Homozygous missense and nonsense mutations in BMPR1B cause acromesomelic chondrodysplasia-type Grebe.

Acromesomelic chondrodysplasias (ACDs) are characterized by disproportionate shortening of the appendicular skeleton, predominantly affecting the middle (forearms and forelegs) and distal segments (hands and feet). Here, we present two consanguineous families with missense (c.157T>C, p.(C53R)) or nonsense (c.657G>A, p.(W219*)) mutations in BMPR1B. Homozygous affected individuals show clinical and radiographic findings consistent with ACD-type Grebe. Functional analysis of the missense mutation C53R revealed that the mutated receptor was partially located at the cell membrane. In contrast to the wild-type receptor, C53R mutation hindered the activation of the receptor by its ligand GDF5, as shown by reporter gene assay. Further, overexpression of the C53R mutation in an in vitro chondrogenesis assay showed no effect on cell differentiation, indicating a loss of function. The nonsense mutation (c.657G>A, p.(W219*)) introduces a premature stop codon, which is predicted to be subject to nonsense-mediated mRNA decay, causing reduced protein translation of the mutant allele. A loss-of-function effect of both mutations causing recessive ACD-type Grebe is further supported by the mild brachydactyly or even non-penetrance of these mutations observed in the heterozygous parents. In contrast, dominant-negative BMPR1B mutations described previously are associated with autosomal-dominant brachydactyly-type A2.

SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer based on bronchial aspirates.

BACKGROUND: This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment. METHODS: Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance. RESULTS: Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]). CONCLUSIONS: Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.

Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses.

A prophylactic vaccine for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity. One current strategy to achieve such long term immune responses is a prime-boost vaccination approach using a DNA priming inoculation, followed by recombinant viral boost. In this report we use a novel prime-boost approach in which the priming injections consist of recombinant HIV-1 Gag protein mixed with cytosine phosphate guanosine oligodeoxynucleotide (CpG ODN), followed by recombinant adenoviral boost expressing HIV-1 Gag. Analysis of the immune responses indicates that HIV-1 Gag protein plus CpG ODN immunization alone induces potent humoral as well as Th1 and CD8+ T cell responses. Boosting with recombinant adenovirus strikingly enhances CD8+, but not Th1, T cell responses, resulting in CD8+ T cell responses far greater in magnitude than Th1 responses. Furthermore, the Th1 and CD8+ T cell responses following prime-boost immunization were seen in both lymphoid and peripheral mucosal organs and were sustained over several months. Together, these data suggest a new immunization approach for elicitation of long term humoral and cellular immune responses.

Contribution of interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 ligand pathways to the development of a pathological T-cell response in IL-10-deficient mice.

The ability of interleukin-10 (IL-10) to suppress accessory cell functions required for optimal T-cell activation makes it an important inhibitor of cell-mediated immunity. Thus, after infection with the protozoan parasite Toxoplasma gondii, IL-10 knockout (KO) mice develop a CD4(+)-T-cell-dependent shock-like reaction with high levels of IL-12 and gamma interferon (IFN-gamma) in serum, leading to death of mice during the acute phase of infection. Previous studies from this laboratory have shown that simultaneous blockade of CD28 and CD40 can prevent this lethal reaction by inhibiting the production of IFN-gamma. However, the blockade of costimulation did not affect systemic levels of IL-12. To better understand the relationship between IL-12 and the CD28 and CD40 pathways in mediating immune hyperactivity, antagonists of these factors were used to determine their effects on the development of a pathological T-cell response in IL-10 KO mice. Blockade of IL-12 or the CD28/B7 interaction alone did not affect survival; however, the combined blockade of both pathways resulted in decreased production of IFN-gamma and the survival of IL-10 KO mice. To assess the role of the two ligands for CD28, B7.1 and B7.2, IL-10 KO mice were treated with alphaIL-12 plus alphaB7.1 or alphaB7.2 or the combination of all three antibodies. These studies revealed that blockade of both B7 molecules is required for decreased production of IFN-gamma and survival of infected IL-10 KO mice, suggesting that B7.1 and B7.2 can contribute to the lethal shock-like reaction in IL-10 KO mice. In contrast, neutralization of IL-12 and blockade of the CD40/CD40 ligand (CD40L) interaction in vivo did not alter the production of IFN-gamma and only resulted in a small delay in time to death of mice. Together, these data suggest that the CD28/B7 interaction has a central role in the development of a pathological T-cell response in IL-10 KO mice, which is distinct from the role of the CD40/CD40L and IL-12 pathways.

Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia.

Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis.