully elucidated. To better understand the role of this lipocalin in the bone/bone marrow system we generated transgenic mice over-expressing LCN2 specifically in bone under the control of a type I collagen promoter. In the bone marrow of these transgenic mice we observed an increased expression of SDF-1 that correlated with an increased number of CD34+/CXCR4+ (SDF-1 receptor) cells. To some extent, this appeared due to an enhanced cell proliferation rate. The higher level of the factor synthesis and the increased number of cells expressing its receptor was maintained during animal aging. Our results show that LCN2 could play a role in determining the number of CD34+/CXCR4+ precursor cells in the bone marrow thus contributing to the control of the bone marrow microenvironment., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Mtfr1/Chppr is a nuclear gene coding for a mitochondrial protein capable of inducing fission of this organelle in a sequence-specific manner. Here we show that in mice, Mtfr1/Chppr is ubiquitously expressed and displays the highest level of expression in pubertal and adult testes and in particular in spermatids and Leydig cells. To investigate Mtfr1 function in vivo, we analyzed homozygous mice null for this gene obtained through a gene trap approach. We show that these mice fail to express Mtfr1 and that in their testes several genes coding for enzymes involved in the defense against oxidative stress are downregulated. Among these, we studied in particular glutathione peroxidase 3 and show its expression in selected testis cell types. Furthermore, we demonstrate oxidative DNA damage specifically in testes of Mtfr1-deficient mice likely resulting from a reduced antioxidant activity. As a whole, these data suggest that Mtfr1 protects the male gonads against oxidative stress.

The RET gene is tightly regulated at the transcriptional level during embryo development, however in vitro experiments in cultured cells have failed to clarify the molecular mechanism of cell-type specificity of RET promoter activity. Therefore, we have generated transgenic mice in which the LacZ reporter gene is controlled by murine Ret promoter sequences to clarify in an in vivo model how this transcriptional regulation is achieved. We describe here the results of reporter gene expression in mice in which the transgene contained 380- and 1962-bp sequence upstream of the ATG start codon, derived from the mouse Ret promoter region, fused to the beta-galactosidase coding sequence. Transgenic mice showed well-defined patterns of beta-galactosidase staining obtained with both transgenes, suggesting that they were able per se to direct the reporter gene expression in specific districts, such as cranial ganglia, dorsal root ganglia, the heart and the kidney, partially recapitulating the profile of the endogenous Ret gene. In particular, proper expression in the developing excretory system seemed quite significant when considering that the appropriate regulation was obtained with a very short, 380 bp, fragment of Ret 5' flanking sequence.

Unlabelled: The role of Hedgehogs (Hh) in murine skeletal development was studied by overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice using the collagen II promoter/enhancer. Overexpression caused a lethal craniorachischisis with major alterations in long bones because of defects in chondrocyte differentiation., Introduction: Hedgehogs (Hhs) are a family of secreted polypeptides that play important roles in vertebrate development, controlling many critical steps of cell differentiation and patterning. Skeletal development is affected in many different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways cause severe abnormalities in the appendicular, axial, and cranial skeleton in man and other vertebrates., Materials and Methods: Genetic manipulation of mouse embryos was used to study in vivo the function of SHH in skeletal development. By DNA microinjection into pronuclei of fertilized oocytes, we have generated transgenic mice that express SHH specifically in chondrocytes using the cartilage-specific collagen II promoter/enhancer. Transgenic skeletal development was studied at different embryonic stages by histology. The expression pattern of specific chondrocyte molecules was studied by immunohistochemistry and in situ hybridization., Results: Transgenic mice died at birth with severe craniorachischisis and other skeletal defects in ribs, sternum, and long bones. Detailed analysis of long bones showed that chondrocyte differentiation was blocked at prehypertrophic stages, hindering endochondral ossification and trabecular bone formation, with specific defects in different limb segments. The growth plate was highly disorganized in the tibia and was completely absent in the femur and humerus, leading to skeletal elements entirely made of cartilage surrounded by a thin layer of bone. In this cartilage, chondrocytes maintained a columnar organization that was perpendicular to the bone longitudinal axis and directed toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was greatly increased in all cartilage, as well as the expression of parathyroid hormone-related protein (PTHrP) at the articular surface; while the expression of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of chondrocyte maturation, was greatly reduced and restricted to the displaced chondrocyte columns. Transgenic mice also revealed the ability of SHH to upregulate the expression of Sox9, a major transcription factor implicated in chondrocyte-specific gene expression, in vivo and in vitro, acting through the proximal 6.8-kb-long Sox9 promoter., Conclusion: Transgenic mice show that continuous expression of SHH in chondrocytes interferes with cell differentiation and growth plate organization and induces high levels and diffuse expression of Sox9 in cartilaginous bones.

Chain-terminating germline APC mutations are responsible for adenomatous polyposis coli (APC). In the present work, we tested the hypothesis that germline APC mutations may be present in some patients with a milder phenotype, i.e., multiple synchronous colorectal adenomas. Eighteen patients with 3 or more colorectal adenomas at endoscopy (within a 6-month period) were ascertained from a series of subjects undergoing endoscopic examination. Their blood DNAs were analysed for the presence of germline mutations in the APC coding region by single-strand polymorphism analysis. Ten unrelated polyp-free subjects and 101 unrelated APC patients were used as controls in the molecular analyses. Five of the eighteen patients carried novel germline APC variants or rare polymorphisms. These were various in site (from the splice acceptor site of intron 7 to the end of exon 15) and type (splice-site, missense, and chain-terminating mutations). Only one of ten polyp-free individuals carried a silent APC variant and none of these variants was found in the 101 APC controls. A first- or second-degree family history of colorectal cancer was reported by 4 of the 5 patients carrying a germline APC variant. In conclusion, novel APC germline variants were detected in patients with multiple synchronous adenomas. This suggests that the development of sporadic adenomas, in some instances, is associated with the presence of minor germline variants of the APC gene and that the spectrum of germline APC functional mutations may be larger than previously thought.

APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor proteins. Both RNA and protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT-PCR strategies were used to characterize mutant and normal APC protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC protein was detected in six of eight cell lines with APC mutations located 5' of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5' of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two- to threefold decrease in RNA concentration is common when APC alleles carry chain-terminating mutations. They also suggest that the severe decrease of truncated APC protein observed in some cell lines is due to mechanisms acting at the protein level.

Fifty-nine colonic adenomas and 6 hyperplastic colonic polyps were analyzed by single-strand conformation polymorphism analysis for mutations in the adenomatous polyposis coli gene (APC). Frameshifts and premature stop codons in at least one copy of APC were detected in 25 of these adenomas. Five adenomas carried 2 APC mutations. No mutations in APC were found in any of the 6 hyperplastic polyps. The detection of APC mutations increased with size and degree of dysplasia and in rectal as compared to colonic adenomas, although the association was not statistically significant. The frequency of detectable APC mutations was higher in tubulovillous and villous adenomas (10 of 13) than in tubular adenomas (15 of 45) (odds ratio, 6.67; 95% confidence limits, 1.39-41.83; P = 0.005). The significance of the association between the detection of APC mutations and a villous architecture was confirmed in multivariate analysis (relative risk, 6.67; 95% confidence limits, 1.54-28.8; P = 0.005). In conclusion, APC mutation plays a role in adenoma progression; its frequency is significantly higher in lesions with a more villous morphology.

Twenty-four sporadic colorectal adenomas were analysed for the presence of allelic loss on the short arm of chromosome 17 as well as mutations in the K-ras and p53 genes. Chromosome 17p13 allelic loss was not present in 14 out of 14 informative cases. K-ras mutations were observed in 15 out of 24 cases. A p53 gene mutation (GGC-->GAC at codon 245) was detected in two biopsies taken at a four year interval from a recurrent rectal villous adenoma. Both biopsies also contained the same K-ras gene mutation (GGT-->GTT at codon 12). The data from the recurrent rectal adenoma provide in vivo evidence that K-ras and p53 heterozygous mutations confer a proliferative advantage but together are not sufficient for malignant transformation.

Adenomatous polyposis coli is an autosomal dominant disease characterized by the development of hundreds of colorectal adenomas in young adults. If prophylactic colectomy is not performed, colorectal cancer develops in virtually all affected individuals by the fifth decade of life. All at-risk relatives older than 10 years of age need to be screened regularly by endoscopy. Recently, the gene responsible for the disease, the APC gene, was cloned. The finding of inactivating mutations of the APC gene in Italian APC patients allowed us to offer DNA-based diagnostic tests to these families.