Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

A TLR5 mono-agonist restores inhibited immune responses to Streptococcus pneumoniae during influenza virus infection in human monocytes.

Influenza A virus (IAV) predisposes individuals to often more severe secondary bacterial infections with Streptococcus pneumonia (S. pneumoniae). The outcomes of these infections may be made worse with the increase in antimicrobial resistance and a lack of new treatments to combat this. Th17 responses are crucial in clearing S. pneumoniae from the lung. We previously demonstrated that early IAV infection of human monocytes significantly reduced levels of S. pneumoniae-driven cytokines involved in the Th17 response. Here, we have further identified that IAV targets specific TLRs (TLR2, TLR4, TLR9) involved in sensing S. pneumoniae infection resulting, in a reduction in TLR agonist-induced IL-23 and TGF-ß. The effect of IAV is more profound on the TLR2 and TLR9 pathways. We have established that IAV-mediated inhibition of TLR9-induction is related to a downregulation of RORC, a Th17 specific transcription factor. Other studies using mouse models demonstrated that TLR5 agonism improved the efficacy of antibiotics in the treatment of IAV/S. pneumoniae co-infections. Therefore, we investigated if TLR5 agonism could restore inhibited Th17 responses in human monocytes. Levels of pneumococcus-driven cytokines, which had previously been inhibited by IAV were not reduced in the presence of the TLR5 mono-agonist, suggesting that such treatment may overcome IAV inhibition of Th17 responses. The importance of our research is in demonstrating the IAV directly targets S. pneumoniae-associated TLR pathways. Additionally, the IAV-inhibition of Th17 responses can be restored by TLR5 agonism, which indicates that there may be a different Th17 signalling pathway which is not affected by IAV infection.

Influenza infection directly alters innate IL-23 and IL-12p70 and subsequent IL-17A and IFN-γ responses to pneumococcus in vitro in human monocytes.

IMPORTANCE: Influenza virus is highly contagious and poses substantial public health problems due to its strong association with morbidity and mortality. Approximately 250,000-500,000 deaths are caused by seasonal influenza virus annually, and this figure increases during periods of pandemic infections. Most of these deaths are due to secondary bacterial pneumonia. Influenza-bacterial superinfection can result in hospitalisation and/or death of both patients with pre-existing lung disease or previously healthy individuals. The importance of our research is in determining that influenza and its component haemagglutinin has a direct effect on the classic pneumococcus induced pathways to IL-17A in our human ex vivo model. Our understanding of the mechanism which leaves people exposed to influenza infection during superinfection remain unresolved. This paper demonstrates that early infection of monocytes inhibits an arm of immunity crucial to bacterial clearance. Understanding this mechanism may provide alternative interventions in the case of superinfection with antimicrobial resistant strains of bacteria.

Comprehensive mutational analysis of a cohort of Swedish Cornelia de Lange syndrome patients.

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a rare multiple congenital anomaly/mental retardation syndrome characterized by distinctive dysmorphic facial features, severe growth and developmental delay and abnormalities of the upper limbs. About 50% of CdLS patients have been found to have heterozygous mutations in the NIPBL gene and a few cases were recently found to be caused by mutations in the X-linked SMC1L1 gene. We performed a mutation screening of all NIPBL coding exons by direct sequencing in 11 patients (nine sporadic and two familial cases) diagnosed with CdLS in Sweden and detected mutations in seven of the cases. All were de novo, and six of the mutations have not been previously described. Four patients without identifiable NIPBL mutations were subsequently subjected to multiplex ligation-dependent probe amplification analysis to exclude whole exon deletions/duplications of NIPBL. In addition, mutation analysis of the 5' untranslated region (5' UTR) of NIPBL was performed. Tiling resolution array comparative genomic hybridization analysis was carried out on these four patients to detect cryptic chromosome imbalances and in addition the boys were screened for SMC1L1 mutations. We found a de novo 9p duplication with a size of 0.6 Mb in one of the patients with a CdLS-like phenotype but no mutations were detected in SMC1L1. So far, two genes (NIPBL and SMC1L1) have been identified causing CdLS or CdLS-like phenotypes. However, in a considerable proportion of individuals demonstrating the CdLS phenotype, mutations in any of these two genes are not found and other potential loci harboring additional CdLS-causing genes should be considered.

Modulation of radiation responses by pre-exposure to irradiated cell conditioned medium.

The aim of this study was to investigate whether exposure of HPV-G cells to irradiated cell conditioned medium (ICCM) could induce an adaptive response if the cells were subsequently challenged with a higher ICCM dose. Clonogenic survival and major steps in the cascade leading to apoptosis, such as calcium influx and loss of mitochondrial membrane potential, were examined to determine whether these events could be modified by giving a priming dose of ICCM before the challenge dose. Clonogenic survival data indicated an ICCM-induced adaptive response in HPV-G cells "primed" with 5 mGy or 0.5 Gy ICCM for 24 h and then exposed to 0.5 Gy or 5 Gy ICCM. Reactive oxygen species (ROS) were found to be involved in the bystander-induced cell death. Calcium fluxes varied in magnitude across the exposed cell population, and a significant number of the primed HPV-G cells did not respond to the challenge ICCM dose. No significant loss of mitochondrial membrane potential was observed when HPV-G cells were exposed to 0.5 Gy ICCM for 24 h followed by exposure to 5 Gy ICCM for 6 h. Exposure of HPV-G cells to 5 mGy ICCM for 24 h followed by exposure to 0.5 Gy ICCM for 18 h caused a significant increase in mitochondrial mass and a change in mitochondrial location, events associated with the perpetuation of genomic instability. This study has shown that a priming dose of ICCM has the ability to induce an adaptive response in HPV-G cells subsequently exposed to a challenge dose of ICCM.

PHIP - a novel candidate breast cancer susceptibility locus on 6q14.1.

Most non-BRCA1/2 breast cancer families have no identified genetic cause. We used linkage and haplotype analyses in familial and sporadic breast cancer cases to identify a susceptibility locus on chromosome 6q. Two independent genome-wide linkage analysis studies suggested a 3 Mb locus on chromosome 6q and two unrelated Swedish families with a LOD >2 together seemed to share a haplotype in 6q14.1. We hypothesized that this region harbored a rare high-risk founder allele contributing to breast cancer in these two families. Sequencing of DNA and RNA from the two families did not detect any pathogenic mutations. Finally, 29 SNPs in the region were analyzed in 44,214 cases and 43,532 controls from BCAC, and the original haplotypes in the two families were suggested as low-risk alleles for European and Swedish women specifically. There was also some support for one additional independent moderate-risk allele in Swedish familial samples. The results were consistent with our previous findings in familial breast cancer and supported a breast cancer susceptibility locus at 6q14.1 around the PHIP gene.

The estrogen receptor alpha C975G variant in familial and sporadic breast cancer: a case-control study.

BACKGROUND: The estrogen receptor alpha (ESR1) mediates the effect of estrogen in target tissues. Estrogen is important in breast cancer development and several polymorphic variants in the ESR1 gene have been investigated for association with breast cancer. The C975G variant is the most extensively studied and has been suggested to be a risk factor. MATERIALS AND METHODS: The frequency of the C975G variant was investigated in 288 sporadic, 197 low-risk non-BRCA1/2 familial and 191 high-risk non-BRCA1/2 familial breast cancer cases and 653 controls. RESULTS: There was a lower frequency of the C975G variant in high-risk familial breast cancer cases compared to the controls (18% versus 22%, p=0.046). The odds ratio (OR) for the GG homozygotes was 0.2 (95% CI: 0.06-0.8) compared to the CC homozygotes. No association was seen with sporadic or low-risk familial breast cancer. CONCLUSION: The results of this study indicated that the common C975G variant may have an effect on familial breast cancer susceptibility.

Apoptosis is initiated in human keratinocytes exposed to signalling factors from microbeam irradiated cells.

PURPOSE: There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionizing track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from gamma irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual. MATERIALS AND METHODS: HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor-induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels. RESULTS: Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 h post-exposure, increased expression of bcl-2 and cytochrome c release at 6.5 h and increased apoptosis at 24 h. CONCLUSION: This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.

Lack of HIN-1 methylation in BRCA1-linked and "BRCA1-like" breast tumors.

We recently identified a candidate tumor suppressor gene, HIN-1, that is silenced due to methylation in the majority of sporadic breast carcinomas and is localized to 5q33-qter, an area frequently lost in BRCA1 tumors and thought to harbor a BRCA1 modifier gene. To establish whether germ-line mutations in HIN-1 may influence breast cancer risk, we sequenced the HIN-1 coding region in 10 familial breast cancer patients with positive logarithm of the odds scores of at least one of the markers flanking HIN-1. We also sequenced the HIN-1 coding region in 15 BRCA1 and 35 sporadic breast tumors to determine whether HIN-1 is the target of the frequent 5q loss in BRCA1 tumors. No sequence alterations were found in any of the cases analyzed. However, analysis of HIN-1 promoter methylation status revealed that in striking contrast to sporadic cases, there is a nearly complete lack of HIN-1 methylation in BRCA1 tumors (P < 0.0001). Sporadic breast tumors with a "BRCA1-like" histopathological phenotype also demonstrated significantly lower frequency of HIN-1 promoter methylation (P = 0.01) compared with other cancer types, and there was also a difference among tumors based on their estrogen receptor and HER2 status (P = 0.006), suggesting that HIN-1 methylation patterns are associated with specific breast cancer subtypes.

Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes.

Exposure of unirradiated human keratinocytes to irradiated cell conditioned medium (ICCM) is known to cause a cascade of events that leads to reproductive death and apoptosis. This study investigates the effect of ICCM on clonogenic survival, mitochondrial mass and BCL2 expression in unirradiated keratinocytes. Exposure to 5 mGy, 0.5 Gy and 5 Gy ICCM resulted in a significant decrease in clonogenic survival. Human keratinocytes incubated with ICCM containing an antioxidant, N-acetylcysteine, showed no significant decrease in clonogenic survival. HPV-G cells incubated with ICCM containing a caspase 9 inhibitor showed no significant decrease in clonogenic survival when the ICCM dose was < or =0.5 Gy. A significant increase in mitochondrial mass per cell was observed after exposure to 5 mGy and 0.5 Gy ICCM. A change in the distribution of the mitochondria from a diffuse cytoplasmic distribution to a more densely concentrated perinuclear distribution was also observed at these doses. No significant increase in mitochondrial mass or change in distribution of the mitochondria was found for 5 Gy ICCM. Low BCL2 expression was observed in HPV-G cells exposed to 5 mGy or 0.5 Gy ICCM, whereas a large significant increase in BCL2 expression was observed in cells exposed to 5 Gy ICCM. This study has shown that low-dose irradiation can cause cells to produce medium-borne signals that can cause mitochondrial changes and the induction of BCL2 expression in unirradiated HPV-G cells. The dose dependence of the mitochondrial changes and BCL2 expression suggests that the mechanisms may be aimed at control of response to radiation at the population level through signaling pathways.

Genetic factors influencing bystander signaling in murine bladder epithelium after low-dose irradiation in vivo.

Radiation-induced bystander effects occur in cells that are not directly hit by radiation tracks but that receive signals from hit cells. They are well-documented in vitro consequences of low-dose exposure, but their relevance to in vivo radiobiology is not established. To investigate the in vivo production of bystander signals, bladder explants were established from two strains of mice known to differ significantly in both short-term and long-term radiation responses. These were investigated for the ability of 0.5 Gy total-body irradiation in vivo to induce production of bystander signals in bladder epithelium. The studies demonstrate that irradiated C57BL/6 mice, but not CBA/Ca mice, produce bystander signals that induce apoptosis and reduce clonogenic survival in reporter HPV-G-transfected keratinocytes. Transfer of medium from explants established from irradiated animals to explants established from unirradiated animals confirmed these differences in bladder epithelium. The responses to the in vivo-generated bystander signal exhibit genotypic differences in calcium signaling and also in signaling pathways indicative of a major role for the balance of pro-apoptosis and anti-apoptosis proteins in determining the overall response. The results clearly demonstrate the in vivo induction of bystander signals that are strongly influenced by genetic factors and have implications for radiation protection, medical imaging, and radiotherapy.

CGH analysis of familial non-BRCA1/BRCA2 breast tumors and mutation screening of a candidate locus on chromosome 17q11.2-12.

Mutations in the known predisposing breast cancer genes BRCA1 and BRCA2 account for only a small proportion (<10%) of breast cancer families in the Stockholm region of Sweden. This study aims to identify novel predisposing genes in non-BRCA1/BRCA2 breast cancer families. We have employed comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) data in combination with data from a recently carried out genome wide linkage scan, in an effort to identify chromosomal regions harboring potential breast cancer genes. CGH revealed loss of chromosome 17 and chromosome 6q to be a frequent event in high-risk breast cancer families, while gain of 8q was most prevalent in low-risk families. The loss of chromosome 17 was consistent with previous LOH studies and so this region was investigated further. Disease was shown to be linked to chromosome 17 in those families exhibiting loss of the chromosome in both CGH and LOH analyses. An overlapping region of linkage was determined to lie between markers D17S1294 and D17S1293, fine mapping of the region delineated a region between markers D17S1880 and D17S1293. Ten genes were determined to lie within this 1.5 Mb region and families were screened for germline mutations in these genes. In conclusion, we have investigated one possible small region on chromosome 17 for its involvement in high-risk non-BRCA1/BRCA2 breast cancer families. No predisposing mutations were identified in the 10 genes investigated in this study, however further analysis of chromosome 17 is warranted.

The in vitro delivery of NSAIDs across skin was in proportion to the delivery of essential fatty acids in the vehicle--evidence that solutes permeate skin associated with their solvation cages?

As part of our investigations into novel dual action topical anti-arthritis systems, the permeation of ibuprofen or ketoprofen plus eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were determined from a fish oil vehicle across pig ear skin in vitro. The steady state fluxes of ibuprofen and ketoprofen were 9.17+/-1.98 microgram cm(-2)h(-1) and 6.12+/-2.39 microgram cm(-2)h(-1), respectively. At 24h, 5.7 microgram cm(-2) EPA and 3.1 microgram cm(-2) DHA permeated when the solute was ibuprofen; 1.4 microgram cm(-2) EPA and 1.0 microgram cm(-2) DHA when ketoprofen was the solute. At 12h, the ketoprofen/ibuprofen ratio of the moles permeated was 0.27, the ratio of EPA permeated simultaneously with ketoprofen and ibuprofen was 0.22 and the ratio of DHA permeated simultaneously with ketoprofen and ibuprofen was 0.24. We believe this is the first time that simultaneous permeation across skin of a solute and its vehicle has been determined purposefully. The data successfully demonstrated that simultaneous permeation of NSAIDs and essential fatty acids, EPA and DHA from a formulation containing fish oil is feasible. In addition, for both NSAIDs, the relative rates of permeation of EPA and DHA, were in proportion to their levels in the fish oil and the permeation rate of either fatty acid was higher when the permeation rate of the solute was greater. This suggested that the greater the rate of permeation of the NSAID, the greater the rate of permeation of the vehicle, and that a solute permeates skin complete with its vehicular solvation cage. This apparent relationship between solute and vehicle fluxes may be of more widespread significance to skin permeation experimentation.

Stimulation of nitric oxide mechanotransduction in single osteoblasts using atomic force microscopy.

Nitric oxide (NO) released from mechanosensitive bone cells plays a key role in the adaptation of bone structure to its mechanical usage. Despite its importance in bone, the mechanisms involved in NO mechanotransduction at the cellular level remain unknown. Using combined atomic force microscopy and fluorescence microscopy, we report both stimulation and real-time monitoring of NO responses in single osteoblasts induced by application of quantified periodic indenting forces to the osteoblast membrane. Peak forces ranging from 17 to 50 nN stimulated three distinct NO responses in the indented osteoblasts: (1) a rapid and sustained diffusion of NO from the perinuclear region, (2) diffusion of NO from localized pools throughout the osteoblast, and (3) an initial increase and subsequent drop in intracellular NO. Force-indentation characteristics showed considerable interosteoblast variation in elasticity. NO responses were associated with application of force to more rigid membrane sites, suggesting cytoskeletal involvement in mechanotransduction.

Morphological and functional differentiation of HSG cells: role of extracellular matrix and trpc 1.

A human salivary intercalated duct cell line (HSG) is capable of morphological change to acinar-type cells, and of salivary amylase (AMY1) expression, by culturing on basement membrane extracts (BME). The aim of this study was to determine the critical conditions for functional and morphological differentiation of HSG cells and to establish if the processes are related. Cells were grown on BMEs that had different protein concentrations and growth factor content, and then examined with respect to morphology and AMY1 expression. To investigate the role of intracellular calcium in amylase expression, a pcDNA3.1-TRPC1alpha construct was used to overexpress htrp1alpha, which mediates the store-operated calcium entry in HSG cells. Expression of the AMY1, TRPC1alpha and beta genes was quantified by means of real time RT-PCR. Growth factor-reduced BME (12.8 mg/ml) induced multicellular acinar structures with lumen formation but without stimulation of either AMY1 or TRPC1. HSG cells cultured on higher concentration BME (17.5 or 16.4 mg/ml) formed reticular networks. AMY1 expression increased both on growth factor-reduced BME (17.5 mg/ml: 3.0-fold, P < 0.001) and on regular BME (16.4 mg/ml: 3.7-fold, P < 0.001) accompanied by a slight increase in expression of TRPC1alpha and TRPC1beta. Overexpression of htrp1alpha did not cause any significant changes in AMY expression, though it attenuated the BME (17.5 mg/ml)-induced AMY1 upregulation. Overall, the higher protein concentration BME favors amylase expression in HSG cells, whereas the lower concentration causes marked morphological changes.

Estrogen receptor beta (ESR2) polymorphisms in familial and sporadic breast cancer.

Estrogen is involved in both normal mammary development and in breast carcinogenesis. A family history of disease and exposure to estrogen are major risk factors for developing breast cancer. Estrogen exerts its biological effects through binding to the estrogen receptors, estrogen receptor alpha (ESR1) and the more recently discovered estrogen receptor beta (ESR2). Genetic variation in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to play a role in breast cancer risk. We therefore tested the hypothesis that common genetic variants of the ESR2 gene may be associated with increased risk for breast cancer and this risk may vary between breast cancer groups. We investigated three common ESR2 polymorphisms, rs1256049 (G1082A), rs4986938 (G1730A) and rs928554 (Cx+56 A-->G) for association to breast cancer risk. A total of 723 breast cancer cases and 480 controls were included in the study. Of the breast cancer cases, 323 were sporadic and 400 were familial, the familial cases were further divided into familial high-risk and familial low-risk breast cancer cases. We found no overall statistically significant association for any of the single polymorphisms studied. Haplotype analysis suggested one haplotype associated with increased risk in sporadic breast cancer patients (OR = 3.0, p = 0.03). Further analysis is needed to elucidate the role of estrogen receptor beta in breast cancer susceptibility.