Hirschsprung associated GDNF mutations do not prevent RET activation

Hirschsprung disease (HSCR) is a complex disorder characterised by aganglia of distal gastrointestinal tracts. The highest proportion of both familial and sporadic cases is due to mutations of the RET proto-oncogene. Five germline mutations in the glial cell-line-derived neurotrophic factor (GDNF) gene, one of the RET ligands, have been detected in HSCR patients. Pedigrees analysis and the observed association between these GDNF alterations and RET variants in the same patients raised the question of whether the GDNF gene plays any causative/predisposing role in HSCR pathogenesis. In the present work, we have studied the ability of GDNF proteins, each bearing one of the reported mutations, to activate RET by performing a functional test in cultured neuroblastoma cells. Consistently with the lack of genotype/phenotype correlation in human subjects, our results indicate absence of detectable alterations of mutant GDNF induced RET activation.     

Immunohistochemical expression of p16(INK4a) is predictive of HR-HPV infection in cervical low-grade lesions.

The p16(INK4a) is a cyclin-dependent kinase inhibitor that decelerates the cell cycle by inactivating the cyclin-dependent kinases involved in the phosphorylation of the retinoblastoma protein (RB). Expression of E6 and E7 oncogenes of high-risk (HR) human papillomavirus (HPV), affecting the RB-p16 pathway, leads to p16 upregulation. Although it is widely reported that p16 is overexpressed in a high percentage of preneoplastic lesions and in almost all carcinomas of the uterine cervix, protein upregulation and its correlation with HPV infection in low-grade lesions is still being debated. In this study, we investigated in parallel, p16 expression and HPV infection in 100 cervical biopsies (17 normal tissues, 54 CIN1, 10 CIN2, 11 CIN3, eight invasive squamous cancers). Results obtained demonstrated that none of the 17 normal cervical tissues, evaluated by immunohistochemistry, presented p16 positivity whereas, starting from CIN1 (31%) to CIN2 (90%), CIN3 (100%) and carcinomas (100%), a constant and significant increase of protein overexpression (P<0.0001) was observed. In addition, p16 overexpression consistently showed elevated sensitivity (84%) and specificity (98%) in detecting HR-HPV infection with a high positive predictive value (97%) and negative predictive value (86%). Of interest, 93% of the p16-positive CIN1 were also HR-HPV infected. Our findings confirmed that p16 overexpression is associated to high-grade precancerous lesions and cervical carcinomas, and further demonstrated that immunohistochemical evaluation of p16 may be a useful biomarker in identifying HR-HPV-infected low-grade lesions.     

Mice lacking the norepinephrine transporter are supersensitive to psychostimulants

The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET), which is a target for antidepressants and psychostimulants. Disruption of the NET gene in mice prolonged the clearance of NE and elevated extracellular levels of this catecholamine. In a classical test for antidepressant drugs, the NET-deficient (NET-/-) animals behaved like antidepressant-treated wild-type mice. Mutants were hyper-responsive to locomotor stimulation by cocaine or amphetamine. These responses were accompanied by dopamine D2/D3 receptor supersensitivity. Thus altering NET expression significantly modulates midbrain dopaminergic function, an effect that may be an important component of the actions of antidepressants and psychostimulants.     

Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients.

BACKGROUND: Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. OBJECTIVE: To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. STUDY DESIGN: Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. RESULTS: Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January-May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. CONCLUSION: HKU1 hCoV strains circulated in northern Italy during the winter-spring season 2005-2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR.     

Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes

The genome of Mycoplasma hyopneumoniae encodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions with M. flocculare and M. hyorhinis reduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific for M. hyopneumoniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with an M. hyopneumoniae infection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection with M. hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection of M. hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.     

Developmental regulation of type 1 and type 3 interferon production and risk for infant infections and asthma development

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Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells

Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system. Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes. We show that monocytes infected with live M. tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt-MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules. These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS-matured DC. In addition, Mt-MoDC produced TNF-alpha and IL-10, but were unable to secrete IL-12. The generation of Mt-MoDC required the infection of monocytes with live M. tuberculosis, since infection with heat-killed bacteria partially abrogated the effects on monocyte differentiation. Interestingly, Mt-MoDC revealed an impaired antigen-presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes. Further, naive T lymphocytes expanded by Mt-MoDC were unable to secrete cytokines, in particular IL-4 and IFN-gamma, suggesting that they could be ineffective in helping the macrophage-mediated killing of intracellular mycobacteria. Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M. tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition.     

Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1−CD8+ Tumor-Infiltrating T Cells

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Bone cement extracts modulate the osteoprotegerin/osteoprotegerin-ligand expression in MG63 osteoblast-like cells

The osteoprotegerin-ligand (OPG-L) has been identified as the essential factor required for osteoclastogenesis, and its effects are prevented by the osteoprotegerin (OPG). The OPG-L/OPG balance plays a crucial role in coordinating the sequence of osteoclast and osteoblast differentiation during the bone remodeling. The aim of the study was to investigate if polymethylmethacrylate-based cements are able to modulate the expression of OPG-L/OPG in MG63 cells, which are known to have high levels of OPG and inducible expression of OPG-L. Four radio-opaque cements. namely Sulfix-60, CMW1, CMW2 and CMW3, were polymerized for either 1 h or 7 d. MG63 were incubated for 24 h with culture medium only, cement extracts and 2 microg/ml of human recombinant IL-1beta as positive control. An RT-PCR was performed to detect the OPG and OPG-L expression, and the house-keeping gene, GAPDH, was used as a reference for the semi-quantitative analysis. An increase in the OPG-L band density was observed for all cements, and consequently, the OPG-L/OPG ratio also increased. The ability of bone cements to induce the expression of OPG-L could be a co-factor in the development of osteolysis at the bone-cement interface.