Determination of Vitamin D Levels in Patients With Neurofibromatosis Type 1 in the Pediatric Age Group.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic disorders. Some clinical manifestations are present at birth, while some develop during childhood, and others can occur at any age. Given the early age at which patients develop clinical features, diagnosis is often made during childhood. The most prevalent features of NF1 are café au lait spots, dermal and plexiform neurofibromas, and learning disability. A variety of skeletal problems may be seen in NF1, including scoliosis, short stature, and pseudoarthrosis. Reduced skeletal bone mass has been documented to be a common phenomenon in children and adults with NF1. Decreased serum 25-hydroxyvitamin D (vitamin D) levels have been noted in adults and children with NF1 and have been reported to be inversely correlated with the number of dermal neurofibromas in adults. However, the actual correlation of vitamin D level to bone density and dermal neurofibroma number in children with NF1 remains unclear. OBJECTIVES: The primary objective of this study was to evaluate vitamin D levels among children and adolescents with NF1. The secondary objective was to describe the levels of vitamin D among children and adolescents with NF1, to verify in which age group there is a higher frequency of vitamin D alterations, and to explore vitamin D level correlations between age, gender, sun exposure, number of neurofibromas, and number of plexiform neurofibromas. METHODS: This was an observational, cross-sectional, hospital-based study. We obtained a convenience sample of individuals with confirmed diagnosis of NF1 from patients attending the Medical Genetics Service of the IPPMG-UFRJ and Santa Casa de Misericórdia of Rio de Janeiro over a 24-month period. We evaluated vitamin D levels in blood samples of patients with NF1 by a chemiluminescent immunoassay method, and we correlated the results with gender, age, number of neurofibromas, number of plexiform neurofibromas, and satisfactory sun exposure. RESULTS: Of the 55 patients, 28 (50.9%) were female and 27 (49.1%) were male. Patient ages ranged from a minimum of 1.2 to a maximum of 19.6 years (mean age 10.95 years) and the median was 11.11 years. Median and mean body mass index (BMI; z score) were -0.09 (minimum value -1.63 and maximum of 4.62) and 0.16, respectively. The mean value of vitamin D was 30.82 ng/mL (±12.31) and the median was 29 ng/mL (minimum value of 10.40 ng/mL and maximum of 79.19 ng/mL). CONCLUSIONS: The levels of vitamin D did not differ according to gender, age group, or the presence or number of cutaneous neurofibromas. Among patients with adequate sun exposure, there was a higher incidence of sufficient serum vitamin D levels. Patients with cutaneous neurofibromas in the 0 to 11 age group had a greater tendency to vitamin D sufficiency in relation to patients aged 11 to 19 years.

Factor VII-activating protease deficiency promotes neointima formation by enhancing leukocyte accumulation.

Essentials Factor VII-activating protease (FSAP) is a plasma protease involved in vascular processes. Neointima formation was investigated after vascular injury in FSAP-/- mice. The neointimal lesion size and the accumulation of macrophages were increased in FSAP-/- mice. This was due to an increased activity of the chemokine (C-C motif) ligand 2 (CCL2). SUMMARY: Background Factor VII-activating protease (FSAP) is a multifunctional circulating plasma serine protease involved in thrombosis and vascular remodeling processes. The Marburg I single-nucleotide polymorphism (MI-SNP) in the FSAP-coding gene is characterized by low proteolytic activity, and is associated with increased rates of stroke and carotid stenosis in humans. Objectives To determine whether neointima formation after vascular injury is increased in FSAP-/- mice. Methods and Results The neointimal lesion size and the proliferation of vascular smooth muscle cells (VSMCs) were significantly enhanced in FSAP-/- mice as compared with C57BL/6 control mice after wire-induced injury of the femoral artery. Accumulation of leukocytes and macrophages was increased within the lesions of FSAP-/- mice at day 3 and day 14. Quantitative zymography demonstrated enhanced activity of gelatinases/matrix metalloproteinase (MMP)-2 and MMP-9 within the neointimal lesions of FSAP-/- mice, and immunohistochemistry showed particular costaining of MMP-9 with accumulating leukocytes. Using intravital microscopy, we observed that FSAP deficiency promoted the intravascular adherence and the subsequent transmigration of leukocytes in vivo in response to chemokine ligand 2 (CCL2). CCL2 expression was increased in FSAP-/- monocytes but not in the vessel wall. There was no difference in the expression of platelet-derived growth factor (PDGF-BB). Conclusions FSAP deficiency causes an increase in CCL2 expression and CCL2-mediated infiltration of leukocytes into the injured vessel, thereby promoting SMC proliferation and migration by the activation of leukocyte-derived gelatinases. These results provide a possible explanation for the observed association of the loss-of-function MI-SNP with vascular proliferative diseases.

Roles of Ki67 in Breast Cancer - Important for Management?

BACKGROUND: The three standard biomarkers used in breast cancer are the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The Ki-67 index, a proliferative marker, has been shown to be associated with a poorer outcome, and despite absence of standardization of pathological assessment, is widely used for therapy decision making. We aim to study the role of the Ki-67 index in a group of Asian women with breast cancer. MATERIALS AND METHODS: A total of 450 women newly diagnosed with Stage 1 to 3 invasive breast cancer in a single centre from July 2013 to Dec 2014 were included in this study. Univariable and multivariable logistic regression was used to determine the association between Ki-67 (positive defined as 14% and above) and age, ethnicity, grade, mitotic index, ER, PR, HER2, lymph node status and size. All analyses were performed using SPSS Version 22. RESULTS: In univariable analysis, Ki -67 index was associated with younger age, higher grade, ER and PR negativity, HER2 positivity, high mitotic index and positive lymph nodes. However on multivariable analysis only tumour size, grade, PR and HER2 remained significant. Out of 102 stage 1 patients who had ER positive/PR positive/HER2 negative tumours and non-grade 3, only 5 (4.9%) had a positive Ki-67 index and may have been offered chemotherapy. However, it is interesting to note that none of these patients received chemotherapy. CONCLUSIONS: Information on Ki67 would have potentially changed management in an insignificant proportion of patients with stage 1 breast cancer.

Contribution of MX dynamin, oligoadenylate synthetase, and protein kinase R to anti-paramyxovirus activity of type 1 interferons in vitro.

OBJECTIVE: To determine the contribution of MX dynamin, oligoadenylate synthetase (OAS), and double-stranded RNA-dependent protein kinase R (PKR) to the antiviral effects of type 1 interferons (IFNs) against bovine parainfluenza-3 virus (PI-3V) infection of Vero cells. SAMPLE POPULATION: Vero cell cultures. PROCEDURES: PI-3V yield was first compared between control and transfected type 1 IFNs-incompetent Vero cells expressing recombinant OAS or MX proteins. Afterwards, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) was used to scale the degree of PKR activation upon infection of Vero cells by PI-3V. RESULTS: Overexpression of OAS did not result in significantly decreased viral replication. Phosphorylated eIF2alpha forms, the hallmark of PKR activation, were not increased in IFNalpha-primed infected Vero cells. Although human MXA contributed to partial blockade of replication of bovine PI-3V, the antiviral effect was not as strong as that of IFNalpha. CONCLUSIONS AND CLINICAL RELEVANCE: The powerful anti-Paramyxovirus activity of type 1 IFNs is mediated by noncanonic pathways.

Genomic structure, promoter analysis, and expression of the porcine (Sus scrofa) Mx1 gene.

Allelic polymorphisms at the mouse Mx1 locus affect the probability of survival after experimental influenzal disease, raising the possibility that marker-assisted selection using the homologous locus could improve the innate resistance of pigs to natural influenza infections. Several issues need to be resolved before efficient large scale screening of the allelic polymorphism at the porcine (Sus scrofa) Mx1 locus can be implemented. First, the Mx1 genomic structure has to be established and sufficient flanking intronic sequences have to be gathered to enable simple PCR amplification of the coding portions of the gene. Then, a basic knowledge of the promoter region needs to be obtained as an allelic variation there can significantly alter absolute levels and/or tissue-specificity of MX protein expression. The results gathered here show that the porcine Mx1 gene and promoter share the major structural and functional characteristics displayed by their homologs described in cattle, mouse, chicken, and man. The crucial function of the proximal interferon-sensitive response elements motif for gene expression is also demonstrated. The sequence data compiled here will allow an extensive analysis of the polymorphisms present among the widest spectrum possible of porcine breeds with the aim to identify an Mx1 allele providing antiviral resistance.

Differential resistance/susceptibility patterns to pneumovirus infection among inbred mouse strains.

Respiratory syncytial virus (RSV) is a prominent cause of airway morbidity in children under 1 yr of age. It is assumed that host factors influence the severity of the disease presentation and thus the need for hospitalization. As a first step toward the identification of the underlying genes involved, this study was undertaken to establish whether inbred mouse strains differ in susceptibility to pneumonia virus of mice (PVM), the murine counterpart of RSV, which has been shown to accurately mimic the RSV disease of children. With this purpose in mind, double-chamber plethysmography and carbon monoxide uptake data were collected daily for 7 days after inoculation of PVM in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values reflected the success of viral replication in the lungs and revealed a pattern of continuous variation, with resistant, intermediate, and susceptible strains. The results suggest that SJL (resistant) and 129/Sv (susceptible) strains should be used in crossing experiments aimed at identifying genes controlling pneumovirus replication by the positional cloning approach. Similarly, crossing experiments using BALB/c or C57BL/6 (resistant) and DBA/2 or 129/Sv (susceptible) will allow the identification of the genes involved in the control of pulmonary inflammation during pneumovirus infection.

Genomic structure, promoter analysis and expression of the porcine (Sus scrofa) TLR4 gene.

Toll-like receptor 4 (TLR4) is essential for initiating the innate response to lipopolysaccharide (LPS) from Gram-negative bacteria by acting as a signal-transducing receptor. As the pig industry faces a unique array of related pathogens, it is anticipated that the genotype of swine TLR4 could be of crucial importance in future strategies aimed at improving genetic resistance to infectious diseases. In order to help in investigating TLR4 as a candidate disease-resistance gene in pigs, we established its genomic structure and produced sufficient flanking intronic sequences to enable simple PCR amplification of the coding portions of the gene. Expression in different porcine tissues was studied and showed splicing variations in mRNA sequences. The cDNA sequence for poTLR4 contains an open reading frame of 2526bp that codes for 841 aa, 98 and 568bp in the 5'- and 3'-UTRs, respectively. Overall, the general organization of porcine, human, murine, and avian TLR4 genes is quite similar: three exons with the third one very long. A high level of conservation of the size and the sequence, especially for the two last exons and particularly in the sequence corresponding to the LRRs and TIR domain, is observed between species. The important antimicrobial properties of these proteins may account for a conservative selection pressure on these TLR4 coding sequences. Several putative binding sites described in the human and murine promoter of TLR4 genes have been identified in the 5'-flanking region of poTLR4. Conversely, this region lacks a TATA box, consensus initiator sequences, or GC-rich regions. The basic sequence data gathered will allow the establishment of an inventory of naturally occurring variation in porcine TLR4, so that alleles can be tested for disease association studies.

Sendai virus-induced alterations in lung structure/function correlate with viral loads and reveal a wide resistance/susceptibility spectrum among mouse strains.

The Paramyxoviridae family includes some of the most important and ubiquitous disease-causing viruses of infants and children, most of which cause significant infections of the respiratory tract. Evidence is accumulating in humans that genetic factors are involved in the severity of clinical presentation. As a first step toward the identification of the genes involved, this study was undertaken to establish whether laboratory mouse strains differ in susceptibility to Sendai virus, the murine counterpart of human type-1 parainfluenza virus which, historically, has been used extensively in studies that have defined the basic biological properties of paramyxoviruses in general. With this purpose in mind, double-chamber plethysmography data were collected daily for 7 days after inoculation of Sendai virus in six inbred strains of mice. In parallel, histological examinations and lung viral titration were carried out from day 5 to day 7 after inoculation. Pulmonary structure/function values closely reflected the success of viral replication in the lungs and revealed a pattern of continuous variation with resistant, intermediate, and susceptible strains. The results unambiguously suggest that BALB/c (resistant) and 129Sv (susceptible) strains should be used in crossing experiments aimed at identifying the genes involved in resistance to Paramyxoviridae by the positional cloning approach.

Genomic structure, organisation, and promoter analysis of the bovine (Bos taurus) Mx1 gene.

Some MX proteins are known to confer a specific resistance against a panel of single-stranded RNA viruses. Many diseases due to such viruses are known to affect cattle worldwide, raising the possibility that the identification of an antiviral isoform of a bovine MX protein would allow the implementation of genetic selection programs aimed at improving innate resistance of cattle. With this potential application in mind, the present study was designed to isolate the bovine Mx1 gene including its promoter region and to investigate its genomic organisation and promoter reactivity. The bovine Mx1 gene is made up of 15 exons. All exon-intron boundaries conformed to the consensus sequences. A PCR product that contained a approximately 1-kb, 5'-flanking region upstream from the putative transcription start site was sequenced. Unexpectedly, this DNA region did not contain TATA or CCAAT motifs. A computer scan of the region disclosed a series of putative binding sites for known cytokines and transcription factors. There was a GAAAN(1-2)GAAA(C/G) motif, typical of an interferon-sensitive responsive element, between -118 and -107 from the putative transcription start site. There were also a NF-kappaB, two interleukin-6 binding sites, two Sp1 sites and five GC-rich boxes. The region also contained 12 stretches of the GAAA type, as described in all IFN-inducible genes. Bovine Mx1 expression was assessed by Northern blotting and immunofluorescence in the Madin Darby bovine kidney cells (MDBK) cell line treated with several stimuli. In conclusion, the bovine Mx1 gene and promoter region share the major structural and functional characteristics displayed by their homologs described in the rainbow trout, chicken, mouse and man.

Effect of somatic growth, strain, and sex on double-chamber plethysmographic respiratory function values in healthy mice.

Double-chamber plethysmography has been recognized since 1979 as a reference technique to measure pulmonary function values in guinea pigs, but it has not gained attention for use in mice. Theoretically, however, this technique combines the advantages of single-chamber plethysmography with a quantitative assessment of flow and/or volume and a calculated resistance, the interpretation of which in terms of bronchoconstriction is not disputed. Here we show that, when appropriately preconditioned, mice are able to gradually grow accustomed to the apparatus and display extremely stable nasal and thoracoabdominal flow tracings. Overall, strain, sex, and somatic growth had a significant effect on pulmonary function values. The changes in specific airway resistance (sRaw) and enhanced pause (Penh) values were never in the same direction, indicating that they measure different things. The respiratory frequency was far higher in C57BL/6 compared with BALB/c mice. Peak flows, minute volume, specific tidal and minute volumes, and sRaw were also higher, but Penh was smaller. Males breathed at a higher frequency than females, leading to a higher minute volume. Nevertheless, the specific volumes were considerably higher among females. Penh was lower in males, whereas sRaw was identical in both sexes. Changes associated with somatic growth were rapid and important between 5 and 9 wk, then slowed down between 9 and 12-13 wk and became almost imperceptible after.