There is mounting evidence to suggest that these opposing roles are therapeutically relevant in that mutation of agr results in increased biofilm formation and decreased antibiotic susceptibility while mutation of sarA has the opposite effect. To the extent that induction of agr or inhibition of sarA could potentially be used to limit biofilm formation, this makes it important to understand the epistatic relationships between these two loci.The accessory gene regulator (sarA and agr mutants in clinical isolates of S. aureus and assessed the relative impact on biofilm formation. Mutation of agr resulted in an increased capacity to form a biofilm in the 8325-4 laboratory strain RN6390 but had little impact in clinical isolates S. aureus. In contrast, mutation of sarA resulted in a reduced capacity to form a biofilm in all clinical isolates irrespective of the functional status of agr. This suggests that the regulatory role of sarA in biofilm formation is independent of the interaction between sarA and agr and that sarA is epistatic to agr in this context. This was confirmed by demonstrating that restoration of sarA function restored the ability to form a biofilm even in the corresponding agr mutants. Mutation of sarA in clinical isolates also resulted in increased production of extracellular proteases and extracellular nucleases, both of which contributed to the biofilm-deficient phenotype of sarA mutants. However, studies comparing different strains with and without proteases inhibitors and/or mutation of the nuclease genes demonstrated that the agr-independent, sarA-mediated repression of extracellular proteases plays a primary role in this regard.We generated isogenic sarA-mediated regulation could be used to limit biofilm formation in S. aureus and that the efficacy of such inhibitors would not be limited by spontaneous mutation of agr in the human host.The results we report suggest that inhibitors of  Staphylococcus aureus infections including endocarditis, osteomyelitis, and infections of implanted medical devices. This is true not only with respect to the pathogenesis of the infection itself but also with respect to antimicrobial therapy. Indeed, the presence of a biofilm limits the efficacy of antimicrobial therapy to the point that surgical intervention is often required to remove infected tissues and/or implanted devices S. aureus biofilm formation. These studies have focused on the role of both individual components and the regulatory factors that modulate the production of these components. To date, over 20 genes have been implicated, with approximately half of these serving a regulatory role agr) and the staphylococcal accessory regulator (sarA) because both of these loci have been shown to play central roles in S. aureus regulatory circuits that includes important but generally opposing roles in biofilm formation. Specifically, while there is one report to the contrary agr limits biofilm formation S. aureus from an established biofilm sarA has consistently been shown to promote biofilm formation in both S. aureus and S. epidermidisBiofilm formation is an important aspect of many sarA and agr in biofilm formation are therapeutically relevant. For instance, we demonstrated that mutation of sarA can be correlated with increased susceptibility to the functionally-diverse antibiotics daptomycin, linezolid and vancomycin in the specific context of an established biofilm in vitro conditions, this increased susceptibility was evident even after taking into account the reduced capacity of a sarA mutant to form a biofilm agr expression was shown to result in the detachment of S. aureus cells from an established biofilm resulting in increased susceptibility to diverse antibiotics including rifampicin and levofloxacin agr mutants accumulate within a biofilm and ultimately become the predominant subpopulation agr function may confer a selective advantage in vivo, particularly under the pressure of vancomycin therapy sarA and agr in biofilm formation can be directly correlated with antibiotic susceptibility, with expression of the first leading to biofilm-associated intrinsic resistance and expression of the second having the opposite effect. This makes it important to define the epistatic relationships between sarA and agr in the context of biofilm formation.There are also reports demonstrating that the opposing roles of sarA and agr play global regulatory roles in S. aureussarA is generally associated with increased expression of agrsarA also modulates expression of many genes independently of agr, examples of which include the genes encoding extracellular proteases and nucleases agr results in reduced production of these enzymes while mutation of sarA has the opposite effect S. aureussarA mutant Because both agr and sarA in biofilm formation are due to the fact that the first induces while the second represses the production of extracellular proteases and/or nucleases. This is consistent with our results comparing the clinical isolate UAMS-1 with the commonly-studied 8325-4 laboratory strain RN6390. Specifically, by comparison to RN6390, UAMS-1 expresses agr at much lower levels, produces reduced amounts of extracellular proteases, and forms a more robust biofilm agr enhances biofilm formation in RN6390 but has little impact in UAMS-1 and, conversely, mutation of sarA results in increased protease production and decreased biofilm formation in UAMS-1 but has little impact in RN6390 S. aureus strains derived from 8325, including the 8325-4 strain RN6390, have genetic defects that have been shown to contribute to its high level of agr expression rsbU defect in RN6390 has been correlated with decreased expression of agr, decreased production of extracellular proteases, and an increased capacity to form a biofilm S. aureus (CA-MRSA) isolates of the USA300 clonal lineage, which are increasingly prominent worldwide agr at high levels Collectively, these studies suggest that the opposing roles of agr, and this suggests that the increased toxin production in USA300 isolates would be correlated with increased protease production and may therefore come at the expense of the ability to form a biofilm. Support for this hypothesis comes from a recent report demonstrating that mutation of agr, the inclusion of inhibitors of extracellular protease activity, and the mutation of specific genes encoding extracellular proteases can all be correlated with an enhanced capacity to form a biofilm in the USA300 isolate LAC agr has a significant impact on biofilm formation in USA300 isolates in general and, if so, whether this limits the regulatory role of sarA in this respect. Based on this, we examined the impact of sarA and agr on biofilm formation in diverse clinical isolates with a specific emphasis on CA-MRSA isolates of the USA300 clonal lineage.The production of extracellular proteases is also under the regulatory control of S. aureus generally form a more robust biofilm than the 8325-4 laboratory strain RN6390 and that biofilm formation in the latter is enhanced by mutation of agragr expression in RN6390 can be functionally defined as excessive at least in the context of biofilm formation. The fact that clinical isolates of the USA300 clonal lineage are genotypically related to isolates of the 8325 lineage agr at high levels agr. To examine this issue, we chose three USA300 isolates, the genomes of two of which  have been sequenced We previously demonstrated that clinical isolates of The levels of RNAIII produced by each strain were dependent on both growth phase and growth medium. RNAIII levels were higher in TSB than in biofilm medium (BM) in all strains except RN6390, which produced comparable levels of RNAIII whether grown in BM or TSB . The redOf the planktonic growth conditions examined in this report, the most applicable by comparison to our biofilm assays is a stationary-phase culture in biofilm medium, and under these conditions the targeted strains could be divided into three groups. The first consisted solely of RN6390, which produced higher levels of RNAIII by comparison to all other strains. The second consisted of the USA300 isolates UAMS-1782 and UAMS-1790, both of which produced RNAIII at levels that exceeded those observed in UAMS-1 or the USA300 isolate UAMS-1625 to a statistically-significant degree . These ragr was reduced in USA300 isolates under our growth conditions to the point that it had no impact on biofilm formation. To the extent that RNAIII levels remained relatively high in RN6390 even under these conditions, this is consistent with the observation that RN6390 was the only strain we examined in which mutation of agr had an impact on biofilm formation (see below).The fact that all three USA300 isolates formed a biofilm in our assay is in contrast to an earlier report demonstrating that the USA300 isolate LAC did not form a biofilm in a microtiter plate assay in vitro results using any assay conditions with in vivo conditions, implanted medical devices are coated with plasma proteins in vivoin vivo by comparison to UAMS-1, it was nevertheless capable of doing so to an extent that could be correlated with reduced antibiotic susceptibility sarA on biofilm formation, and the positive impact of this on antibiotic susceptibility, in both UAMS-1 and UAMS-1625 under both in vitro and in vivo conditions Coating with plasma was also shown to play an important role. Specifically, we repeated our experiments using the same microtiter plate assay but without plasma coating and found that, as with the other clinical isolates we have examined agr did not enhance biofilm formation in any of the USA300 isolates irrespective of whether plasma coating was employed  while in methicillin-resistant strains the more important consideration was the impact of sarA on the production of surface-associated protein adhesins. We previously demonstrated that mutation of sarA in the methicillin-sensitive strain UAMS-1 does result in reduced PIA production, but an isogenic ica mutant retained the capacity to form a biofilm sarA mutant. Rather, our results to date suggest that the more important consideration even in UAMS-1 is the increased production of extracellular proteases Unlike s of agr . O'NeillS. aureusagr both represses the production of these adhesins and induces the production of extracellular proteases agr and biofilm formation. At the same time, transcription of the genes encoding extracellular proteases, including aureolysin (aur) and sspA, is directly repressed by both SarA and Rot sarA, rot and agr appears to be dependent on the relative concentrations of their products with respect to each other rather than the concentration of any individual product alone agr expression, and we found that this was in fact the case. Specifically, by comparison to RN6390, all three USA300 isolates produced reduced amounts of all extracellular proteases that could be detected using either casein or gelatin zymography  and protein A mography . In factmography , this wamography .sarA and agr on both biofilm formation and protease production, and in all cases we found a direct and inverse relationship between these two phenotypes. Specifically, the only strain derived from RN6390 that produced a biofilm in our microtiter plate assay was an agr mutant, which also exhibited decreased production of extracellular proteases   of sarA . This nosarA mutants to repress the production of extracellular proteases plays a predominant role with respect to their dominant-negative phenotype by comparison to agr. However, the inclusion of protease inhibitors did not fully restore biofilm formation in any of the sarA mutants (sarA mutants by comparison to their respective parent strains (sarA mutants. Because extracellular DNA (eDNA) also contributes to biofilm formation in S. aureussarA in UAMS-1 resulted in increased production of nuclease and that mutation of nuc partially restored the ability of a UAMS-1 sarA mutant to form a biofilm sarA also results in increased production of extracellular nucleases in USA300 isolates  at levels that would be expected to promote biofilm formation and one (protease) at levels that would be expected to limit biofilm formation. Taken together, these results provide further support for the hypothesis that the sarA-mediated repression of extracellular proteases plays a predominant role in S. aureus biofilm formation.Concomitant mutation of A mutant . In this mutants . Thus, on RN6390  and 11. isolates . Thus, onuc gene designated as open-reading frame SA0746 in the N315 genome, mutation of which was shown to abolish nuclease production even in a UAMS-1 sarA as assessed using DNase agar S. aureus, one of which is encoded by SA0746 while the other is encoded by SA1160 nuc1 and nuc2 respectively. In these experiments we confirmed that mutation of nuc1 in UAMS-1 essentially eliminated the production of extracellular nuclease even in a sarA mutant and that mutation of nuc2 had no effect as assessed using DNase agar  known to impact S. aureus regulatory circuits rsbU in an 8325-4 strain (SH1000) resulted in reduced production of extracellular proteases agr resulted in an enhanced capacity to form a biofilm  and sarS, neither of these mutations is present in any of the clinical isolates examined in this report  in UAMS-1 and its isogenic sarA mutant was done using pKOR1 as previously described nuc mutant (designated here as nuc2) was generated by allele replacement mutagenesis. Specifically, 498 and 469 bp fragments were independently amplified from the upstream and downstream regions of the targeted gene using the primers shown in erm gene using the EcoRI and KpnI restriction sites for the upstream fragment and XbaI and PstI restriction sites for the downstream fragment. The resulting nuc2-erm-nuc2 cassette, which contains all of the SA1160 gene except the 115 bp region that was replaced with the 1.3 kb erm gene, was then excised by EcoRI/PstI digestion and subcloned into pCL52.2 nuc2 mutation was subsequently introduced into the isogenic nuc1, sarA and sarA/nuc1 mutants by phage-mediated transduction The All strains were maintained as stock cultures at −80°C in tryptic soy broth (TSB) containing 25% (v/v) glycerol. For each experiment, each strain was retrieved from cold storage by plating on tryptic soy agar (TSA) with appropriate antibiotic selection. Antibiotics were used at the following concentrations: erythromycin , tetracycline , kanamycin , neomycin , and chloramphenicol . Kanamycin and neomycin were always used together to avoid the spontaneous generation of resistant strains. To ensure that the results of all phenotypic assays were consistent, unless otherwise noted all assays were done using cultures grown in TSB supplemented with 0.5% glucose and 3.0% sodium chloride (biofilm medium) without antibiotic selection as previously described asp23, sarS and RNAIII expression, total bacterial RNA was isolated using the Qiagen RNeasy Mini Kit as previously described To assess levels of in vitro using a static, microtiter plate biofilm assay as previously described Biofilm formation was assessed 2, 1 mM DTT). To visualize protease bands, gels were then stained with SimplyBlue SafeStain  at RT for 2 hrs before destaining overnight in distilled water. Assays employing the E-64, 1-10-phenanthroline  and dichloroisocoumarin (DIC)  protease inhibitors were done as previously described using the highest concentration of each inhibitor that did not limit growth (4).Protease activity was assessed by zymography using 4–16% Zymogram (Blue Casein) Gels and 10% Zymogram (Gelatin) Gels . In both cases, supernatants were harvested from overnight (15 hr) cultures and normalized with respect to each other prior to filter sterilization. Sterile supernatants were then concentrated 15-fold using Centricon YM-3 filter units  before loading equivalent samples using a buffer containing DTT but not β-mercaptoethanol. After electrophoresis, gels were first incubated for 30 min at room temperature (RT) in renaturing buffer (2.5% TritonX-100) and then overnight at 37°C in developing buffer . Briefly, supernatants were harvested from overnight (15 hr) cultures and normalized with respect to each other prior to filter sterilization. Sterile supernatants were then concentrated 15-fold Centricon YM-3 filter units . 15 µl aliquots were then placed into wells cut into the D'NASE test agar. Plates were incubated overnight at 37°C. Nuclease activity was then assessed by overlaying the agar with 1N HCl to precipitate undigested DNA and define the zone of clearance around the supernatant harvested from each strain.To assess the production of PIA, overnight cultures were normalized with respect to each other before harvesting cells by centrifugation. Cells were resuspended in 50 µl of 0.5 M EDTA (pH 8.0) and boiled for 5 min. Cellular debris was removed by centrifugation before incubating the supernatant with proteinase K (20 mg/ml) at 37°C for 60 minutes. After the addition of 10 µl of Tris-buffered saline , each extract was spotted onto a nitrocellulose membrane using a BIO-Dot microfiltration apparatus . After drying, the presence and amount of PIA was assessed using anti-PIA antiserum and the WesternBreeze chemiluminescence immunodetection kit .sarA and agr mutants and their sarA-complemented derivatives was done by ANOVA based on all pairwise comparisons. In both cases p values <0.05 were considered significant.Statistical analysis of results comparing wild-type strains was done using the Students t-test. Statistical analysis of results comparing different strains with their isogenic


ETS gene fusion analyses, we have gained insights into the molecular basis of CRPC. We envision that a better understanding of the mechanisms underlying resistance to abiraterone acetate, as well as the development of validated predictive and intermediate endpoint biomarkers to aid both patient selection and monitor response to treatment, will improve the outcome of CRPC patients.The limited prognosis of patients with castration-resistant prostate cancer (CRPC) on existing hormonal manipulation therapies calls out for the urgent need for new management strategies. The novel, orally available, small-molecule compound, abiraterone acetate, is undergoing evaluation in early clinical trials and emerging data have shown that the selective, irreversible and continuous inhibition of CYP17 is safe with durable responses in CRPC. Importantly, these efficacy data along with strong preclinical evidence indicate that a significant proportion of CRPC remains dependant on ligand-activated androgen receptor (AR) signalling. Coupled with the use of innovative biological molecular techniques, including the characterisation of circulating tumour cells and  Although the majority of patients diagnosed with prostate cancer are cured with definitive primary treatment, prostate cancer remains the second leading cause of male cancer-related death in the western world . The cende novo synthesis) has long been recognised. In fact, contemporaneous ADT does not eliminate androgen synthesis within prostate cancer cells. On the contrary, intraprostatic (in situ and metastatic) levels of DHT and testosterone have been shown to remain elevated despite castrate serum levels  that renders these cells exquisitely sensitive to extremely low levels of exogenous androgens (α (androgenic and oestrogenic) that may impact CRPC biology.The recognition that the state of androgen resistance is relative is certainly not new. Clinical experience in the management of breast and prostate cancers have clearly illustrated that repeated hormonal manipulations can be used successfully . Moreovendrogens . Furtherα-hydroxylase and C17, 20-lyase, in the biosynthesis pathway, as illustrated in α-hydroxylase activity converts pregnenolone to 17α-hydroxypregnenolone and progesterone to 17α-hydroxyprogesterone, whereas C17,20-lyase converts 17α-hydroxypregnenolone to dehydroepiandrosterone (DHEA) and 17α-hydroxyprogesterone to androstenedione  occurs at low doses of ketoconazole, whereas much higher doses are required for the non-specific inhibition of CYP17  working group (PSAWG) criteria , was developed as part of a series of potent, inhibitory 17-(3-pyridyl) steroids at The Institute of Cancer Research, UK  with minimal side effects in other organs when administered daily.First-in-man phase I studies reported that abiraterone acetate was safe when administered daily for 12 days to men with non-progressing prostate cancer, and resulted in >50% suppression of baseline testosterone in non-castrate patients  use. Significantly, tumour responses to abiraterone acetate were observed in castrate patients who had failed several prior lines of AR-targeting therapy . Subsequent phase I/II studies in the UK and US have treated more than 100 patients and provide additional support to the efficacy of this agent. These preliminary efficacy data compare favourably with established second-line ADT and novel molecularly targeting agents, and mandate further evaluation of this agent in randomised studies.The results of the phase I studies have led to the phase II evaluation of abiraterone acetate in CRPC patients in the UK and US (vs prednisolone (or prednisone) plus placebo, with a primary endpoint of overall survival. The combination of steroids and abiraterone acetate should potentially prevent the syndrome of secondary mineralocorticoid excess and maximise efficacy.A placebo-controlled randomised phase III study with the aim of securing FDA licensing approval in the post-docetaxel setting is now open to accrual; 1180 patients will be randomised 2 : 1 for abiraterone acetate plus prednisolone (or prednisone) The antitumour activity reported with abiraterone acetate could be explained, at least in part, by the durable and profound suppression of serum androstenedione and DHEA, in addition to that of testosterone and oestradiol  with two of these patients having had progressive disease earlier on the same dose of dexamethasone, were successfully salvaged using this approach -α  loss; and/or activation of translocated r (ER)-α . In fact acetate . TranslaTo maximise benefit to patients and accelerate drug approval by regulatory authorities, the evaluation of robust biomarkers that can serve as predictive and intermediate end-points is urgently required in CRPC therapeutic studies. We describe the preliminary experience we have of the two of the most promising biomarkers that are emerging in this field; the results of their prospective verification in larger cohorts of subjects are eagerly awaited.TMPRSS2, with ERG occurs in up to 60% of prostate cancers and is likely to account for the majority of ETS oncogene rearrangements in prostate cancer  samples (five out of six patients in the published phase I study with an ERG gene rearrangement had a decline in PSA ⩾50%). However, due to ETS gene rearrangement heterogeneity within a single prostate, the use of single TRBP cores may miss areas with ERG rearrangements and fusion of other ETS genes with androgen-regulated partners can also occur.Fusion of the androgen-regulated gene, We have also recently shown that the presence of ⩾5 circulating tumour cells at baseline is associated with a poor outcome and that a fall in CTC count to <5 following treatment is associated with an improved prognosis , is a 17-benzoimidazole that has direct inhibitory properties against CYP17 and AR (The novel small molecule AR antagonist, MDV3100, was rationally designed for its ability to overcome resistance to conventional bicalutamide in the setting of increased AR expression (To summarise, these agents work directly to reduce or inhibit AR-mediated signalling by ligand depletion and/or receptor antagonism. Overall, these data provide further mechanistic and clinical evidence of the importance and relevance of AR-mediated signalling pathway in CRPC therapeutics.The promising efficacy of abiraterone acetate stemming from these early clinical trials in CRPC setting attests to our renewed recognition of the importance of the AR-signalling pathway in CRPC biology. It is sobering to realise that up to 70% of patients who had disease progression on previous lines of hormonal therapies, are still hormone dependant. Indeed, the recent revision of misnomers, such as ‘androgen independence’ and ‘hormone resistance’, by the Prostate Cancer Clinical Trials Working Group is a reflection of this. The successful reversal of resistance to abiraterone acetate using low-dose dexamethasone in some patients also points to the likely importance of steroids upstream of CYP17, and indicate that AR, although likely mutated and promiscuous, remains key in this process.ETS gene fusion products and CTCs provide further, albeit preliminary, molecular biological corroboration of the importance of AR-mediated signalling mechanisms. These novel molecular platforms may have additional utility in improving our ability to select patients more likely to benefit from abiraterone acetate treatment.Although the numbers of subjects and biological specimens involved are small, the evidence from the translational studies involving There are numerous questions emerging from the use of this compound. For instance, how are the intratumoural androgenic steroid levels affected by abiraterone acetate? What are the mechanisms of resistance to it? In what clinical settings ought the drug be used? With which agents, cytotoxic and novel, should abiraterone acetate be used? What about the role of abiraterone acetate in other hormonally driven malignancies?Overall, these data support the development of further novel inhibitors of AR signalling. However, sceptics will trumpet the acknowledgement that we have been in similar positions before with other ADT modalities. We argue that these new clinical and translational seeds are being sowed in research soil more fertile than ever before, enriched by an improved understanding of the pathways of treatment resistance and the availability of new technologies in the evaluation of tumour biology. Hence, not only do we have more effective compounds, there are unparalleled opportunities for these to be combined with emerging novel biological agents, supported by the presence of new molecular and imaging methodologies.


Viruses are the predominant infectious cause of asthma exacerbations in the developed world. In addition, recent evidence strongly suggests that viral infections may also have a causal role in the development of childhood asthma. In this article, we will briefly describe the general perception of how the link between infections and asthma has changed over the last century, and then focus on very recent developments that have provided new insights into the contribution of viruses to asthma pathogenesis. Highlighted areas include the contribution of severe early life viral infections to asthma inception, genetic determinants of severe viral infections in infancy, the differences in innate and adaptive immune system cytokine responses to viral infection between asthmatic and nonasthmatic subjects, and a potential vaccine strategy to prevent severe early life virally-induced illness. Infections have been recognized to be a cause of asthma exacerbations for over 100 years. In his landmark textbook that heralded modern medicine, Sir William Osler noted that among other asthma exacerbating factors such as allergens and environmental changes, "every fresh cold" could induce a paroxysm of disease . This coDespite the inability of bacterial skin testing to confirm hypersensitivity to these organisms, immunotherapy containing bacterial extracts was used substantially in the prophylaxis against asthma exacerbations from the 1920s to the late 1950s ,5. PhysiTo further determine if the presence of bacteria was associated with worsening asthma symptoms, investigators sought to identify if bacteria could be cultured from the upper respiratory tract more often when patients were symptomatic with asthma exacerbations, compared to times when they were not. There was no difference in the presence of bacterial infections when subjects were symptomatic compared to when they were not, further suggesting that bacteria were not responsible for asthma worsening ,11. In cBy the mid 1990s, polymerase chain reaction (PCR) technology was being used in clinical research studies and revealed that viruses could be detected in the respiratory tract secretions of 80–85% of school children who were experiencing asthma exacerbations . StudiesThe role of viral infections in asthma inception in infants and young children has been debated for many years. Wu and colleagues found that birth timing in relationship to the peak of hospitalization for bronchiolitis determines the risk of severe bronchiolitis during infancy, as well as the risk of developing asthma . These fFirst, the timing of infant birth in relationship to the winter virus peak, as defined as the first day of the week with the highest number of bronchiolitis hospitalizations for that winter season, predicted the likelihood of developing clinically significant bronchiolitis . ClinicaIn the third analysis, compared to children who were 365 days old at the winter virus peak, there was a 29% increase in odds of developing high-risk childhood asthma for children who were 121 days of age at the winter virus peak, the almost identical infant age at winter virus peak that conferred the greatest risk of bronchiolitis . The impFifth, despite the fact that smoking during pregnancy and maternal asthma increased the odds of developing high risk asthma by 7% and 82%, respectively, neither of these factors significantly changed the relationship between infant age at winter virus peak and childhood asthma . There wThe authors speculate that there are two possible explanations for the relationship between the timing of the peak of the viral season and the increased risk of developing both significant bronchiolitis and high risk asthma . The firWu and colleagues did not determine the viruses responsible for the peak of the viral season in their study ; howeverAnalysis of respiratory tract secretions revealed that viruses were present in these fluids 90% of the time that wheezing illnesses occurred in the first three years of life . RhinoviThe authors also analyzed the data based on the viruses detected at each year up to age 3 and the subsequent risk of developing asthma at age 6 . They foAs noted above, early life rhinovirus and RSV are associated with the subsequent development of asthma. The recognition that rhinovirus may have an important role in the inception of asthma is a novel concept, first put forward by Jackson and colleagues as just reviewed . TherefoVDR), the signal transduction molecule Jun (JUN) which composes part of the AP-1 signaling complex, interferon (IFN)-α (IFNA5) which is a type I IFN involved in early cytokine responses to viral infection, inducible nitric oxide synthase (NOS2A), and the high affinity IgE receptor (FCER1A) important in IgE binding to mast cells, basophils, dendritic cells, and other immune cells.Janssen reported that 22 single nucleotide polymorphisms (SNPs) in 21 genes were identified that were statistically significantly associated with severe RSV disease at either the genotype or allele level . Those SIFNA13, IL15, STAT1, and TLR8), chemotaxis , adaptive immunity (CD28 and STAT1), and allergic asthma . Those SNPs that had an association with RSV at the genotype level only were present in genes involved in innate immunity (TNF and NCF2) and adaptive immunity .SNPs were also discovered that were associated with severe RSV disease at the allele level . Based oThis study is an important contribution to our scientific knowledge of RSV-induced illness because it provides candidates for future genetic analysis, haplotype determination, and functional studies to further define the contributing factors that are responsible for severe disease. In addition, the data reveal that genetic susceptibility to RSV bronchiolitis is a very complex trait with host response genes being strong contribution factors.IFNA13, IL15, IL10, and IL17) and cytokine receptor signaling . Since there is a very high percentage of asthmatic children who are atopic, one could hypothesize that those children who have RSV bronchiolitis in infancy and who subsequently develop asthma have increased levels of Th2 cytokines compared to children who experience RSV bronchiolitis, but that are not diagnosed with asthma. Castro and colleagues tested this hypothesis in a prospective cohort of 206 previously healthy children who had been enrolled in the RSV Bronchiolitis in Early Life (RBEL) study [As just mentioned above, genes associated with severe RSV disease in infancy, and perhaps asthma inception, include those that are involved in cytokine production (L) study . As oppoL) study , these cL) study . DespiteL) study . HoweverL) study .1) and peak expiratory flow (PEF), and heightened methacholine reactivity in response to experimental rhinovirus infection compared to the nonasthmatic subjects. In regard to cytokine responses, persons who had greater IFN-γ and IL-10 production by peripheral blood CD4 cells had decreased viral loads and decreased upper respiratory tract symptoms. Similarly, those subjects with greater IFN-γ production by CD4 cells obtained by BAL were protected from falls in PEF after rhinovirus infection, while those subjects whose BAL CD4 cells produced higher levels of the Th2 cytokines IL-4, IL-5, and IL-13 all had increased lower respiratory symptom scores. In addition, after rhinovirus infection, BAL cells from asthmatic subjects were deficient in the production of IFN-γ, IL-10, and IL-12, while producing greater Th2 cytokine levels. In contrast rhinovirus infection resulted in induced IFN-γ, IL-10, and IL-12 from the nonasthmatics' BAL CD4 cells, while there was no increase in Th2 cytokines in this group [The cytokine profile produced in bronchiolitis is very different from that which occurs with virally-induced asthma exacerbations. Message and colleagues performed a study in which they experimentally infected 10 asthmatic subjects and 15 nonasthmatic subjects with rhinovirus and then examined pulmonary function and cytokine responses in CD4 cells from peripheral blood and bronchoalveolar lavage fluid . They reis group . This dais group  and typeis group  in respois group . In thisis group .As early life severe viral infection has been linked to asthma inception, preventing such episodes would perhaps decrease the incidence and prevalence of asthma. Unfortunately there is no currently available FDA-approved vaccine against RSV. However, a recently completed trial provides hope that such a vaccine approach may soon be available. Wright and colleagues report that 388 study subjects were examined and divided into one of three groups: children that received one of several live, attenuated RSV vaccines, children administered placebo vaccine, and age-matched controls . SubjectWhile, live attenuated RSV vaccines may present a potential novel therapy, other studies suggest that prophylactic anti-RSV immunoglobulin may affect the later development of asthma. Simoes reported that palivizumab, an anti-RSV monocolonal antibody, reduced subsequent wheezing in preterm infants . In a reAlthough bacterial infections were long considered to the primary infectious agents linked to asthma pathogenesis, over the last decade, new data supports that virus infections are the cause of a great majority of asthma exacerbations. New data published in the last year strengthens earlier reports that early life viral infections may have a significant role in asthma inception. Gene association studies reveal that persons with deficiencies in innate immunity may be more predisposed to these severe early life episodes of viral bronchiolitis that predispose to the later development of asthma. Deficiencies in innate immunity are also present in adult asthmatics compared to their nonasthmatic counterpart as witnessed by the difference in cytokine responses in peripheral blood and BAL CD4+ cells following rhinovirus infection and in the epithelial cells responses to viral challenge. Prevention of early life severe bronchiolitis episodes is a major priority and vaccine approaches are being developed to fill this therapeutic need. Further research and therapeutic strategies are needed to advance our understanding of the contribution of viruses to asthma pathogenesis and protect against both the inception and exacerbations of this disease.PCR: polymerase chain reaction; RSV: respiratory syncytial virus; TLR: toll like receptor; TNF: tumor necrosis factor; IL: interleukin; SNP: single nucleotide polymorphism; VDR: vitamin D receptor; IFN: interferon, NOS2A: nitric oxide synthase; FEV1: forced expiratory volume in 1 second; PEF: peak expiratory flow; RV: rhinovirus.The authors declare that they have no competing interests.MMH and RSP both wrote the paper and contributed equally.


Under physiological conditions, hepatic stellate cells store 80 % of the total vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm, and regulate both transport and storage of vitamin A -3.It has been demonstrated that animal or human individuals exposed to drugs such as methadone, prednisone and phenobarbital, antiepileptics , and xenRecent data demonstrate that top predators among Svalbard mammals and birds like polar bear, arctic fox and glaucous gull accumulate relatively large amounts of persistent organic pollutants . Since iAfter getting permission to hunt the animals from the district governor of Svalbard, 11 arctic foxes and 14 bearded seals, were caught in the Svalbard archipelago near Longyearbyen  in the period from August 1996 to September 2001. Three polar bears were shot in self-defense at Svalbard February and August 1998 in Ny Ølesund and Hornsund. Distribution and content of vitamin A in livers and other organs were analyzed by morphological methods such as transmission electron microscopy, fluorescence microscopy for detection of autofluorescence of vitamin A and gold chloride staining [The amounts of vitamin A stored in livers of arctic animal are shown in Table The concentration of total retinol in kidney is normally less that 1% of the concentration in liver. However, following an intake of excessive amounts of vitamin A or after experimental treatment with xenobiotics like HCB, TCDD and PCB, the concentration in kidneys may increase several-fold ,11. KidnStrong autofluorescence and gold chloride staining were present in hepatic stellate cells of polar bear, arctic fox, and bearded seal (data not shown). The distribution of stored vitamin A in the arctic animals was essentially the same as that published previously in normal rat and human liver ,3. In liIncreased kidney concentrations of total retinol in arctic fox most likely are a sign of pollutant-induced vitamin A-toxicity. It is interesting to note that the highest accumulation of organic pollutants in arctic animals occur in the arctic fox . Relativ


Congenital Bicuspid Aortic Valve (BAV) is a significant risk factor for serious complications including valve dysfunction, aortic dilatation, dissection, and sudden death. Clinical tools for identification and monitoring of BAV patients at high risk for development of aortic dilatation, an early complication, are not available.This paper reports an investigation in 18 pediatric BAV patients and 10 normal controls of links between abnormal blood flow patterns in the ascending aorta and aortic dilatation using velocity-encoded cardiovascular magnetic resonance. Blood flow patterns were quantitatively expressed in the angle between systolic left ventricular outflow and the aortic root channel axis, and also correlated with known biochemical markers of vessel wall disease.The data confirm larger ascending aortas in BAV patients than in controls, and show more angled LV outflow in BAV (17.54 ± 0.87 degrees) than controls (10.01 ± 1.29) (p = 0.01). Significant correlation of systolic LV outflow jet angles with dilatation was found at different levels of the aorta in BAV patients STJ: r = 0.386 , AAO: r = 0.536 , and stronger correlation was found with patients and controls combined into one population: SOV: r = 0.405 , STJ: r = 0.562 , and AAO r = 0.645 . Dilatation and the flow jet angle were also found to correlate with plasma levels of matrix metallo-proteinase 2.The results of this study provide new insights into the pathophysiological processes underlying aortic dilatation in BAV patients. These results show a possible path towards the development of clinical risk stratification protocols in order to reduce morbidity and mortality for this common congenital heart defect. Bicuspid aortic valve (BAV) is the most common congenital cardiovascular malformation with an estimated prevalence of 1-2% ,4. An asin vivo by 4-D cardiovascular magnetic resonance (CMR) visualization in case reports involving BAV in association with a coarctation [Even though the association between bicuspid aortic valves and aortic dilatation is well known, clinical predictors for the development and progression of aortic dilatation are lacking. Abnormal blood flow patterns and the consideration of BAV as part of a connective tissue disease may be important factors, but their relative importance remains unclear. Excised BAVs have been shown associated with abnormal flow patterns and turbulence aimed toward the right anterolateral wall (convexity) of the aorta in flow loop experiments with phantom models of aorta . An echorctation  and in arctation . Patholorctation ,26. Locarctation . In BAV This paper reports on a prospective CMR study to define quantifiers of abnormal aortic blood flow patterns in a group of pediatric BAV patients, in comparison to normal control subjects in the same age range. The main purpose of the study is to investigate the hypothesis that more eccentric systolic aortic flow jets in BAV patients are associated with more dilated aortic dimensions. The study was motivated by the need for a robust risk stratification protocol for development of clinically significant complications in pediatric BAV patients. Support for the stated hypothesis could help justify more extensive investigation of blood flow phenomena in this context. The same motivation also prompted us to add to the experimental protocol a confirmatory investigation of serum levels of Matrix Metalloproteinases  and associated tissue inhibitors . MMPs are associated with various forms of vascular remodeling -32, and Time-resolved structural and 3-D velocity-encoded CMR images were visualized jointly to create a dynamic 3-D view of blood flow patterns as vector fields in an anatomical context using CMR techniques similar to those explored previously for observation of blood flow patterns in the great arteries -40, braiFrom October 2006 until July 2008 a total of 18 asymptomatic BAV patients and 11 healthy control subjects in the age group 10-18 years were recruited and successfully underwent CMR. All subjects gave informed consent/assent per IRB-approved protocol. Patient exclusion criteria were: systemic hypertension for age and height, treatment with cardiac or vasoactive medications, subjects suffering from possibly confounding conditions such as connective tissue diseases including Marfan or Turner syndrome, greater than mild aortic stenosis (defined by a mean gradient by Doppler echocardiography of ≥ 17 mmHg), more than mild aortic regurgitation (defined by a regurgitant jet resulting in a left ventricular end-diastolic dimension >2 standard deviations above the mean for body surface area (BSA), or the presence of pan-diastolic retrograde flow on pulsed Doppler evaluation of the abdominal descending aorta). One control subject was found to have dilatation of the ascending aorta, considered unrepresentative of the normal pediatric population, and was excluded from analyses. General patient characteristics are summarized in Table All subjects underwent a single non-contrast CMR which included structural and phase velocity-encoded imaging of the left ventricular outflow tract (LVOT) and ascending aorta. Structural breath-held SSFP ciné images were acquired on a GE Signa Twinspeed 1.5 T scanner, running Release 12M4. Scan parameters for the FastCINE/FIESTA pulse sequence included TR = 4 ms, TE = 2 ms, flip angle 45 degrees, typical FOV 300-350 mm, 128-192 phase encodings, RFOV 70-100%, reconstruction matrix 256 × 256, pixel BW 1 kHz, VPS = 8-12 , 20 cardiac phases reconstructed using retrospective VCG gating, 1 slice acquired per breath-hold, ASSET parallel imaging acceleration used as SNR allowed, acquisition time 6-14 s/slice. Views included an oblique sagittal LVOT view Figure , an obliDynamic structural images and blood flow velocity patterns were visualized using the in-house developed Heartviz/Flowviz 3-D software. Example visualizations are shown in Figure Time points of systole and diastole were determined by observation of dynamics of cardiac contraction and blood flow. A vector representing direction and velocity of the peak systolic blood flow jet was calculated by averaging of flow vectors in all forward flow pixels inside the aorta  in a slice located centrally in the sinuses of Valsalva at peak systole. Figure Aortic diameters were measured at the AOV, SOV, STJ and AAO during peak systole. At each location the largest observed perpendicular cross-sectional diameter was used for analysis. For diameters at the level of AOV, SOV, and STJ axial images at t hose locations were analyzed, angled perpendicular to the aorta Figure . The maxFrom the BAV patients blood samples were obtained prior to CMR from peripheral veins and collected in EDTA tubes. These measurements were not obtained in control subjects in this preliminary study for reasons of ethics and recruitment logistics. Plasma was separated by centrifugation at 3300 rpm for 10 min at 4°C and immediately frozen and stored at -80°C. Plasma MMP2, MMP9, TIMP1 and TIMP2 levels were determined by two-site enzyme-linked immunosorbent (ELISA) assays using commercially available kits with monoclonal antibodies against the proteins . The assays measured total MMP 2, MMP 9 and TIMP1 and TIMP 2 levels in the samples. All samples were run in duplicate and results were averaged.Distribution characteristics are presented as mean +/- standard error of the mean, unless otherwise noted. Distributions were tested for normality using Q-Q plots and the Shapiro-Wilk test. Group comparisons between BAV patients and controls used the unpaired Student's t-test. Pearson's correlation coefficients were used to determine correlations between normal distributions. Spearman's non-parametric rank correlation coefficients were used for very small samples and non-normally distributed populations.Prior to all correlation analyses data were screened on multivariate outliers using informal identification in scatter plots and calculation of Mahalanobis distance. Outliers were not removed from data. All tables indicate the number of subjects on which results are based.Intra-observer variability in aortic dimension and flow angle measurements was calculated by evaluation of duplicate measurements by the same observer. Inter-observer variability was calculated by evaluation of the same measurements by two observers. These measurement sets were evaluated for the entire study population, and thus represent the entire encountered range of sizes and blood flow. Calculated statistics included Lin's concordance correlation coefficients (CCC) , the estStatistics were evaluated using Microsoft Excel 2003  and SPSS 16.0 for Windows . A p-value < 0.05 was considered to establish statistical significance.Characteristics of all BAV patients and control subjects are summarized in Table Comparison of aortic diameters, corrected for BSA, revealed that on average BAV patients had a statistically significant larger diameter at the level of the SOV, STJ and AAO compared to controls Figure , Table 2The inter- and intra-observer variability for diameter measurements is summarized in Table The average angle between the systolic blood flow jet direction at the level of the AOV and the left ventricular outflow tract axis was significantly larger in BAV patients as a group (17.54 +/- 0.87 degrees) than in control subjects   and approached significant correlations at the levels of the SOV  and STJ . TIMP2 plasma levels correlated significantly to aortic diameter at the level of the STJ  and moderately at the level of the SOV . MMP9 and TIMP1 plasma concentrations were not correlated to aortic diameter at any anatomical level.Plasma MMP2 levels were moderately correlated to blood flow jet angle . No significant correlation was observed between blood flow jet angle and MMP9 or TIMP1 and TIMP2 plasma levels.We found significant correlations between the blood flow jet angle, representing a quantitative measure of misdirected blood flow, and aortic diameters at the level of the SOV, STJ and AAO. The significantly positive correlation coefficients suggest that the larger the angle of misdirected flow with the aortic axis, the larger aortic diameter and thus the more severe the observed dilatation is. These results favor the hypothesis that abnormal, misdirected blood flow patterns associated with BAV are directly implicated in the development of aortic dilatation and that dilatation is not solely a manifestation of a connective tissue disease as some investigators have proposed . CorrelaIn addition to the flow data, we also found significant correlations between aortic diameters and MMP 2 and TIMP 2 but not MMP 9 and TIMP 1 plasma levels in BAV patients. These results confirm previous studies that demonstrated significantly increased tissue levels of MMP 2 but not MMP 9 in aneurysms of BAV patients compared to TAV patients ,49,50, aThe hypothesis of a direct relation between abnormal flow patterns and aortic dilatation is consistent with our observations that the correlation between the blood flow jet angle and aortic diameter is stronger when BAV patients and control subjects are pooled into a single population. A hypothesized effect of dilatation as a result of eccentric systolic blood flow need not necessarily be specific to BAV subjects, but may occur similarly in other individuals with similarly disturbed flow, and absent in any subjects with well-aligned flow patterns. In this respect it is a logical step to consider both groups as different classes within one population, who are all subject to this same effect. With the relatively small number of subjects included in the present study significant correlations were found at 2 of 4 levels of the aorta when observing BAV patients alone. The normal population alone mostly has normal flow, hence lower spread in aortic sizes, and thus shows no correlation. Pooling the normal control subjects and the BAV population will show larger numbers as well as an increased range of both phenomena, evidenced by a stronger correlation, which is now significant at 3 out of 4 levels of the aorta. Again larger studies in a wider age range are required to actually determine cause and effect and to differentiate between BAV patients at risk for rapid aortic dilatation and lower-risk slow progressors. The causality question may of course not be straightforward to answer, since it is well-known that BAV is also associated with connective tissue disease ,51,52. BShould a causal link between misdirected blood flow and severity of aortic dilatation be confirmed, a combination of imaging tests and protein assays may be envisioned for use in a BAV management protocol to allow for clinical risk monitoring of BAV patients. For instance, regular sampling of plasma levels of extracellular matrix proteins in previously diagnosed BAV may be used as an indication of the presence of vessel wall injury. In this population such a finding puts the immediate suspicion on injury localized in the aorta, and imaging tests may be indicated in patients with consistently elevated plasma levels to diagnose dilatation, and/or risk for accelerated dilatation if highly abnormal flow patterns are observed. Clinical risk stratification of BAV patients is much desired considering the variation in development and progression of aortic dilatation and the increased risk for serious complications including sudden cardiac death -20. DetaDue to the relatively small number of samples and lack of age stratification in our data the results do not yield insight into whether misdirected flow patterns are a cause or a consequence of aortic dilatation. This preliminary study aimed at investigating 3-D CMR flow analysis as a promising discriminating test for at-risk patients. Despite the small sample some important significant correlations were elucidated, but a larger study, spanning a more complete spectrum of ages and flow patterns, and aortic dimensions, will be required to investigate causality of these phenomena.Another limitation of the study is the relatively simplistic and naïve quantification of abnormality the aortic flow jet by calculation of the flow angle. The flow jet angle, investigated in this work, quantifies mis-direction of aortic flow, presumably due at least in part by incomplete opening of the aortic valve. While this phenomenon is readily observed and quantified by velocity-encoded CMR as we have shown in this paper, it only quantifies an important but limited and simple aspect of the relevant fluid mechanics at work. More complete and sophisticated characterization of the hemodynamic stresses and forces acting on the aorta in this (and other) congenital disease are quite challenging from aspects of both mathematics and imaging technology.Increasing availability of 4-D flow imaging methods on commercial platforms, along with emerging research thrusts integrating CMR flow data with computational flow modeling, are anticipated to further improve on the accuracy and precision of the ad-hoc methods employed in this early investigation. We believe that combination of computational fluid modeling and 4-D blood flow CMR, although currently still in an early stage of development, carries great potential to contribute important new knowledge and answers to many important questions involving blood flow in congenital heart disease.In conclusion, the results of this study suggest that abnormal flow patterns, abnormal protein expression and aortic dilatation are all directly related in BAV patients. Our findings support the hypothesis that both factors are acting simultaneously in the development of dilatation . DespiteThe authors declare that they have no competing interests.PMdR performed much of the final data analysis, the MMP analyses, and drafted the manuscript; DSIII performed/supervised and read most CMR scans, was responsible for clinical direction and project design, and participated in scan and analysis protocol design, and contributed in manuscript writing and review; PvdV participated in design of image analysis protocols, performed early image and statistical analysis work; ERZ participated in scan protocol and project design, and early image analysis efforts; WJP performed/supervised/read CMR scans, and participated in scan protocol design; SR performed all off-line image reconstructions, image transfer and management, and 4-D flow acquisition; TQR contributed software/methods development for flow visualization and analysis and HIPAA compliance; GD performed all final image analysis work; CL was responsible for patient recruitment and IRB matters; RPB contributed as research advisor for PMdR, PvdV, and ERZ; MEB served as project director, finalized manuscript preparation, designed scan and image analysis software and methods, and performed the final statistical analyses.


Transient receptor potential vanilloid type 1 (TRPV1) receptor is a non selective ligand-gated cation channel activated by capsaicin, heat, protons and endogenous lipids termed endovanilloids. As well as peripheral primary afferent neurons and dorsal root ganglia, TRPV1 receptor is also expressed in spinal and supraspinal structures such as those belonging to the endogenous antinociceptive descending pathway which is a circuitry of the supraspinal central nervous system whose task is to counteract pain. It includes periaqueductal grey (PAG) and rostral ventromedial medulla (RVM) whose activation leads to analgesia. Such an effect is associated with a glutamate increase and the activation of OFF and inhibition of ON cell population in the rostral ventromedial medulla (RVM). Activation of the antinociceptive descending pathway via TPRV1 receptor stimulation in the PAG may be a novel strategy for producing analgesia in chronic pain. This review will summarize the more recent insights into the role of TRPV1 receptor within the antinociceptive descending pathway and its possible exploitation as a target for new pain-killer agents in chronic pain conditions, with particular emphasis on the most untreatable pain state: neuropathic pain. Drosophila melanogaster .2+-mediated mitochondrial damage and cytochrome C release [2+ influx and the activation of p38 [Until recently, pain had been thought to arise primarily from the dysfunction of neurons. Recent evidence, however, suggests that neuro-immune changes might also contribute to pain following injury to the nervous system. Glial cells involved in mediating inflammatory processes are resident within the spinal cord and include both astroglia and microglia, the latter of which has been directly implicated in the initiation of peripheral injury-induced pain . Moreove release  and thatn of p38 . Thus, bn of p38 . Howeverendovanilloids within the PAG-RVM counteract nociception. Thus, TRPV1 receptor  may offer a suitable target for enhancing the activity of the endogenous antinociceptive descending pathway and producing analgesia. Moreover, hybrid molecules with a dual mechanism, such as AA-5-HT, which blocks FAAH activity and TRPV1 receptors, do not induce hyperthermia as "classic" TRPV1 antagonists do. As well as the PAG-RVM antinociceptive axis, other regions of the brain known to influence descending pain modulation, such as the amygdala, thalamus and LC show TRPV1 receptor localization, although the exact contribution of such receptors, if any, has yet to be determined. Further studies to clarify the role of the supraspinal TRPV1 receptor and in particular, within the antinociceptive descending pathway, may open the way to a novel strategy of pain relief in central nervous system disorders, such as neuropathic pain, which does not yet have an appropriate therapy.The few findings about the role of supraspinal TRPV1 receptors in controlling pain appear encouraging. The possible exploitation of TRPV1 receptor for activating the antinociceptive descending pathway at PAG level may be a strategy of interest. Indeed a direct agonist such as capsaicin, an indirect one such as URB597 or an hybrid anadamide hydrolysis and TRPV1 receptor blocker such as AA-5-HT, lead to analgesia. TRPV1 receptor stimulation is associated with increased glutamate release and activation of group I mGlu and NMDA receptors and with modulation of downstream RVM ON and OFF neurons. Immunohistochemical data strongly support the hypothesis that the activation/inhibition of TRPV1 receptor expressed on glutamatergic neurons following intra-PAG injection of capsaicin/I-RTX leads to an increase/decrease in glutamate release and behavioural analgesia/hyperalgesia. Exogenous compounds capable of activating TRPV1 channels or strategy increasing The authors declare that they have no competing interests.EP has written the manuscript. EP and SM have conceived and conceptualized the manuscript. LL has written the paragraph on microglia. VdN, LB and FR contributed to the drafting of the paper. All authors have read and approved the final manuscript.


Panic disorder (PD) is characterised by sudden attacks of intense fear with somatic symptoms including palpitations and tachycardia. Reciprocally, palpitations caused by paroxysmal supraventricular tachycardia (PSVT) are commonly associated with anxiety and may therefore be misdiagnosed as PD. As demonstrated by two case reports, PSVT and PD can occur comorbidly in a chronological sequence, with PSVT possibly precipitating and maintaining PD via interoceptive processes or, alternatively, with PD increasing the risk for PSVT by elevating stress levels. As both PSVT and PD require different treatments, potentially helpful differential clinical diagnostic criteria are proposed. Panic disorder (PD) is characterised by a lifetime prevalence of 1% to 3% and sudden attacks of intense fear accompanied by somatic, particularly cardiac symptoms such as palpitations, chest pain, and tachycardia: 89% of patients with PD complain of palpitations, with up to 25% of patients initially referred to cardiac clinics with atypical chest pain or palpitations being later diagnosed with PD . In patiBased on two case reports of patients with comorbid PSVT and PD, neuropsychophysiological processes potentially driving this comorbidity will be discussed. Additionally, as both PSVT and PD require different treatments, potentially helpful differential clinical criteria will be proposed.A 34-year-old female patient presented to the department of psychiatry in 2002 with a history of panic attacks since the age of 18, occurring two to three times per week lasting for about 10 to 30 min and presenting with somatic symptoms including palpitations and tachycardia not terminable by vagal manoeuvres, as well as feelings of derealisation/depersonalisation and fear of losing control, going crazy or dying. In 1996, the patient additionally began to suffer from palpitations diagnosed as PSVT with a sudden onset and duration of 6 h, terminable by vagal manoeuvres. PSVT attacks were not accompanied by the other panic-related symptoms described above and the patient could clearly differentiate between PSVT and a tachycardic state within a panic attack. During an invasive electrophysiological study in July 2000, a rapid typical AV nodal re-entrant tachycardia was diagnosed, with induced cycle lengths of 280 to 330 ms (corresponding to heart rates during the tachycardia of 180 to 220 bpm). Using radiofrequency catheter ablation, the slow pathway of the AV node was successfully ablated. Thereafter, PSVT attacks subsided while the patient continued to suffer from increasingly severe panic attacks corresponding to a pathological Hamilton Anxiety Scale (HAMA) score of 36 and an increased score of 33 on the Anxiety Sensitivity Index (ASI). Increased sensitivity to cardiac sensations was reflected by an elevated Body Sensation Questionnaire (BSQ) mean score of 2.35 at the time of referral in 2002. Antidepressive pharmacological treatment with mirtazapine (15 to 30 mg) was administered for 6 months. Additionally, the patient underwent cognitive-behavioural psychotherapy (CBT) (20 sessions) including psychoeducation about the role of interoceptive cues within the vicious circle leading to panic attacks as well as interoceptive exposure  that terminated PSVT symptoms. However, panic attacks continued to occur with an even increased frequency of about 1 per day, which corresponded to a HAMA score of 39, an increased ASI score of 40. Again, an elevated mean BSQ score of 2.53 in December 2003 mirrored increased interoceptive sensitivity particularly towards cardiac activity. After cognitive-behavioural psychotherapy (25 sessions) including interoceptive exposure as described above the patient was completely symptom free after 12 months of treatment and also at a long-term follow-up (November 2007: HAMA: 1).The present cases demonstrate that PSVT and PD can co-occur as two different diagnostic entities. Both patients could clearly differentiate between PD and PSVT symptoms: Differential diagnostic clues were that in contrast to panic attacks PSVT attacks were not accompanied by symptoms such as feelings of derealisation/depersonalisation or a fear of losing control, going crazy or dying. Conversely, panic attacks were not terminable by vagal manoeuvres. The possibility of true comorbidity of PSVT and panic disorder thus necessitates reciprocal inclusion of both nosological entities in the diagnostic evaluation of the respective diseases.Given the chronological sequence of onset of the two comorbid diseases in the present cases, two different neuropsychophysiological interactions of PSVT symptoms and specific PD symptoms may be postulated.Firstly, as in patient 1, PD might trigger PSVT as has previously been shown for ventricular premature beats or arrhythmias possibly via chronic mental and somatic stress .Secondly, as in patient 2 and also in patient 1 after ablation, PSVT might initiate or aggravate anxiety states as an internal cue of panic and anxiety. This latter hypothesis is in line with anxiety disorder patients exhibiting increased interoceptive sensitivity, particularly to heart beat ,9, as miAssuming a reciprocal neuropsychophysiological interaction between PSVT and PD via either chronic mental and somatic stress or interoceptive processes, immediate and focused differential treatment such as radiofrequency ablation in PSVT or cognitive behavioural psychotherapy, if necessary in combination with pharmacological treatment  or noradrenaline and selective serotonin agonists (NaSSA)) in PD is warranted in order to possibly avoid the precipitation of the respective other disorder.The authors declare that they have no competing interests.KD contributed to the acquisition and interpretation of data  and conceived and designed the case report. JD initiated the case report after treating the patients in a psychiatric/psychotherapeutic setting. PK performed radiofrequency catheter ablation in both patients, AG was involved in psychotherapeutic interventions and psychometric description; both together with GB and PZ contributed to the manuscript with important intellectual content. All authors contributed to drafting the manuscript and gave approval to the final version.Written informed consent was obtained from the patients for publication of this case report.


The contribution of host-derived growth factors to tumour growth in vivo was studied using the transplantable murine mammary carcinoma, MT1, grown in syngeneic mice. Promotion of growth of the mammary carcinoma by a factor(s) from the host was evident in experiments in which the carcinoma cells were inoculated intraperitoneally. In this environment, tumours develop as multiple solid nodules, each probably arising from an individual cell or a small cluster of cells. Tumour growth was found to occur in the peritoneal cavity following inoculation of 10(3) cells, but an inoculum of as few as ten cells grew if a leucocyte-rich exudate had first been induced. To determine which host-derived growth factors might contribute to growth of MT1, extracts of the tumour were first examined for growth factor activity. Fractionation of tumour extracts by either ion-exchange chromatography or gel filtration revealed several peaks of mitogenic activity, but none of this could be attributed to epidermal growth factor (EGF). Accordingly, an anti-EGF antibody was tested as a putative inhibitor of tumour growth as any effect of this antibody could be ascribed to removal of EGF derived from the host. The antibody was found to have potent anti-tumour activity when tested against MT1 tumours that had been inoculated into the peritoneal cavity. In contrast, the antibody had little effect on growth of the discrete tumour mass which formed when MT1 was transplanted subcutaneously. The results suggest that host-derived EGF contributes to establishment of microcolonies of MT1 carcinoma within the peritoneal cavity. This may be directly, by providing growth factors to supplement those produced by the tumour until it reaches a certain critical mass to sustain autocrine growth, or indirectly, by affecting the production of other growth-stimulatory factors or cytokines.


The pathogenesis of sporadic Parkinson’s disease (PD) remains enigmatic. Mitochondrial complex-I defects are known to occur in the substantia nigra (SN) of PD patients and are also debated in some extracerebral tissues. Early sequencing efforts of the mitochondrial DNA (mtDNA) did not reveal specific mutations, but a long lasting discussion was devoted to the issue of randomly distributed low level point mutations, caused by oxidative stress. However, a potential functional impact remained a matter of speculation, since heteroplasmy  at any base position analyzed, remained far below the relevant functional threshold. A clearly age-dependent increase of the ‘common mtDNA deletion’ had been demonstrated in most brain regions by several authors since 1992. However, heteroplasmy did hardly exceed 1% of total mtDNA. It became necessary to exploit PCR techniques, which were able to detect any deletion in a few microdissected dopaminergic neurons of the SN. In 2006, two groups published biochemically relevant loads of somatic mtDNA deletions in these neurons. They seem to accumulate to relevant levels in the SN dopaminergic neurons of aged individuals in general, but faster in those developing PD. It is reasonable to assume that this accumulation causes mitochondrial dysfunction of the SN, although it cannot be taken as a final proof for an early pathogenetic role of this dysfunction. Recent studies demonstrate a distribution of deletion breakpoints, which does not differ between PD, aging and classical mitochondrial disorders, suggesting a common, but yet unknown mechanism. The central pathological feature is a profound loss of melanin-rich dopaminergic neurons in the substantia nigra (SN) pars compacta, which may exceed one half of the total neuronal population even at the beginning of symptoms. Since these neurons form the nigrostriatal pathway, which delivers dopaminergic input to the striatum, the severe loss of the neurotransmitter dopamine explains the central neurological symptoms of PD. Any disease causing striatal dopamine deficiency or striatal damage may elicit parkinsonism, characterized by tremor at rest, slow volontary movements, rigidity and poor balance, PD being the most common cause of parkinsonism (around 80% of cases). About 95% of PD cases are sporadic and the primary cause of neuronal death in the SN is not known. Only the small remainder is inherited and some monogenic forms can be explained by mutations of a handful of genes involved in protein folding, proteasome function, mitochondrial function and oxidative stress , which severely affect the quality of life. It would thus be an intriguing challenge to uncover the early events of dopaminergic cell death at the molecular level. Although the discussion of PD is mainly focused on the central feature of dopaminergic cell loss in the SN, it should not be forgotten, that the pathology exceeds the nigrostriatal axis by far (for review see ). Neurod2.Besides a basic role of protein misfolding, altered proteasomal degradation and cellular deposition of these proteins in the form of inclusion bodies (Lewy bodies), two other main hypotheses of nigral neurodegeneration focus on mitochondrial dysfunction and oxidative stress , 3, 4. SShortly after the discovery of the parkinsonism inducing properties of MPTP, several reports in the late 1980’s and early 1990’s detected a moderately decreased activity of complex I and sometimes of other ETC complexes in the SN and in extracerebral tissues of human patients suffering from sporadic PD. Schapira and colleagues  analyzedcytoplasmic hybrids) were generated to uncouple potential effects of a damaged mtDNA from confounding effects of the nuclear background. In these cells, mtDNA of a tumor cell line (recipient) has been replaced by that one of a donor cell, derived from either a PD patient or a control. This goal is achieved by culturing the recipient cellline in subtoxic concentrations of an agent, which depletes the cells of their endogenous mtDNA, such as ethidium bromide or rhodamine-6-G. These so called rho-zero cells are then fused by polyethylene glycol treatment to either blood platelets of the donor (no nucleus) or to enucleated fibroblasts. In this way series of cell clones can be generated with identical nuclear background, but differing mtDNA. Using this technique, Swerdlow and colleagues demonstrated moderately decreased complex I activity (-20%), enhanced ROS production and enhanced MPTP sensitivity of neuroblastoma-derived cybrids, containing mtDNA from PD platelets [A further line of evidence implicating mitochondria and actually the mitochondrial genome (mtDNA) in PD pathogenesis, comes from ‘cybrid’ studies. While most polypeptides of all ETC complexes are transcribed from chromosomal genes, 13 of them are encoded in the small circular 16.6 kb mtDNA molecule, located in several copies within the mitochondrial matrix. This genome contains seven subunits of complex I and at least one subunit of all other ETC enzymes (with the exception of complex II) and of the mitochondrial ATP synthetase. Mitochondrial genes exhibit a much higher mutation rate as compared to nuclear genes and mtDNA is not protected by histones and exposed directly to ROS species generated during respiration. It was thus speculated that oxidative damage to mitochondrial DNA and resulting mutations may play a role for mitochondrial dysfunction in PD. So called ‘cybrid cells’ , undetectable by direct sequencing. The mutations were thought to be caused by ROS (reactive oxygen species) generated in the ETC, a process which may be enhanced in dopaminergic neurons of the SN due to additional ROS sources in their metabolism. 3.10, CoQ10). While it indeed protects primary dopaminergic neurons in vitro against MPTP induced cell death [While oxidative stress is commonly accepted to occur in the SN of PD patients, its pathogenetic impact remains uncertain. It may just accompany a late stage of cellular degeneration as an unspecific feature of dying neurons, or it may reflect or actually elicit mitochondrial dysfunction at a relatively early time, when the decision of an individual cell’s fate is being made. In the latter scenario, it would be worth to test pharmacological interventions, capable of restoring ETC function and scavenging ROS. A theoretically promising molecule for this purpose is the flexible electron carrier and antioxidant ubiquinone . Malonaldehyde, a marker of lipid peroxidation, was found to be increased, while the total amount of polyunsaturated fatty acids was decreased . The comOxidative stress markers have been meanwhile described by several studies even in extracerebral tissues and bodily fluids of PD patients , 42-45, Besides the possible pathogenetic impact of oxidative stress in the dopaminergic neurons, a second important question cannot be answered sufficiently at present: the origin(s) of oxidative stress. Is it mainly caused by enhanced ROS production of inhibited ETC complex I or by enhanced ROS generation from other metabolic sources, such as dopamine metabolism? Or may it be caused by a lowered antioxidative defense, allowing higher ROS concentrations even under conditions of unaltered production? Is the primary pathogenic role of complex I in the dopaminergic neurons that one of an important ROS source or of an important ROS target? What comes first: ETC damage or oxidative stress? One early biochemical alteration observed in the SN of PD patients, a loss of reduced glutathione (GSH), may support an important role of weakened antioxidative defense , 49. In 2O2 [If ETC dysfunction and oxidative stress occur also at extracerebral sites in PD, as suggested by several reports, why do mainly dopaminergic neurons die? One important factor to explain selective vulnerability of the dopaminergic cell population, is the relatively high baseline of ROS production due to dopamine (DA) metabolism. Both, enzymatic DA degradation by monoamine oxidase and intracellular autooxidation of DA yield H2O2 , 53, whi2O2 , 55. The2O2 , 57. Rea2O2 . Since ROS generation in the ETC may induce mtDNA mutations, which in turn may inhibit ETC complexes and enhance mitochondrial ROS production, a long debate dealt with a possible ‘vicious cycle’, which may lead to a self-accelerating feedback between oxidative DNA damage and mutagenesis, leading to an accelerated (non-linear) accumulation of mutated mtDNA in aging dopaminergic neurons. Since accumulation of such mutations, widely distributed over the mitochondrial genome, offered an attractive explanation for the age-dependent onset and progressive worsening of PD and some other neurodegenerative diseases, mtDNA came into the focus of research. 4.In the 1990s some sequenced mtDNA mutations, i.e. inherited homoplasmic or heteroplasmic mutations representing a relatively high percentage of total mtDNA, had been suspected to be more frequent in Caucasian PD patients as compared to haplogroup-matched controls (belonging to the same branch of the mtDNA evolutionary tree). However, studies with larger numbers of well characterized or pathologically proven PD cases could not confirm any of these predicted PD associated mtDNA mutations , 60, rejHowever, these results did not exclude a higher burden of widely distributed heteroplasmic mutations in PD tissues, which may reach only extremely low heteroplamy levels at any given base position and thus require highly sensitive detection techniques. Such mutations of low abundance would actually be expected, if somatic mutagenesis was driven by oxidative stress. PCR-cloning of mtDNA with a proofreading polymerase and subsequent sequencing of clones is a technique, which allows to further enhance the sensitivity of mutation detection. It is mainly limited by the endogenous error rate of the chosen proofreading polymerase and by the fussiness of this rather time-consuming method. Due to the latter circumstance, the method usually allows only small sample sizes, and results must be interpreted with caution. A few studies used this technique to analyze various tissues of PD patients and age-matched controls. In their first study, Smigrodski and colleagues compared all seven complex I genes of the mtDNA in six PD patients versus six age-matched controls . The autStimulated by these results, our laboratory analyzed with a similar cloning technique skeletal muscle from five idiopathic PD cases of the akinetic-rigid type, which had suffered from PD between two and thirteen years, and had previously been shown to exhibit a complex I dysfunction in a skeletal muscle sample, a small frozen part of which was now used for DNA preparation . The meaAnother study used the so called ‘double PCR and digestion (DPD)’ method to measure specific base exchanges in mildly ETC deficient skeletal muscle of nine PD cases and nineteen controls at two specific positions of the mtDNA (np 7445 and 8993). Both base substitutions are known to be pathogenic due to their proven role in classical mitochondrial disorders, but both sites are located outside of complex I genes, i.e. in subunits of cytochrome c oxidase and ATP-synthetase. The method was originally used to investigate mtDNA point mutations in aging muscles  and had 5.5.1.Aging is the main risk factor for developing idiopathic PD. Another type of mtDNA mutation of low abundance in the human brain had been clearly associated since the year 1992 with the process of aging: the so called ‘5 kb common deletion’. Using dilution-PCR, Corral-Debrinski and co-workers demonstrated that this large scale deletion accumulates dramatically in the putamen of aged persons, but to a lesser extend also in occipital, frontal and temporal cerebral cortex, while the cerebellum was largely devoid of deleted mtDNA . A simil5.2.Fourteen years after the initial studies described above, a major breakthrough was achieved independently by two groups in the year 2006, who demonstrated the age-dependent accumulation of clonal somatic mtDNA deletions in individual nigral neurons, which reached relevant levels to cause ETC dysfunction and eventually neuronal death.Bender and colleagues  examinedTo allow reliable quantification of ∆mtDNA, the authors took advantage of the fact that nearly all deletions of the mitochondrial genome occur in one half of the circular molecule. This allowed a relative PCR-quantification of two short fragments, representing either the deletion-prone major arc of mtDNA (complex I gene ND4) or the commonly unaffected half of the molecule (ND1). Twenty-five microdissected nigral neurons with normal COX activity from PD patients were compared with age-matched controls. High levels of the mutations were observed, which seemed to be somewhat higher in PD patients  as compared to controls (43.3%), although the difference was not significant (p = 0.06). These mean levels were near to the suggested functional threshold around 60%, which was exceeded in some cells. Moreover, the mean level of deletions was significantly higher in COX-negative cells as compared with COX-positive neurons, underlining the functional impact. The formerly described age-dependency of deletion burden in the brain , 69 was Largely the same results with respect to aging and functional COX decline were obtained in an independent study of Kraytsberg and colleagues, who analyzed brains of individuals between 33 years and 102 years of age with similar methods . The stuTaken together, the reports clearly established a connection between progressive mitochondrial genome deletion during aging and developing ETC dysfunction in the SN, which may participate in neuronal cell loss in this brain region They deliver exiting hints, but do not demonstrate unequivocally that this process of deletion-driven mitochondrial dysfunction is accelerated in PD patients.6.The nature of mitochondrial DNA deletions in nigral neurons still remains unclear. A comparison of breakpoints was performed in laser-microdissected individual neurons of brains from PD patients, aged individuals and one patient suffering from autosomal progressive external ophthalmoplegia (PEO) due to mutations in the polymerase-γ (POLG) gene. Polymerase-γ copies the mitochondrial genome. Mutations of this gene in autosomal PEO cause a fragility of mtDNA, which leads to multiple ∆mtDNA-species e.g. in skeletal muscle of the patients. Moreover, the disease is sometimes associated with parkinsonism. In all groups, most deletions  were flanked by perfect or imperfect short direct repeats, suggesting a common mechanism , irrespeThe elegant mouse model, recently developed by the group of C. Moraes, allowed to generate DSBs in the mouse brains in a timely and spatially controlled manner and to observe accumulation of ∆mtDNA-species and functional defects over time . The autDespite the exciting progress made possible by sophisticated single-cell PCR techniques and mouse models in the last three years, important questions arising from those studies cannot be answered at present. Currently, one of the most important of these unresolved questions is: what may drive a slightly faster accumulation of ∆mtDNA in a minority of individuals, who are then condemned to develop full-blown PD? May a short incidental increase in oxidative stress and DSBs  be sufficient to initiate a fatal process of ∆mtDNA accumulation, leading to ETC dysfunction and finally neuronal death? What makes the difference between PD and normal aging?


After basal and squamous cell skin cancers, prostate cancer is the most frequent cancer in men in the United States, with 186,320 men estimated to be diagnosed with the disease and 28,660 expected to die from it in 2008 Although skeletal metastases from prostate cancer are usually considered osteoblastic by radiologic analysis, histologic and biochemical studies suggest that osteolytic and osteoblastic responses are sequentially linked mmps) in bone metastasis has long been suspected. Because mmps contribute to the process of normal bone remodelling, and because enhanced turnover of bone matrix occurs when tumour cells metastasize to bone, a role can be predicted for these enzymes in metastasis-associated bone modification.A role for matrix metalloproteinases  proteins that have the ability to mediate proteolytic events at both the cell surface and in the immediate pericellular milieu. The mmps consist of an N-terminal signal sequence, a propeptide domain, a catalytic domain, and a C-terminal domain known as the hemopexin-like domain. In the case of the mt-mmps, a C-terminal transmembrane domain or a glycosylphosphatidylinisotol link domain is added.The mmps are synthesized as inactive zymogens and are maintained as such by coordination of a cysteine residue within the propeptide domain, with a Zn2+ ion present in the catalytic domain, which prevents binding and cleavage of the substrate. The dissociation of cysteine from the Zn2+ atom, known as “cysteine switch,” is a complex process, the details of which have emerged only recentlymmps is specifically inhibited by tissue inhibitors of mmps , which usually bind to the active species, inhibiting catalysis. In some mmps, the timps can bind to the zymogens.All timps to the latent form of some mmps was unclear. It is now known that, for example, low concentrations of timp-2 can promote activation of pro-mmp-2 through a mechanism in which timp-2 acts as a linker between the pro-mmp-2 zymogen and mt1-mmp at the cell surface. In this way, the propeptide of the pro-mmp-2 present in this ternary complex formed on the cell surface is cleaved by an adjacent active mt1-mmp. Conversely, high levels of timp-2 relative to mt1-mmp would inhibit activation by blocking all free mt1-mmp molecules.For a long time, the significance of the binding of certain mmps were recognized as proteinases exclusively committed to ecm remodelling during invasive, angiogenic, and metastatic processes. They are now known to have additional roles, such as activation of growth factors, cleavage of cell surface receptors, release of angiogenic factors, release of apoptotic ligands, and generation of angiostatic molecules, to mention just a few. These multiple roles demonstrate the complexity and multiplicity of the substrates of mmps and their ample range of biologic activity in normal and pathologic conditions alike.Initially, mmps are involved in the physiologic turnover of ecm and in bone remodelling, a contribution of mmps to prostate cancer bone metastasis seems to be reasonable. In fact, the participation of mmps in the pathogenesis of osteolytic prostate cancer bone metastasis was confirmed by several experimental studies.Cancer cells that disseminate to bone alter the normal skeletal remodelling process, upsetting the balance between bone formation and bone resorption. Because scid) mice by intravenous inoculation of human PC-3 ML cancer cells. This effect was mainly the result of a complete abrogation of the production and secretion of mmps In vitro, it was found that PC-3 cells produce s when placed on bone surfaces mmp and that MMP2 and MMP9 are among a set of genes altered when prostate cancer cells and bone marrow stromal cells interact mmp-2 mmp-9.15More than 10 years ago, it was shown that the combined administration of alendronate (a bisphosphonate compound) and of paclitaxel inhibits bone metastases produced in severe combined immunodeficient  inhibited the proliferation of prostate tumour cells growing within the human bone implants. That inhibition was accompanied by reduced degradation of bone marrow trabeculae and decreased osteoclast recruitment Using the mmp-9 activity shortly after establishment of PC-3 cells in human bone xenografts—an increase that coincided with a wave of osteoclast recruitment and vigorous bone degradation mmp-9 that occurs during prostate cancer–bone interaction is species-specific, because active mmp-9 was found when human prostate cancer cells grew within human bone tissue, but not when they grew within mouse bones Recently, we showed an upregulation of net ecm by using proteases. Although mmp-9 does not degrade type i collagen, the most abundant organic component in bone, we found that the protease is expressed mainly by osteoclasts. That finding suggests that active mmp-9 induced by prostate cancer–bone interaction most likely contributes to osteolysis by indirect mechanisms. For instance, mmp-9 may cause the release of ecm-bound vascular endothelial growth factor into a soluble and bioactive form Tumour-associated osteoclasts are known initially to dissolve the mineralized bone matrix by acid secretion and then to disrupt the exposed non-mineralized mmp inhibitor with high selectivity for mmp-9 scid mice bearing prostate cancer bone tumours resulted in significant inhibition of angiogenesis and intraosseous tumour growth, together with reduction in osteolysis mmp-9 to neovascularization of expanding bone metastases and to subsequent bone degradation. Moreover, pro-mmp-9 has been shown to play a crucial role in osteoclast recruitment MMP9 mmp-9 not only in normal but in pathologic processes occurring in the skeleton.The foregoing hypothesis was confirmed by experimental therapy with SB-3CT, a novel mechanism-based mmp knockout mice also present severe skeletal abnormalities, mostly as a result of their incapacity to degrade crosslinked fibrillar type i collagen prevalent in the bone matrixmt1-mmp expression correlates with a more aggressive behaviour and has been shown to promote invasion and metastasis mt1- mmp mt1-mmp expression observed in all cases of prostate cancer bone metastases analyzed mt1-mmp–expressing prostate cancer cells may have more tendency to metastasize to skeleton. Alternatively, the bone microenvironment might induce the expression of mt1-mmp in prostate cancer cells after their arrival in the marrow.Membrane-tethered 1 mt1-mmp to bone metastasis. We overexpressed mt1-mmp in LNCaP human prostate cancer cells (which have baseline non-detectable expression of the protease), while its expression was attenuated in DU145 cells (which have baseline high mt1-mmp expression) using small interfering rna. We showed that intratibial injection of those cells resulted in completely opposite phenotypes in terms of intraosseous tumour growth and bone response. Compared with controls, tibiae injected with LNCaP cells overexpressing mt1-mmp showed increased osteolysis (as demonstrated by radiography and histomorphometry) and enhanced intraosseous tumour growth. In contrast, mt1-mmp downregulation in high-expressing mt1-mmp DU145 prostate cancer cells led to diminished intraosseous tumour growth and a mixed bone reaction, in which osteosclerotic responses predominated mt1-mmp upregulation in cancer cells resulted in the release of one or more factors that promoted osteoclast differentiation in vitro. That effect was abrogated by pharmacologic inhibition of mt1-mmp activity and by osteoprotegerin, a soluble decoy receptor of the osteoclastogenic receptor activator of nuclear factor κB ligand (rankl). Our results strongly suggest the possibility that prostate cancer–associated mt1-mmp promotes an osteolytic response by shedding membrane-bound rankl (mrankl) from the cancer cell surface. Recently, mmp-7, produced mainly by osteoclasts at the prostate tumour–bone interface, has also been reported as a rankl “sheddase,” promoting osteolysis mmps in prostate cancer bone metastasis may be therapeutically beneficial.We conducted a series of studies to assess the contribution of prostate cancer cell–derived mmp inhibitors in animal tumour models has been mitigated by a lack of therapeutic efficacy and undesired side effects observed in clinical trials with cancer patients Much of the initial excitement associated with the use of broad-spectrum testing of patients with advanced high-volume disease refractory to other treatments;mmps with unspecific and reversible effects;use of broad-spectrum inhibitors of unknown repertoire of proteases expressed by the patients’ tumours (protease “degradome” mmps with important physiologic roles (anti-targets), probably resulting in neutralization of the effects of the inhibitors on actual mmp “targets” that truly contribute to disease unintended inhibition of mmp inhibition during treatment; andlack of studies to monitor mmp inhibitors and diminished therapeutic index because of forced dose reduction to tolerable levels.unknown effective doses of mmp inhibition were peptidomimetic hydroxamate compounds with potent inhibitory effects, but no selectivity . The second-generation mmp inhibitors exhibited some marginal selectivity . Because those compounds failed in clinical trials, a third generation of selective mmp inhibitors is now being developed and considered for cancer therapy mmp target is involved with minimal adverse reactions. These selective inhibitors are hoped to have a ratio of at least 1000 between the iK (the dissociation constant for binding of inhibitor) values for mmp anti-targets and those for mmp targets The first drugs developed for mmps at low nanomolar or picomolar levels in vitro, despite the “factor of 1000” selectivity, might not prove selective in vivo because a low micromolar or high nanomolar level of activity against the anti-target mmps will still foster in vivo consequences. The challenge is not necessarily an issue of affinity for the target, but rather of the mechanism for discrimination other than mere recognition events between the drug and the target. In that vein, the mechanism-based gelatinase inhibitor SB-3CT and its new structural variants stand out. This inhibitor class takes advantage of the active site of the enzyme to undergo a specific chemical transformation facilitated by the target enzyme itself. Whether a given mmp might be able to perform this reaction, or whether it might not, a process that leads to potent enzyme inhibition is at the root of its ability to serve as a selective mmp inhibitor to the given target. The concepts pertinent to inhibition of mmp have been discussed in a recent review For example, potent linear competitive inhibition of target mmp inhibitors on bone metastasis. We believe that the knowledge obtained in recent years using animal models has provided validated mmp targets that, together with the development of third-generation mmp inhibitors, would justify use of those inhibitors in the treatment of prostate cancer patients with skeletal metastasis. For instance, prostate cancer patients with locally advanced disease who have a high probability of developing bone metastasis and no current prospect of curative treatment could benefit from therapy with novel mmp inhibitors.In the particular case of prostate cancer patients, no clinical trials have been performed to evaluate the therapeutic efficacy of mmp being targeted be confirmed by appropriate methods during treatment. Alternatively, combination therapy using inhibitors for mmp targets and agents that can block osteoclastic action (such as bisphosphonates or anti-rankl drugs) could reasonably be employed in clinical trials involving prostate cancer patients with potential to develop bone metastases. The experimental evidence described herein showing a key role for mmps in bone metastasis suggests that targeting those mmps could have therapeutic value. New approaches must be explored, especially given that current approaches for treating bone metastasis in prostate cancer patients are still limited and only palliative.For successful treatment, it is crucial that the protease degradome for tumours in each patient be assessed and that inhibition of the


To the Editor: Since the first identifications of avian influenza (H5N1) in Europe in late 2005 and early 2006, Eurobarometer survey data obtained during April–May 2006 have provided a unique opportunity to examine the knowledge of respondents across the European Union, Croatia, and Turkey about the risks and transmission of avian influenza. The H5N1 strain of avian influenza virus has caused >240 human deaths in central and Southeast Asia, the Middle East, and Africa  were found in October 2005 in Turkey; additional cases were found later that month in Romania, Croatia, and the United Kingdom. Therefore, the period would have included media coverage about avian influenza as well as any targeted public health efforts to inform residents about avian influenza risks. By the end of this survey’s fieldwork period, 17 of the 29 countries surveyed had reported influenza virus (H5N1) in birds, 3 in mammals, and 1 in humans (Respondents were asked 7 questions about their knowledge of the risks humans face regarding avian influenza . When weThese results support previous findings that knowledge about avian influenza, especially about prevention and human-to-human transmission, has scope for improvement (The state of knowledge about avian influenza in Europe during the outbreak in the spring of 2006 leaves room for further public health information campaign efforts, especially those that increase consumers’ understanding of consumption-related avian influenza risks. Persons in Europe appear to be aware of culling procedures and the risks of touching infected birds but have a more limited understanding of how avian influenza in their region should influence their consumption patterns.


This report gives the 5-year findings of a double-blind study of long-term cytotoxic chemotherapy as an adjuvant to surgery in patients receiving busulphan or cyclophosphamide for carcinoma of the bronchus compared with a group receiving a placebo. Of 243 patients initially allocated busulphan, 234 cyclophosphamide and 249 placebo, 28%, 27% and 34% respectively were alive at 5 years. There were significant associations between mortality from bronchial carcinoma and histological involvement of the resected intrathoracic nodes and the histology of the tumour. Haematological toxicity, especially thrombocytopenia, was frequent and severe in the busulphan series, and low platelet counts continued long after chemotherapy was stopped.


Plasmodium Berghei Anka (PbA)infected. In non infected mice, incidence of apoptosis was lower in the lymph nodes of CD18-/- and uPAR -/- than in +/+ mice, as seen by FACS analysis to count the number of hypodiploidand Annexin-V binding cells. Infection of mice with PbA resulted in a marked increasein the size of spleen and lymph nodes 7–8 days after infection, which was slightly higher inuPAR -/- and CD18 -/- than in +/+ mice. PbA infection increased about 7 fold the incidence ofapoptosis in the lymphoid organs of +/+, especially in the white pulp and germinal centers ofthe spleen and lymph nodes, while in contrast it was unchanged in PbA infected CD18 -/- oruPAR -/- mice. Serum IgG levels, and number of circulating leukocytes were significantlyhigher in both uPAR and CD18 -/- than in +/+ mice. These results indicate that the CD18 anduPAR surface molecules, which are known to be associated in the cell membrane, have animportant influence upon the incidence of cell survival in both normal or stimulated lymphoidorgans.Incidence of apoptosis was investigated in the spleen and lymph nodes of +/+, CD18 -/- andurokinase receptor  -/- mice, untreated or