Progression of atherosclerosis in ApoE-deficient mice that express distinct molecular forms of TNF-alpha

TNFalpha (TNF) critically regulates inflammation-driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno-modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE(-/-) mice were studied: one expressing wild-type TNF (WT-TNF); one expressing exclusively a mutated non-cleavable form of TNF (KI-TNF); and one deficient in TNF (KO-TNF). Mice aged 5 weeks were fed the high-fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high-fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO-TNF mice compared with those in WT-TNF mice, whereas in KI-TNF mice, they were intermediate between KO- and WT-TNF mice but not significantly different from these two groups. In aortas, lesions in KI-TNF were comparable to those of KO-TNF, both being significantly lower than those in WT-TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL-6, IFNgamma, IL-10, and Foxp3 mRNAs in spleens and production of IL-6, IL-10, MCP-1, RANTES, and TNFR-2 by peritoneal macrophages at T15 of the high-fat diet showed a decrease in pro-inflammatory status, more marked in KO-TNF than in KI-TNF mice. Apoptosis was reduced only in KO-TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild-type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF-dependent progression of atherosclerosis is probably linked to the differential production of pro-inflammatory mediators whether tmTNF is preponderant or essentially cleaved. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley ; Sons, Ltd.

Femoral morphology differs between deficient and excessive acetabular coverage

Structural deformities of the femoral head occurring during skeletal development (eg, Legg-Calvé-Perthes disease) are associated with individual shapes of the acetabulum but it is unclear whether differences in acetabular shape are associated with differences in proximal femoral shape. We questioned whether the amount of acetabular coverage influences femoral morphology. We retrospectively compared the proximal femoral anatomy of 50 selected patients (50 hips) with developmental dysplasia of the hip (lateral center-edge angle [LCE] < or = 25 degrees ; acetabular index > or = 14 degrees ) with 45 selected patients (50 hips) with a deep acetabulum (LCE > or = 39 degrees ). Using MRI arthrography we measured head sphericity, epiphyseal shape, epiphyseal extension, and femoral head-neck offset. A deep acetabulum was associated with a more spherical head shape, increased epiphyseal height with a pronounced extension of the epiphysis towards the femoral neck, and an increased offset. In contrast, dysplastic hips showed an elliptical femoral head, decreased epiphyseal height with a less pronounced extension of the epiphysis, and decreased head-neck offset. Hips with different acetabular coverage are associated with different proximal femoral anatomy. A nonspherical head in dysplastic hips could lead to joint incongruity after an acetabular reorientation procedure. LEVEL OF EVIDENCE: Level IV, retrospective comparative study. See the Guidelines for Authors for a complete description of levels of evidence.

L-Selectin-deficient SJL and C57BL/6 mice are not resistant to experimental autoimmune encephalomyelitis

L-selectin has been suggested to play a role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Here we demonstrate that L-selectin(-/-) SJL mice are susceptible to proteolipid protein (PLP)-induced EAE because the compromised antigen-specific T cell proliferation in peripheral lymph nodes is fully compensated by the T cell response raised in their spleen. Transfer of PLP-specific T cells into syngeneic recipients induced EAE independent of the presence or absence of L-selectin on PLP-specific T cells or in the recipient. Leukocyte infiltration into the central nervous system parenchyma was detectable independent of the mode of disease induction and the presence or absence of L-selectin. In addition, we found L-selectin(-/-) C57BL/6 mice to be susceptible to myelin oligodendrocyte glycoprotein-induced EAE. Taken together, we demonstrate that in SJL and C57BL/6 mice L-selectin is not required for EAE pathogenesis. The apparent discrepancy of our present observation to previous findings, demonstrating a role of L-selectin in EAE pathogenesis in C57BL/6 mice or myelin-basic protein (MBP)-specific TCR-transgenic B10.PL mice, may be attributed to background genes rather than L-selectin and to a unique role of L-selectin in EAE pathogenesis in MBP-TCR-transgenic mice.

Determination of carbohydrate-deficient transferrin in human serum with capillary zone electrophoresis. Sample preparation strategies for the removal of interferences caused by increased levels of immunoglobulins

Capillary zone electrophoresis (CZE) in fused-silica capillaries is an effective analytical approach for the separation and determination of the transferrin (Tf) isoforms and thus carbohydrate-deficient transferrin (CDT) in human serum. Sera of patients with progressed liver cirrhosis are prone to interferences in the beta region which prevent the proper determination of CDT by CZE without additional sample preparation. Efforts to identify, reduce or even eliminate these interferences have been undertaken. Data obtained by ultrafiltration, affinity subtraction procedures using protein A, protein L and antibodies against immunoglobulins or Tf, and immunopurification of Tf suggest that the interferences in the patient sera are caused by increased levels of IgA and IgM and are best eliminated by immunopurification. Avian IgY antibody spin column immunocapture of serum Tf followed by CZE analysis of the stripped and concentrated fraction is shown to provide an attractive approach for CDT monitoring in sera with beta region interferences.

Determination of carbohydrate-deficient transferrin in human serum by two capillary zone electrophoresis methods and a direct immunoassay: comparison of patient data

Data obtained with two CZE assays for determining carbohydrate-deficient transferrin (CDT) in human serum under routine conditions, the CAPILLARYS CDT and the high-resolution CEofix (HR-CEofix) CDT methods, are in agreement with patient sera that do not exhibit interferences, high trisialo-transferrin (Tf) levels or genetic variants. HR-CEofix CDT levels are somewhat higher compared to those obtained with the CAPILLARYS method and this bias corresponds to the difference of the upper reference values of the two assays. The lower resolution between disialo-Tf and trisialo-Tf observed in the CAPILLARYS system (mean: 1.24) compared to HR-CEofix (mean: 1.74) is believed to be the key for this difference. For critical sera with high trisialo-Tf levels, genetic variants, or certain interferences in the beta-region, the HR-CEofix approach is demonstrated to perform better than CAPILLARYS. However, the determination of CDT with the HR-CEofix method can also be hampered with interferences. Results with disialo-Tf values larger than 3% in the absence of asialo-Tf should be evaluated with immunosubtraction of Tf and possibly also confirmed with another CZE method or by HPLC. Furthermore, data gathered with the N Latex CDT direct immunonephelometric assay suggest that this assay can be used for screening purposes. To reduce the number of false negative results, CDT data above 2.0% should be confirmed using a separation method.

Accelerated hypertrophic chondrocyte kinetics in GDF-7 deficient murine tibial growth plates

The Growth/Differentiation Factors (GDFs) are a subgroup of the Bone Morphogenetic Proteins (BMPs) well known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling molecules, GDF-5, have recently been shown to exhibit a decreased rate of endochondral bone growth in the proximal tibia due to a significantly longer hypertrophic phase duration. GDF-7 is a related family member, which exhibits a high degree of sequence identity with GDF-5. The purpose of the present study was to determine whether GDF-7 deficiency also alters the endochondral bone growth rate in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-7 -/- mice and wild type control littermates were examined. GDF-7 deficiency resulted in a statistically significant increase in growth rate (+26%; p = 0.0084) and rate of cell loss at the chondrosseous junction (+25%; p = 0.0217). Cells from GDF-7 deficient mice also exhibited a significantly shorter hypertrophic phase duration compared to wild type controls (-27%; p = 0.0326). These data demonstrate that, in the absence of GDF-7, the rate of endochondral bone growth is affected through the modulation of hypertrophic phase duration in growth plate chondrocytes. These findings further support a growing body of evidence implicating the GDFs in the formation, maturation, and maintenance of healthy cartilage.

Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs

BACKGROUND: Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-alpha(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. METHODS: Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by chromium-release assays using human serum and freshly isolated NK cells. RESULTS: Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. CONCLUSIONS: The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.

Altered hypertrophic chondrocyte kinetics in GDF-5 deficient murine tibial growth plates

The growth/differentiation factors (GDFs) are a subgroup of the bone morphogenetic proteins best known for their role in joint formation and chondrogenesis. Mice deficient in one of these signaling proteins, GDF-5, exhibit numerous skeletal abnormalities, including shortened limb bones. The primary aim of this study was determine whether GDF-5 deficiency would alter the growth rate in growth plates from the long bones in mice and, if so, how this is achieved. Stereologic and cell kinetic parameters in proximal tibial growth plates from 5-week-old female GDF-5 -/- mice and control littermates were examined. GDF-5 deficiency resulted in a statistically significant reduction in growth rate (-14%, p=0.03). The effect of genotype on growth rate was associated with an altered hypertrophic phase duration, with hypertrophic cells from GDF-5 deficient mice exhibiting a significantly longer phase duration compared to control littermates (+25%, p=0.006). These data suggest that one way in which GDF-5 might modulate the rate of endochondral bone growth could be by affecting the duration of the hypertrophic phase in growth plate chondrocytes.

The identification of cerebral volume changes in treated growth hormone-deficient adults using serial 3D MR image processing

A pilot study to detect volume changes of cerebral structures in growth hormone (GH)-deficient adults treated with GH using serial 3D MR image processing and to assess need for segmentation prior to registration was conducted.

A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic

Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation. CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders. The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2. Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients. Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients. The mutation cosegregated with the disease in a Mendelian recessive manner. Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S. cerevisiae alg5 and alg6 deficiency. By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype. The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.

The etiology of cleft palate formation in BMP7-deficient mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.

Proteasome inhibitor (MG132) rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx (5cv) mice

The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (l Na) were both decreased in cardiomyocytes of dystrophin-deficient mdx (5cv) mice. In this study, wild-type and mdx (5cv) mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and l Na but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

Loss of β1-integrin-deficient cells during the development of endoderm-derived epithelia

Beta1-integrins (beta1) represent cell surface receptors which mediate cell-matrix and cell-cell interactions. Fässler and Meyer described chimeric mice containing transgenic cells that express the LacZ gene instead of the beta1 gene. They observed beta1-negative cells in all germ layers at embryonic day E 8.5. Later in development, using a glucose phosphate isomerase assay of homogenized tissue samples, high levels of transgenic cells were found in skeletal muscle and gut, low levels in lung, heart, and kidney and none in the liver and spleen (Fässler and Meyer 1995). In order to study which cell types require beta1 during development of the primitive gut including its derivatives, chimeric fetuses containing 15 to 25% transgenic cells were obtained at days E 14.5 and E 15.5. They were LacZ (beta-galactosidase) stained "en bloc" and cross-sectioned head to tail. In esophagus, trachea, lung, stomach, hindgut, and the future urinary bladder, we observed various mesoderm-derived beta1-negative cells (e.g. fibroblasts, chondrocytes, endothelial cells, and smooth muscle cells) but no beta1-negative epithelial cells. Since the epithelia of lung, esophagus, trachea, stomach, hindgut, and urinary bladder are derived from the endodermal gut tube, we hypothesize that beta1 is essential for the development and/or survival of the epithelia of the fore- and hindgut and its derivatives.