We used the nation-wide Swedish Family-Cancer Database to examine the risk for testicular cancer in offspring through parental and sibling probands. Among 0–68-year-old offspring, 4082 patients had testicular cancer in years 1961–2000, among whom 68 (1.67%) had an affected father/brother. Standardized incidence ratios (SIRs) for familial risk were four-fold when a father and nine-fold when a brother had testicular cancer. Histology-specific risks (for the testicular cancer) were similar for sons of affected fathers, but were higher among brothers for teratoma and seminoma than for mixed histologies. Standardized incidence ratios for either histology depended on the age difference between the brothers: 10.81 when the age difference was less than 5 years compared to 6.69 for a larger age difference. Parental colorectal, pancreatic, lung and breast cancer and non-Hodgkin's lymphoma and Hodgkin's disease were associated with seminoma among sons. Seminoma risk was also increased when a sibling had melanoma. Teratoma was associated with parental lung cancer and melanoma. The high familial risk may be the product of shared childhood environment and heritable causes. Familial cases of fraternal pairs with an early-onset teratoma represent a challenge for gene identification. The dathttp://snomed.org) was used, referred to here as ‘SNOMED’.The registered site of cancer is as a four-digit diagnostic code based on the 7th revision of the International Classification of Diseases (ICD-7). The following ICD-7 codes were grouped: ‘upper aerodigestive tract’ cancer codes 161 (larynx) and 140–148 , except for code 142 , ‘lymphoma’ codes 200, 202 (non-Hodgkin lymphoma), 201 (Hodgkin's disease) and ‘leukemia’ codes 204–207 (leukemias), 208 (polycytemia vera) and 209 (myelofibrosis). Rectal cancer, ICD-7 code 154, was subdivided into the anus  and mucosal rectum (154.0). Basal cell carcinoma of the skin is not registered in the Cancer Registry. Up to 1992, the histology of testicular cancers as in the Cancer Registry (WHO/HS/CANC/24.1 Histology Code) was used, to define seminoma (pathology codes 066) and teratoma . From 1993, ICD-O-2/ICD with histopathological data according to the Systematized Nomenclature of Medicine  were used to measure cancer risks for sons  according to the occurrence of cancers in their families. When more than two affected sons were found in any family, they were counted as independent events. Standardized incidence ratios were calculated for sons whose parents or brothers had the same, concordant cancer, that is, using parents or brothers as probands. Follow-up started for each offspring at birth, immigration or January 1, 1961, whichever came latest, and terminated on diagnosis of the first cancer, death, emigration, or the closing date of the study, December 31, 2000.Parents’ ages were not limited but sons were 0–68 years of age. All tumour incidence rates were based on the data in the Family-Cancer Database, and they were essentially similar to rates in the Swedish Cancer Registry. Rates were standardized to the European population. Standardized incidence ratios were calculated as the ratio of observed (O) to expected (E) number of cases. The expected numbers were calculated from 5-year-age-, sex-, tumour type-, period- (5-year bands), socioeconomic status- (six groups) and residential area- (three groups) specific standard incidence rates for all sons lacking a family history (The kappa statistic was used as the measure of agreement between histologies: (observed number of cases−expected number of cases)/(1−expected number of cases) . The kapvs 27 in offspring and 39–33 in fathers). According to the SNOMED histology, covering years 1993–2000, embryonal carcinoma accounted for 14.8% in sons and 10.9% in fathers. The age-specific incidence rates of testicular cancer among sons are shown in Figure 1The Family-Cancer Database, which covered years 1961–2000 from the Swedish Cancer Registry, included 4082 testicular cancers in sons of ages 0–68 years and 3878 fathers with testicular cancer . Among brothers, both SNOMED types showed peak SIRs at 30–44 years . No familial cases were found for embryonal carcinoma.We analysed using SNOMED histopathology, available only from 1993 (data not shown), that teratoma showed an age peak at 15–29 years in sons of fathers with testicular cancer . The overall value was 0.01 between sons and fathers, and 0.26 among brothers; among brothers, the kappa value of seminoma was 0.23 and that of teratoma was 0.31.The Swedish Family-Cancer Database contains national family data linked to the Swedish Cancer Registry. The inability to link some 10% of deceased offspring diagnosed with cancer to their parents (see Subjects and Methods) may cause small errors in the familial risks of fatal cancers. However, testicular cancer has had a good prognosis during the past decades. The missing links predominantly influence those born in the 1930s and who died before 1991, and we have not observed a difference in familial risks in comparing different diagnostic periods . We concFamilial occurrence of testicular cancer is well recognized but rare. The proportion of sons with testicular cancer who had an affected father or brother was 1.67% in the present study, consistent with previous studies, reporting affected first-degree relatives in 1.0–2.8% of cases . Even thThe higher familial risk for testicular cancer among brothers than father–son pairs may suggest the involvement of a recessive mode of inheritance or an X-linked susceptibility locus in the aetiology of testicular cancer, consistent with the segregation analysis and the gene-mapping findings . IdentifAge-specific familial risks of In families of seminoma patients, associations were found with colorectal, pancreatic, lung and breast cancer and non-Hodgkin's lymphoma and Hodgkin's disease among parents. Among brothers, there was an association with seminoma and melanoma. Teratoma was associated with parental lung cancer and melanoma. However, no association has been found for primary melanoma or lung cancer following first testicular cancer (In summary, the present study may offer some explanation to the inability in finding susceptibility genes for testicular cancer. The high familial risk may be the product of shared childhood environment and heritable causes and so may be difficult to untangle. Identifying any relevant environmental factors will be challenging but may explain some of the changes in testicular cancer incidence. For gene identification, fraternal pairs with teratoma below age 25 may be particularly useful.


Aging reviews research papers published in 2009, which they                        believe have or will have a significant impact on aging research.  Among many                        others, the topics include genes that accelerate aging or in contrast promote                        longevity in model organisms, DNA damage responses and telomeres, molecular                        mechanisms of life span extension by calorie restriction and pharmacologic                         interventions into aging.  The emerging message in 2009 is that aging is not                        random but determined by a genetically-regulated longevity network and can be                        decelerated both genetically and pharmacologically.                                    The editorial board of  Aging, the rate of telomere shortening in peripheral                            leukocytes predicts mortality from cardiovascular disease in elderly men .                                        The                            most striking event of the year was the demonstration that rapamycin,                            administrated to middle-aged (600 day old) mice, significantly extended their                            life span . The effIt was shown that clioquinol, a metal                            chelator that has beneficial effects in several models of neuro-degenerative                            diseases, inhibits the activity of the mitochondrial enzyme CLK-1 in mammalian                            cells. Clioquinol-treated nematodes and mice presented a variety of phenotypes                            produced by mutational reduction of CLK-1. Given that reduction of CLK-1 slows                            down aging in these organisms, these results suggest that clioquinol (by                            inhibiting CLK-1) may slow down the aging process .       Finally,                            as a follow-up of the work on the anti-aging effects of mitochondria-targeted                            antioxidant SkQ1 , it was In                            2009, several lines of evidence suggested that overactivation of signaling                            pathways might cause exhaustion of stem cells and that vice versa ‘longevity                            genes' could prevent stem cell exhaustion.  Thus, mTOR mediated Wnt-induced epidermal                            stem cell exhaustion and aging phenotypes in skin .  FurtheAging, once-monthly infusions                            of bone marrow (BM)-derived cells from young adult female mice sustained the                            fertility of aging females long past their time of normal reproductive failure                            [In                            addition, functions of aging organs can be rejuvenated by young supporting stem                            cells.  As published in the first issue of          .  The fe         ,49. In INK4a tumor suppressor proteins were shown to be important in                            limiting reprogramming [INK4a appeared                            the predominant barrier in human cells [Much                            interest has also been devoted in the past year to nuclear reprogramming of                            differentiated cells into induced pluripotent stem (iPS) cells by using defined                            factors. Understanding which factors facilitate the reprogramming process is                            thought to give clues to the process of carcinogenesis. Inversely, nuclear reprogramming                            could be also envisioned as a "rejuvenation process". In this regard, p53 and                            p16gramming -55.  Actan cells .       INK4a tumors suppressor gene, whose                            expression is increased markedly with aging in several human and murine tissues                            [CDKN2a/b locus  with human atherosclerotic                            disease [CDKN2a/b transcripts is decreased in                            individuals harboring the risk alleles, suggesting that atherosclerotic disease                            may result from aberrant, unrestrained proliferation. In this regard, studies                            on mice overexposing the CDKN2a/b locus were found to have delayed aging and                            extended longevity [Of                            particular importance to the field of regenerative medicine, which will need                            patient-specific stem cells derived from older patients, is reprogramming                            efficiency in fibroblasts from aged humans versus young humans.  There is an                            age-associated decline in reprogramming efficiency, which is largely reversed                            by inactivation of the p16         ,55.  Alo         . The sam disease .  Expresongevity .       Drosophila                            [Drosophila [In 2009, numerous publications extended                            our knowledge on the role of sirtuins , TOR sig         .  Moreovosophila .  Finallosophila .       C                                    elegans and Drosophila the aging of the soma is influenced by the                            germline: namely, when germline-stem cells are removed, aging slows and                            lifespan is increased. In 2009, it was published that a predicted transcription                            elongation factor, TCER-1, plays a key role in this process [In                            2009, Kenyon and co-workers further uncovered mechanisms of their previous                            observations made in 1999  that in  process . When th process . In mice process .       A                            prior  work by Willcox et al  showed that genetic                            variation within the FOXO3A gene was strongly associated with human longevity.                            Long-lived men also presented several additional phenotypes linked to healthy                            aging, including lower prevalence of cancer and cardiovascular disease, and                            high physical and cognitive function. Long-lived men also exhibited greater                            insulin sensitivity associated with homozygosity for the FOXO3A GG genotype. In                            2009, confirming the Willcox observation, the flurry of papers showed the                            association between SNPs in the FoxO3A gene and extreme longevity in Japanese,                            German, American, Italian, and Chinese populations -71.    Drosophila                                    melanogaster, p53 exerted developmental stage-specific and sex-specific                            effects on adult life span, indicative of sexual antagonistic pleiotropy [ There                            were intriguing publications on the complex role of p53 in longevity.  In eiotropy ,73.  Fureiotropy .       Interestingly,                            SNPs in the p21 gene correlated with longevity in an Italian population .       WRN gene defective in the premature aging disorder                            Werner syndrome encodes a protein with both helicase and exonuclease activities                            [sgs1-related phenotypes.  WRN was shown to genetically                            interact with topoisomerase 3 and restore the slow growth phenotype of sgs1                                    top3.  WRN helicase but not exonuclease activity was genetically required                            for restoration of top3 growth phenotype, demonstrating separation of                            function of WRN catalytic activities.  In a top3 mutant background, DNA                            unwinding by WRN helicase may be deleterious to cell growth and genome                            homeostasis [Model                            systems continue to be instrumental in understanding the genetics of                            longevity.  The          .  To diseostasis .       INK4a tumor suppressor causes an age-dependent decline                            in beta-cell replication.  In 2009, it was reported that loss of Polycomb (PcG)                            repression of p16INK4a mediated by the EZH2 histone methytransferase occurred                            with aging in humans and mice [INK4a and Arf.                                        In 2009, a few studies delved into the                            genetics of the insulin-producing pancreatic beta-cell aging in humans and mice                            -83.  A land mice .  In micINK4a expression with aging by the Bmi-1 PcG                            protein, which functions in concert with EZH2 to repress p16INK4a                            expression [NK4a                            with aging in beta-cells, suggesting a possible pharmacologic approach to                            regulating aging of this tissue [This                            work linked alterations of chromatin architecture with aging to expression of                            anti-proliferative molecules.  Bhushan and colleagues also reported a similar                            regulation of p16pression . Lastly,s tissue .       Drosophila                            [C. elegans [In                            2009, the simple dogma that autophagy is always associated with or causes                            senescence was challenged. Although autophagy remains a crucial anti-aging                            mechanism, the relationship is likely to be complex.  Thus, autophagy was shown                            to be activated during cellular senescence, and activation correlated with                            negative feedback in the PI3K-mTOR pathway. A subset of autophagy-related genes                            was up-regulated during senescence: overexpression of one gene, ULK3, induced autophagy                            and senescence.  Furthermore, inhibition of autophagy delayed the senescence                            phenotype, including senescence-associated secretion.  These data suggest that                            autophagy, and its consequent protein turnover, may mediate acquisition of the                            senescence phenotype . Inhibit          or C. el elegans  was foun elegans -90, sugg elegans .  In 200 elegans -94. Furt elegans .       C. elegans). Spermidine was found to act as a potent inducer                            of autophagy in all species tested, including yeast, Drosophila, C. elegans                            [Drosophila and C.                                    elegans [The                            natural polyanion spermidine can extend the chronological and replicative life                            span in yeast and increase the median and maximal longevity of fruit flies and                            nematodes ,                            culture in nutrient-free media (in human cells) and caloric restriction (in                            nematodes). In C. elegans, it was found that activation of Sirtuin-1                            extended longevity in an autophagy-dependent fashion. Thus, the knockdown of                            the essential autophagy gene Beclin1/ATG6 abolished life span extension by                            Sirtuin-1 activation [Sirtuin-1                            and that of its tivation . These rtivation .       In                            fact, 2009 saw an escalation in interest in microRNAs and other non-coding RNAs                            implicated in aging and replicative senescence.  A prominent example of this                            regulation came studies of the mitogen-activated protein kinase (MAPK)                            signaling component MKK4 (MAPK kinase kinase 4).  MKK4 levels were elevated in                            aging tissues and in senescent cells thanks to reductions in the abundance of                            four microRNAs  that interacted with the                            5'- and 3'-untranslated regions of the MKK4 mRNA and repressed its translation                            .       The                            other major class of post-transcriptional regulatory factors, RNA-binding                            proteins (RBPs), were also the focus of important age-related studies in 2009.                             Several RBPs that affect the turnover and translation of proteins implicated in                            proliferation, survival, inflammation, neurodegeneration, and cancer  displayed elevated abundance in a broad array of human                            tissues and in all ages, suggesting that their influence extends throughout the                            human life span .  The RBperiod extended the health                            span of aging in Drosophila melanogaster [There is growing evidence for a link                            between circadian rhythm,  signal-transduction genes, metabolism, cancer and                            aging ,104.  Thnogaster .  Furthenogaster .       nogaster . In ratsnogaster . In micenogaster .       CR                            is known to slow aging and delay cancer.  In 2009, it was reported that fasting                            abrogates side effects caused by chemotherapy in cancer patients.  Importantly,                            for those patients in whom cancer progression could be assessed, fasting did                            not prevent chemotherapy-induced reduction of tumor volume or tumor markers                            .  The liReduced                            incidence and delayed occurrence of fatal neoplastic diseases in growth hormone                            receptor/binding protein knockout mice.  These changes of fatal neoplasms are                            similar to the effects observed with calorie restriction and therefore could                            possibly be a major contributing factor to the extended life span observed in                            the GHR/BP KO mice.  Overall,We                        apologize to the authors whose important publications were not discussed due to                        space limitations or were simply overlooked. Here we referenced only papers published                        in the 2009 calendar year. That was not an easy task given that most of                        publications are the continuation of or based on previous research. We expect                        to make this a tradition to publish ‘the year overview' every year. The next                        year, the task will be easier, given that "Aging in 2010" will be the                        continuation of "Aging in 2009".                                Aging at papers@impactaging.com.                                Please                        e-mail your reprints to us in December 2010 (editors@impactaging.com) or, even                        better, please submit your best manuscripts for publication in


Chlamydia replicate within an intracellular vacuole, termed an inclusion. A large family of bacterial proteins is inserted in the inclusion membrane, and the role of these inclusion proteins is mostly unknown. Here we identify SNARE-like motifs in the inclusion protein IncA, which are conserved among most Chlamydia species. We show that IncA can bind directly to several host SNARE proteins. A subset of SNAREs is specifically recruited to the immediate vicinity of the inclusion membrane, and their accumulation is reduced around inclusions that lack IncA, demonstrating that IncA plays a predominant role in SNARE recruitment. However, interaction with the SNARE machinery is probably not restricted to IncA as at least another inclusion protein shows similarities with SNARE motifs and can interact with SNAREs. We modelled IncA's association with host SNAREs. The analysis of intermolecular contacts showed that the IncA SNARE-like motif can make specific interactions with host SNARE motifs similar to those found in a bona fide SNARE complex. Moreover, point mutations in the central layer of IncA SNARE-like motifs resulted in the loss of binding to host SNAREs. Altogether, our data demonstrate for the first time mimicry of the SNARE motif by a bacterium.Many intracellular pathogens rely on host cell membrane compartments for their survival. The strategies they have developed to subvert intracellular trafficking are often unknown, and SNARE proteins, which are essential for membrane fusion, are possible targets. The obligate intracellular bacteria  Chlamydia trachomatis, is the leading cause of preventable blindness and of bacterial sexually transmitted diseases worldwide. Chlamydiae multiply inside a membrane-bound compartment, the inclusion. The exchanges between the membrane of the inclusion and other intracellular membranes are tightly controlled by the bacteria, for example avoiding fusion with some degradation compartments, while acquiring lipids. Inclusion proteins, made by the bacteria and secreted into the inclusion membrane, are thought to play a central role in controlling these interactions, although their exact function is mostly unknown. We have identified, in three inclusion proteins, a motif common to proteins that are essential for the fusion of two compartments in eukaryotic cells, the SNARE proteins. Via this motif, inclusion proteins interact specifically with a subset of SNAREs of the host, which leads to the selective recruitment of intracellular compartments around the inclusion. This study thus provides a striking example of mimicry of the host by an intracellular pathogen.Chlamydiae are obligate intracellular bacteria that have co-evolved with eukaryotic cells and adapted to a wide range of hosts, causing several diseases in humans and animals. For example, one species pathogenic to humans,  Chlamydia are obligate intracellular bacterial pathogens of eukaryotic cells. They infect a variety of animals, including humans, and cause acute and chronic diseases Chlamydia replicate primarily within epithelial cells, in a membrane-bound compartment called the inclusion. The membrane of the inclusion is of dual origin, reflecting its position at the interface between host and pathogen. The bacteria use a type III secretion process to translocate into it the Inc proteins, a large family of Chlamydia specific proteins of mostly unknown function Chlamydiae control their interactions with the host intracellular traffic, allowing some fusion events while avoiding others. In eukaryotic cells, SNARE  attachment protein receptors) proteins play an essential role in compartment fusion Legionnella effector proteins Chlamydia inclusion protein might interact with itself to form a complex similar to that of the SNARE complex, and thus facilitate the homotypic fusion of inclusions Chlamydia proteins, we used a bioinformatic approach. SNARE motifs are made of heptad repeat sequences that form coil-coiled structures. Position a and d of the heptads are occupied by hydrophobic residues, except at the centre of the motif, where a very conserved glutamine or arginine residue occupies position d, defining the zero layer. Many coiled-coil regions meet these criteria, hampering the identification of SNARE-like motifs from genomic data. However, in this case, we reasoned that the access to several different Chlamydia genome sequences should allow us to identify SNARE-like motifs in Chlamydia proteins with a high level of confidence. We restricted the search to proteins containing a large bilobed hydrophobic motif, which is the hallmark of Inc proteins Chlamydia genome encodes more than 40 putative Inc proteins. Among them, we found that the C. trachomatis genome encodes 11 proteins predicted to engage in coiled-coil interactions  and C. caviae (CcaIncA) shared biochemical properties C. caviae IncA (Δ75IncA) was not enriched around the inclusion and had no effect on the recruitment of Vamp3 and Vamp7 to the inclusion . Interaction of CT813 with GFP-Vamp3 could not be assessed because the expression levels of both constructs were low . This re strains . C. caviImportantly, targeting the host SNARE machinery is not the sole method for Inc proteins to interfere with host trafficking: members of the family of rab proteins, which also participate in recognition and fusion of cell compartments C. trachomatis IncA classifies as a Q-SNARE, and aligned better with Q-SNAREs than R-SNAREs  InR-SNAREs . We modeR-SNAREs . The modR-SNAREs . In partR-SNAREs , inset. Our models, and numerous reports on eukaryotic SNARE complexes, suggest that, if the IncA SNARE-like motif functions as a bona fide SNARE motif, forming complexes with R-SNAREs, introduction of a large charged amino acid, such as an arginine, in the zero layer of the IncA SNARE-like motif might destabilize the SNARE complex sufficiently to lose the interaction between IncA and host SNAREs. To test this hypothesis, we introduced point mutations in the zero layer of IncA SNARE-like motif Nter (IncAT126R), motif Cter (IncAQ244R) or both (IncAT126RQ244R). All constructs were expressed at a level comparable to wild type IncA when transfected in HeLa cells. However, coexpression of the Q244R mutant together with GFP-Vamps constructs resulted in very weak expression of both transfected genes. The reasons for this phenomenon are unclear, and we could not assay the ability of this mutant to co-immunoprecipitate with GFP-SNAREs. We performed co-immunoprecipitation experiments in cells co-expressing different GFP-SNAREs and either IncA wild type, or mutated in motif Nter, or in both SNARE-like motifs. The single mutant (IncAT126R) co-immunoprecipitated with GFP-Vamp3, GFP-Vamp7 and GFP-Vamp8, to the same extent as IncA wild-type. However, IncA mutated in both SNARE-like motifs (IncAT126RQ244R) did not co-immunoprecipitate with any of the Vamps . This exTo confirm this important result by a different approach, we investigated the effect of the point mutations in IncA SNARE-like motif on the ability of IncA to engage in homotypic interactions. We have previously shown that heterologous expression of IncA, which localizes at the endoplasmic reticulum, inhibits inclusion development. This effect requires a direct interaction between IncA molecules at the inclusion and on the endoplasmic reticulum, and we hypothesized that it might be due to the formation of homotypic SNARE complexes between IncA molecules present on the two compartments Altogether, point mutations in IncA SNARE-like motifs impaired its ability to associate with host SNAREs and with itself. These results show that IncA SNARE-like motifs behave as bona fide SNARE motifs, and strongly support our hypothesis that IncA interaction with host SNAREs is mediated by the formation of SNARE complexes.Chlamydia recruit a specific subset of host SNAREs. We have identified SNARE-like motifs in the inclusion protein IncA, and showed that a mutant strain that does not express IncA presents reduced ability to recruit SNAREs around its inclusions. Our data also suggest that other inclusion proteins may use SNARE motif mimicry to interact with SNAREs. These conclusions open two important questions: in which SNARE complexes is IncA engaged, and what are the consequences in term of membrane fusion?In agreement with previous reports from several laboratories, we have observed that a variety of cellular compartments accumulate around the inclusion. Here we bring evidence that SNARE mimicry is one mechanism by which the in vivo, interactions between the inclusion membrane and the host SNAREs involve a combination of SNARE-like motifs from several different inclusion proteins and/or of SNARE motifs from several different host SNAREs present in the same compartment. Dissecting the complexes in which IncA is involved therefore remains a challenging task for the future.To start answering the first question, we have tested a variety of IncA/SNARE interactions. By co-imunoprecipitation experiments, we found that IncA was able to interact with several different SNAREs, but not all. We observed a good correlation between this result and the specific recruitment of a subset of SNAREs around the inclusion membrane. The basis of the specificity of interaction between IncA and a subset of host SNAREs is not yet known. A model between the SNARE motif of Sec22 and a trimer of IncA motif Cter resulted in an unstable complex (data not shown), a finding that correlates with the absence of recruitment of Sec22 around the inclusion membrane. However, modelling predicted a stable association of Vamp4 with IncA motif Cter, while we observed no or very weak interaction between IncA and Vamp4 . This reChlamydia genome. The three SNAREs recruited to C. trachomatis inclusion membrane are SNAREs involved in endosomal trafficking, while Vamp4 and Sec22, which are more involved in the secretory pathway, were not, suggesting that SNARE mimicry may have preferentially evolved in C. trachomatis to target the endosomal pathway. Depletion of host SNAREs Vamp3, Vamp4, Vamp7 or Vamp8 using siRNA had no impact on the growth of the bacteria  was from ATCC. C. trachomatis serotype D(s)5058 is a clinical isolate, which does not express IncA, and was kindly given by Drs. D. Rockey and W. Stamm C. caviae and the CWL029 strain from C. pneumoniae were obtained from Drs. R. Rank (University of Arkansas) and G. Christiansen  respectively. HeLa cells were used in all experiments except for infection with C. pneumoniae, which was performed in Hep2 cells. Infections were performed as described E. coli. Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden , rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli , rabbit anti-Sec22 was described previously HeLa cells were transfected by electroporation with the indicated constructs, and lysed 24 h later on ice in 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 10 mM EDTA, pH 7.5 and cocktails of inhibitors. Immunoprecipitation of GFP-tagged proteins was performed using anti-GFP monoclonal antibodies . Immunoprecipitated proteins were incubated in sample buffer, boiled and loaded on acrylamide gel for western blot detection of IncA or of the histidine tag . Expression level and immunoprecipitation of SNAREs proteins were checked by stripping the membrane and incubating it with anti-GFP antibodies (#sc-8334 Santa Cruz Biotechnology) followed by horseradish peroxidase-linked (HRP) anti-rabbit antibodies (Amersham Biosciences).E. coli for 20 hrs at 16°C, and subsequently purified in buffer A  using the protocol described in GST-Vamp3, GST-Vamp4, and GST-Vamp8 were expressed, purified and reconstituted into a first population of clear liposomes as described Transfected and infected HeLa cells were fixed with a mixture of 2% (wt/vol) paraformaldehyde and 0.5% (wt/vol) glutaraldehyde in a 0.2 M phosphate buffer (PB) pH 7.4, 4 h at room temperature. After many washing with 0.1 M glycin in PBS, cells were processed for ultracryomicrotomy as described Construction of CtrIncA and CcaIncA with a carboxyterminal His tag was described elsewhere AAGAUUUGGACCUAGAAAUGA) was designed by Dr. Andrew A. Peden . 5×106 cells were electroporated in the presence of 2 pmoles of siRNA at day 1 and again at day 4, plated in 24-well plates and one 10-cm dish at day 5 and infected at day 6 in the 24-well plates. On the day of infection, cell lysates were prepared from the 10 cm dish and normalized to equal protein concentration. The level of expression of each SNARE was assessed by western blot using specific antibodies. Nitrocellulose membrane were incubated with ECL western blotting reagents (Amersham) and processed for detection using a Luminescent Image Analyzer LAS-3000 (Fujifilm). Digital images were acquired using the software Image Reader LAS-3000 v2.2 (Fujifilm) and analyzed by MultiGauge v3.0 (Fujifilm). All the quantification were performed using the expression of β-tubulin to normalize the samples, and for each SNARE, the level of expression is expressed as the percentage of expression in control cells transfected with the si-β-globin. One day after infection, the cells were detached from 24-well plates in 0.5 mM EDTA in PBS, fixed for one hour in 70% ethanol, centrifuged and washed in PBS. Bacteria were then stained using anti-EfTu antibody from Dr Y-X Zhang (Boston) followed by Cy5-coupled anti-mouse antibody (Amersham) and the samples were analyzed by flow cytometry.The selection of specific oligonucleotides and the procedures for RNAi experiments to silence the expression of endosomal v-SNAREs  will be published elsewhere , Vamp4 siRNA Click here for additional data file.Figure S2IncA is involved in the recruitment of Vamp3 and Vamp7 to the inclusion.(8.90 MB TIF)Click here for additional data file.Figure S3C. caviae IncA inhibits the recruitment of Vamp3 and Vamp7 to C. trachomatis inclusions.Expression of (9.99 MB TIF)Click here for additional data file.Figure S4C. caviae and C. pneumoniae inclusions.Recruitment of GFP-SNAREs around (1.91 MB TIF)Click here for additional data file.Figure S5Modelling.(6.23 MB TIF)Click here for additional data file.Figure S6C. trachomatis.Depletion of Vamp3, Vamp4, Vamp7 or Vamp8 does not affect the development of (2.63 MB TIF)Click here for additional data file.


Stanleya pinnata and Astragalus bisulcatus against two cell disrupting herbivores, the western flower thrips  and the two-spotted spider mite (Tetranychus urticae).Hyperaccumulation, the rare capacity of certain plant species to accumulate toxic trace elements to levels several orders of magnitude higher than other species growing on the same site, is thought to be an elemental defense mechanism against herbivores and pathogens. Previous research has shown that selenium (Se) hyperaccumulation protects plants from a variety of herbivores and pathogens. Selenium hyperaccumulating plants sequester Se in discrete locations in the leaf periphery, making them potentially more susceptible to some herbivore feeding modes than others. In this study we investigate the protective function of Se in the Se hyperaccumulators Astragalus bisulcatus and S. pinnata with high Se concentrations (greater than 650 mg Se kg-1) were less subject to thrips herbivory than plants with low Se levels (less than 150 mg Se kg-1). Furthermore, in plants containing elevated Se levels, leaves with higher concentrations of Se suffered less herbivory than leaves with less Se. Spider mites also preferred to feed on low-Se A. bisulcatus and S. pinnata plants rather than high-Se plants. Spider mite populations on A. bisulcatus decreased after plants were given a higher concentration of Se. Interestingly, spider mites could colonize A. bisulcatus plants containing up to 200 mg Se kg-1 dry weight, concentrations which are toxic to many other herbivores. Selenium distribution and speciation studies using micro-focused X-ray fluorescence (μXRF) mapping and Se K-edge X-ray absorption spectroscopy revealed that the spider mites accumulated primarily methylselenocysteine, the relatively non-toxic form of Se that is also the predominant form of Se in hyperaccumulators.This is the first reported study investigating the protective effect of hyperaccumulated Se against cell-disrupting herbivores. The finding that Se protected the two hyperaccumulator species from both cell disruptors lends further support to the elemental defense hypothesis and increases the number of herbivores and feeding modes against which Se has shown a protective effect. Because western flower thrips and two-spotted spider mites are widespread and economically important herbivores, the results from this study also have potential applications in agriculture or horticulture, and implications for the management of Se-rich crops. For many organisms, including mammals and many species of bacteria and algae, selenium (Se) is an essential trace element . These o-1 dry weight (DW) .,22.-1 DWs Figure . This fo-1 [-1 DW [These results have important implications for managing Se-rich agricultural or natural areas and Se phytoremediation or biofortification crops. Crops in seleniferous habitats and plants used for Se phytoremediation often do not accumulate more than 150 mg Se kg-1 . While t1 [-1 DW , likely -1 may allow transfer of Se into higher trophic levels, which is an area that needs to be further investigated.Herbivores with different feeding modes may respond differently to hyperaccumulation in plants, as was suggested by Jhee et al. . BecauseCFQ, JLF, RJBR and EAHPS conceived and coordinated the experiments in this study. SCF performed μXRF and μXAS data analyses. All authors assisted with the experiments. CFQ, JLF and EAHPS drafted the manuscript. All authors read and approved the final manuscript.This research was supported by grants #IOB-0444471 and #IOS-0817748 from the National Science Foundation to EAHPS. We thank David Steingraeber, Arathi Seshadri and Mark Paschke for helpful comments on the manuscript. We would also like to thank 2 anonymous reviewers for helpful comments that improved the manuscript. The Advanced Light Source is supported by the Office of Science, Basic Energy Sciences, Division of Materials Science of the U.S. Department of Energy (DE-AC02-05CH11231).


Pretreatment 28% of all patients were classified as ‘high-anxiety’; their average anxiety scores decreased significantly post-treatment, that is towards less anxiety. At all assessments, high-anxiety men treated by prostatectomy reported less depression than high-anxiety men treated by radiotherapy. Of men treated by radiotherapy, 27% reported clinical significant levels of depression while 20% is expected in a general population. The improvement in mental health at 6-months follow-up was statistically significant and clinically meaningful in all respondent groups. Sensitivity of anxiety at baseline as a screening tool was 71% for anxiety and 60% for symptoms of depression. We recommend clinicians to attempt early detection of patients at risk of high levels of anxiety and depression after prostate cancer diagnosis since prevalence is high. STAI-State can be a useful screening tool but needs further development.To document anxiety and depression from pretreatment till 5-year follow-up in 299 men with localized prostate cancer. To assess, if baseline scores were predictive for anxiety and depression at 1-year follow-up. Respondents completed four assessments  on anxiety, depression and mental health. Respondents were subdivided according to therapy (prostatectomy or radiotherapy) and high  Prostate cancer is highly prevalent in most Western countries  referral to watchful waiting or advanced disease therapy; (2) noncompletion of the STAI-State scores (see below), since this score was crucial in assessing the respondents' anxiety level at baseline. The prospective study consisted of four measurements: 1 month before initiation of primary therapy, and at 6 months, 12 months and 5-year follow-up. The nonrandomly allocated treatment consisted of  radical prostatectomy or external beam radiotherapy (comprising an average of 33 radiation sessions over 7 weeks). Further details on the study design and the inclusion of respondents were published previously , intermediate or high (college/university degree). To assess the prevalence of comorbidity, we used a standardized list of 28 chronic conditions, such as heart failure, asthma, diabetes and asked respondents to report which condition they currently experienced or had experienced during the previous year .−1 after prostatectomy, confirmed at least once, or as a rise in PSA-level of at least 0.5 ng ml−1 after radiotherapy, confirmed at least once.Baseline clinical information on tumour stage , histopaThe questionnaires contained three validated self-report psychological measures.Anxiety was assessed by the State Trait Anxiety Inventory (STAI-State). This scale contains 20 items on, for instance, feeling at ease or upset . Scale sThe Center for Epidemiologic Studies Depression Scale (CES-D) was used to assess the frequency and severity of symptoms of depression. We applied the 20-item version with items relating to, for instance, feeling depressed or fearful, being happy, and enjoying life. Scores range from 0 to 60 with higher scores indicating higher levels of symptoms of depression . A scoreThe Mental Health scale of the RAND 36-item Short-Form Health Survey (SF36-MH) was used as a general measure of mental health. The scale consists of five items on being nervous, down, peaceful, depressed and happy. Item scores are transformed to ranges of 0–100 with higher scores indicating better mental health . Between-group and within-group differences in background characteristics and descriptive statistics were calculated using SPSS for Windows, release 10.0.7. The χ2 test was used for categorical variables, the t-test or Mann–Whitney U for continuous variables. P-values <0.05  were considered significant.Respondents were subdivided into four groups, defined by therapy  and by level of anxiety  using proc mixed from the SAS system for Windows release 8.2. Random intercept models were applied that allowed for the use of all available data, including incomplete records. These models comprised the main effects of ‘anxiety’ and ‘time’ and the interaction between anxiety and time. Time was included as a factor with four levels – one for each assessment - to account for possible nonlinearities in the course of scale scores. Comorbidity and PSA-level were included in the models as covariates.To compare the course of anxiety and symptoms of depression in subgroups stratified by baseline anxiety scores, we calculated mean scores at 6 and 12-months follow-up for respondents with low (⩽44) and high (>44) anxiety scores at baseline. To evaluate the diagnostic performance of baseline anxiety screening, we calculated the sensitivity, specificity, positive (PPV) and negative predictive value (NPV) using data of all patients who completed the first three assessments. This complete case analysis is a valid strategy, because missing values at 1-year follow-up were not related to levels of anxiety or symptoms of depression at baseline nor to age.x-axes in our graphs – analogous to earlier reported disease-specific and generic quality of life scores of the same cohort (Average age differed considerably between treatment groups (±6 years). Age-adjustment was not appropriate, because the relation between age and the physical functions in particular was found to be nonlinear . Therefore, the average age per treatment group is presented on the n=25) or advanced disease therapy (n=48), or had not completed the STAI-State at the first assessment (n=15) were excluded. The final cohort consisted of 299 primary prostate cancer patients treated with radical prostatectomy (n=118) or external radiotherapy (n=181). The response rate to all four questionnaires was 78% . Median time to long-term follow-up was 52 months, and mean time 51 months (range: 44–56 months).Between June 1996 and January 1999, 415 men met the inclusion criteria, of whom 387 consented to participate and completed the first questionnaire (93%). Men who were referred to watchful waiting (n=29) and 30% of the radiotherapy group (n=55) were classified as high-anxiety. Characteristics did not differ significantly between high-anxiety and low-anxiety individuals within the two treatment groups, except that in the radiotherapy group the percentage of singles was higher among high-anxiety men compared to low-anxiety men .Information on clinical or biochemical recurrence at the 5-year assessment was available for 94% of the 5-year respondents; prostate cancer had recurred in 5% (5/91) of the prostatectomy patients and in 21% (26/123) of the radiotherapy patients.Scores of men treated by surgery were more favourable than those treated by radiotherapy for all three psychological measures and at all four assessments, that is, less anxiety and feelings of depression and better mental health.P<0.0001): although high-anxiety men improved substantially at 6-months follow-up, they still reported more anxiety than low-anxiety men , that is, 25% of the surgery group and 30% of the radiotherapy group. Average STAI-State scores in high-anxiety men of 52 (surgery group) and 54 (radiotherapy group) decreased significantly after treatment, that is, less anxiety, and remained at the lower level through follow-up . At all iety men .Compared to pretreatment, all groups except the high-anxiety men treated by radiotherapy reported significantly lower CES-D scores at 6-months follow-up, that is, less symptoms of depression . At all lower percentages of prostatectomy men reported clinically significant levels of symptoms of depression than the general population (9–18% vs 20%), pretreatment and at 5-year assessments in the radiotherapy group these percentages were higher than 20% (27 and 22%).At all assessments Compared to pretreatment, the improvement in post-treatment SF36-MH was not only statistically significant, but also clinically meaningful . RepeateWe performed a prospective, longitudinal study on mental health in 299 prostate cancer patients, using validated instruments. At 1 month before treatment roughly one in every four patients was classified as ‘high-anxiety’. After 6 months, after initiation of treatment, men reported significantly less anxiety and feelings of depression and a significantly better mental health. Average scale scores remained at the improved levels through follow-up.Before interpreting the results from a clinical perspective, two methodological issues have to be discussed. First, there is no unequivocal cutoff value for the STAI to define high-anxiety. We used the earlier reported cutoff value of 45 and higher  predict anxiety and depression  and STAI to identify the most suitable instrument for screening a population at in-hospital intensive rehabilitation on anxiety and depression. The sensitivity of the STAI (52%) was less than that of the HADS (72%), but its specificity (99%) was greater than that of HADS (84%). The authors concluded that both instruments can be recommended for psychological screening of patients in an in-hospital intensive rehabilitation (In our study, using the STAI-State baseline score as a screening tool resulted in the early detection of 71, respectively, 60% of patients who were to experience high levels of symptoms of anxiety, respectively, feelings of depression at 6 or 12-months follow-up. The sensitivity might be improved by expanding the tool with, for instance, disease characteristics as the Gleason score, or by applying other measures, for instance the HADS .Treating clinicians may not always realize that, in spite of a comparably favourable prognosis, so many patients experience high levels of anxiety and symptoms of depression after a diagnosis of localized prostate cancer. We recommend clinicians to attempt early detection of patients at risk of such high levels and provide them with psychological support. STAI-State can be a useful screening tool but needs further development.


PTPN22 genetic susceptibility variants predict response to therapy in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor (TNF) agents.To determine whether rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) antibodies, or carriage of shared epitope (SE) and PTPN22 R620W genotyping was performed by Sequenom MassArray. Linear regression analyses were performed to investigate the role of these four factors in predicting response to treatment by 6 months, defined as the absolute change in 28-joint Disease Activity Score (DAS28).UK-wide multicentre collaborations were established to recruit a large cohort of patients treated with anti-TNF drugs for RA. Serum RF, anti-CCP antibody and SE status were determined using commercially available kits. PTPN22 620W carriage.Of the 642 patients analysed, 46% received infliximab, 43% etanercept and 11% adalimumab. In all, 89% and 82% of patients were RF and anti-CCP positive, respectively. Patients that were RF negative had a 0.48 (95% CI 0.08 to 0.87) greater mean improvement in DAS28 compared to patients that were RF positive. A better response was also seen among patients that were anti-CCP negative. No association was demonstrated between drug response and SE or PTPN22.The presence of RF or anti-CCP antibodies was associated with a reduced response to anti-TNF drugs. However, these antibodies only account for a small proportion of the variance in treatment response. It is likely that genetic factors will contribute to treatment response, but these do not include the well established RA susceptibility loci, SE and  Several small studies (sample sizes <130) have investigated the utility of autoantibodies, including rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP), for predicting response to treatment with biological agents but results have been inconsistent.7UK-wide multicentre collaborations were established to recruit patients treated with anti-TNF drugs for RA. Eligible patients from each centre were subsequently identified from the British Society of Rheumatology’s (BSR) Biologics Register (BR).Eligible patients from each collaborating centre were invited to take part in the study. Additional blood samples were obtained from consenting patients when they required a blood test as part of routine care. The additional blood samples and signed consent forms were posted to the Arthritis Research Campaign (arc) Epidemiology Unit for processing and storage. For the majority of patients, two samples of blood were taken: one for serum and one for DNA extraction. DNA was isolated using a standard phenol/chloroform extraction method. Serum and DNA samples were stored at −80°C. UK Central Office of Research Ethics Committees (COREC) approval (04/Q1403/37) was obtained for the study.Clinical and demographic data held on the BSRBR database was extracted, with the consultants’ permission, and compiled for each consenting patient. Disease activity was measured using the 28-joint count disease activity score (DAS28).19PTPN22 R620W (1858C/T) genotyping was performed using mass spectrometry  as recommended by the manufacturer.Serum RF and anti-CCP antibody titre were measured using commercially available kits ). Patients with titres ⩾40 U/μl and ⩾5 U/μl were defined as positive for RF and anti-CCP antibodies, respectively. HLA-DRB1 typing was performed using commercially available kits ). The SE was defined as the presence of any of the following alleles: human leukocyte antigen (HLA)-DRB1*0101, *0102, *0104, *0401, *0404, *0405, *0408 or *1001. In addition, PTPN22*620W carriage. For the purposes of this analysis, the recorded 6-month DAS28 score was used whether patients had discontinued therapy or not. Analyses were adjusted for baseline DAS28, baseline HAQ score, administration of concurrent DMARDs and gender, as these factors have previously been shown to be significant independent predictors of response in the BSRBR cohort as a whole and were also associated in the current cohort .http://hydra.usc.edu/gxe).21The primary outcome measure was absolute change in DAS28 between baseline and 6 months. Linear regression analyses were performed to investigate association between change in DAS28 and RF, anti-CCP status, SE and Collaborations were established with 20 rheumatology centres across the UK from which 1485 patients receiving anti-TNF therapy for RA satisfied the study inclusion criteria. Of these, 1292 responded to the invitation letter (87%) with 1195 patients willing to take part (80%). DNA samples were extracted and available for the first set of 642 patients to be recruited, which were utilised in the current analysis.Baseline characteristics for the group of 642 patients are presented in 22PTPN22 R620W (C1858T) polymorphism and SE was successfully performed in 96% and 83% of patients, respectively . These findings were expected as associations to these factors have been previously reported in the BSRBR data, from which the current cohort was recruited.Linear regression analyses were first performed to investigate association between drug response at 6 months, defined as the absolute change in DAS28, and the baseline factors listed in PTPN22*620W status  . Furthermore, there was no interaction between these two factors and their association with drug response . However, as the majority of patients were positive for both antibodies, these analyses may be underpowered.The effects of RF and anti-CCP antibodies were investigated further by performing multivariate linear regression combining both antibodies, together with previously known predictors . Being positive for RF and anti-CCP did not better predict response to anti-TNF therapy . As the study aims to identify predictors of response by 6 months rather than predictors of response only in those who remain on treatment, these patients were included in the analysis although we recognise that this may have resulted in underestimations of observed effects.et al, patients with more severe disease as a result of irreversible joint damage may be less likely to respond to treatment.Thirdly, as one of the requirements for prescribing anti-TNF agents in the UK includes failure of at least two previous DMARDs, the patients recruited have severe, long-standing RA with a mean duration of 14 years. As discussed by Hyrich Lastly, the serology was measured cross-sectionally at the time of sample collection, which may have been some time after commencement of treatment. Previous studies have shown that, although titres are affected by treatment, status generally is not.Conversely, our study has several advantages over previous investigations. Importantly, the use of the BSRBR to identify suitable patients has meant that the subgroup studied is comparable to the BSRBR in its entirety. As, until relatively recently, almost all patients receiving an anti-TNF drug in the UK for RA were included on this register, the cohort studied is likely to reflect the characteristics of patients treated with anti-TNF as a whole, at least in the UK. Furthermore, a wealth of clinical and demographic data had already been collected, creating a well characterised cohort. In addition, this is a large cohort, allowing robust inferences to be drawn. Finally, the use of the DAS28 measure rather than the EULAR response criteria enhances the power of the study to detect association with genetic predictors of response in patients with RA treated with anti-TNF.et al, RF and anti-CCP antibodies are independent markers of disease severity for RA.As no correction for multiple testing was applied in the current analyses, these results will require validation in similar-sized cohorts. Nonetheless, the findings support those of smaller studies in which similar trends between drug response and baseline RF and anti-CCP antibody titres have been demonstrated.–PTPN22 gene and, in keeping with most previous studies, no association of treatment response was observed with SE carriage, a well established RA severity and susceptibility locus. Several novel RA susceptibility loci have recently been reported , which may also warrant investigation.25–It is salient to note that combining information on clinical markers of anti-TNF treatment response previously identified, 2PTPN22.In summary, the presence of RF or anti-CCP antibodies was associated with a reduced response to anti-TNF drugs in patients with RA treated with anti-TNF. However, the presence of these antibodies only accounts for a small proportion of the variance in treatment response. It is likely that genetic factors will contribute to determining the response to treatment with these agents but do not include the well established RA susceptibility loci, SE or


A 54-year-old man was admitted to the hospital presenting with a 3-month history of sclerosing dermal lesion in the external genitalia. A scrotal skin biopsy revealed a poorly-differentiated adenocarcinoma, immunohistochemically positive for cytokeratin 7 (CK7) and for thyroid transcription factor 1 (TTF-1), and negative for CK20. One month after admission, he died of respiratory failure. At autopsy, a consolidating lesion with vague margin was noted in the left lung as well as a well-circumscribed nodule in the right lobe of the thyroid. Histopathologically, pulmonary lesion was adenocarcinoma with a micropapillary component. On the other hand, thyroid tumor was diagnosed as a follicular variant of papillary carcinoma with foci of micropapillary adenocarcinoma. Positive immunohistochemistry for surfactant protein on micoropapillary component was useful to confirm that micropapillary component was of lung adenocarcinoma origin. Carcinomas with micropapillary components have been reported as rare but poorly prognostic tumors in various organs including the breast, urinary bladder, lung, ovary, and major salivary glands -4. Not oA 54-year-old Japanese man was admitted to our hospital complaining of a 3-month history of a sclerosing dermal lesion on the external genitalia Fig. . ComputeGrossly, massively spreading lesion (approximately 3 × 3 × 3 cm) with vague margin was noted in the right lung accompanying lymphangitic carcinomatosis, as well as a well-circumscribed nodule (8 mm in diameter) in the right lobe of the thyroid. We confirmed aggressive metastatsis at various loci including liver, bilateral adrenals, peritoneum, cardiac muscle of the right atrium, both thoracic (Th5) and lumbar (L5) vertebra, and lymph nodes within trunk except for head and neck. Microscopic examination revealed that the lung tumor was composed of adenocarcinoma presenting characteristic morphological structure of small tumor clusters with no fibrovascular cores surrounded by clear spaces Fig. , so-callImmunohistochemical analysis of tissue samples taken at autopsy confirmed that tumor cells in the lung MPCs, thyroid papillary carcinoma, and thyroid MPC-like clusters were all positive for CK7 but negative for CK20. TTF-1 staining demonstrated weakly positive in lung MPC and in thyroid papillary carcinoma follicular variant, whereas thyroid MPC-like clusters were negative. Furthermore, thyroglobulin was positive in thyroid papillary carcinoma with follicular variant, while negative in the MPC-like clusters in both thyroid and lung. On the other hand, lung adenocarcinoma and metastatic MPCs, including MPC in thyroid tumor Fig. , were exCarcinomas with MPC were rare but aggressive tumors with poor prognosis. According to the review article, these tumors have predilections for mammary gland, urinary bladder, lung and salivary gland . Also coIn this case, because immunohistochemical analysis for TTF-1 and CK7/CK20 suggested that primary focus can either be thyroid cancer or lung adenocarcinoma, we used surfactant protein as a selective marker for the lung cancer. With positive immunohistochemistry for the surfactant protein and systemic analysis by autopsy, we concluded that this case is a primary lung adenocarcinoma with MPC with cancer-to-cancer metastasis. Lack of obvious head and neck lymph node metastasis  in spite of massive lymph nodes metastases seen in thoracoabdominal cavity also supported that MPC is derived from the lung.In summary, we reported an autopsy case of lung cancer with MPC metastasizing to the thyroid papillary carcinoma. Wide spreading systemic metastasis clearly illustrates the aggressive character of cancers with this particular histopathological feature.The authors declare that they have no competing interests.KM performed the autopsy and histological examination, and was a major contributor in writing the manuscript. RK assisted the histological examination. TK assisted immunohistochemical evaluations. SK supported case analysis and writing the manuscript. All authors read and approved the final manuscript."Written informed consent was obtained from the patient' family for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Multiple sclerosis (MS) is considered an autoimmune disease of the central nervous system and therapeutic inhibition of leukocyte migration with natalizumab, an anti-alpha4 integrin antibody, is highly effective in patients with MS. Recent studies performed in experimental animal models with relevance to human disease suggested a key role for blood-brain barrier damage and leukocyte trafficking mechanisms also in the pathogenesis of epilepsy. In addition, vascular alterations and increased leukocyte accumulation into the brain were recently documented in patients with refractory epilepsy independently on the disease etiology.Here we describe the clinical course of a 24-year-old patient with MS in whom abrupt tonic-clonic generalized seizures manifested at disease onset. Although MS had a more favorable course, treatment with glatiramer acetate and antiepileptic drugs for 7 years had no control on seizure generation and the patient developed severe refractory epilepsy. Interestingly, generalized seizures preceded new MS relapses suggesting that seizure activity may contribute to MS worsening creating a positive feedback loop between the two disease conditions. Notably, treatment with natalizumab for 12 months improved MS condition and led to a dramatic reduction of seizures.Our case report suggests that inhibition of leukocyte adhesion may represent a new potential therapeutic approach in epilepsy and complement the traditional therapy with anti-epileptic drugs. Multiple sclerosis (MS) is considered a T cell-mediated autoimmune disease of the central nervous system (CNS) with a complex genetic background . It is aSeizures can occur in MS patients and the risk of epilepsy seems to be three-times higher in patients with MS than in the general population . SeizureRecent evidence suggests that inflammation mechanisms play a role in the pathogenesis of epilepsy -12. MoreCurrent anti-inflammatory and immunosuppressive MS-treatments include β-interferons, glatiramer acetate (GA) and different chemotherapies. Recently, natalizumab, a monoclonal antibody directed against the α4 chain of integrin VLA-4, an adhesion molecule controlling leukocyte adhesion to brain endothelium, was approved by the U.S. Food and Drug Administration and the European Medicines Agency as monotherapy for highly active relapsing-remitting MS. Despite the occurrence of progressive multifocal leukoencephalopathy (PML) as adverse reaction, natalizumab represents the most potent drug approved thus far for the treatment of relapsing-remitting MS ,14.Here we describe a dramatic reduction of seizures after treatment with natalizumab in a patient with severe refractory epilepsy and MS. For better clarity we split the description of seizures and epilepsy from the non-epileptic MS course.status epilepticus) and frequency of seizures, type and dose of antiepileptic drugs (AEDs) are also reported. In the time interval 1999-2000 the patient suffered from 1 partial and 1 generalised seizure (those at disease onset) as described below in the text. Abbreviations and symbols are the followings: n.d. = not done; * = mild decrease in mentation; ** = marked decrease in learning and memory; EDSS = Kurtzke's expanded disability status scale; VPA = valproate; CBZ = carbamazepine; LMT = lamotrigine; TOP = topiramate; OXC = oxcarbazepine; KEP = levetiracetam; GBP = gabapentin; § = LMT discontinued after 1 month for allergic dermatitis. Each non-epileptic MS relapse has been treated with methyl-prednisolone 1 gr/daily for 3-5 days without tapering-off.The 10-years follow-up, from MS onset (October 1999) to January 2010 is summarised on Table In October 1999, a previously healthy 24-year-old man complained of the sudden appearance of a brainstem syndrome (multiple oculomotor nerves involvement and gait instability) followed, 24 hours later, by an abrupt tonic-clonic generalized seizure. Routine laboratory exams were normal. Family and personal history were all negative for neuro-psychiatric, hereditary and autoimmune diseases. MRI showed multiple supra-and infra-tentorial T2-w hyper-intense and two Gd-enhanced T1-w lesions. Cerebrospinal fluid (CSF) composition was normal in protein level and cell count. Post-critical EEG was also normal.Differential diagnosis was made with conditions causing seizures and mimicking MS either of inflammatory , infectious , genetic , metabolic , and neoplastic origin (CNS lymphoma). Anti-voltage-gated potassium channel and onconeural antibodies for limbic encephalitis were also tested. All investigations performed to seek for signs of these diseases resulted negative. A subsequent spinal MRI performed a week after disease onset showed cervical T2-w hyper-intense lesions with no Gd-enhancement. Six CSF oligoclonal bands were found, which allowed a diagnosis of laboratory-supported definite MS according to the Poser criteria in use at that time . A coursThe first MS relapse occurred during 2002 and was characterized by a mild cerebellar ataxia and dysmetria Table . RombergThe second MS relapse occurred in May 2004 and was characterized by diplopia. Neurological examination disclosed a mild right internuclear ophthalmoparesis and MRI revealed the absence of new T2-w or Gd-enhancing lesions Table . A new cThe third non-epileptic MS relapse occurred three years later (April 2007) and was characterized by urinary urgency. MRI was not performed. At examination, a left Babinski sign was evident together with nystagmus and dysmetria. EDSS score was 3 Table . A new cIn October 2008 new clinical (right hemiparestesia) and subclinical MRI activities emerged, showing new T2-w lesions in both brain and spinal cord and Gd-enhancing T1-w brain lesions Figure . PatientNatalizumab was administered at a dose of 300 mg every 4 weeks from February 2009 till January 2010 . The cognitive status improved and the EDSS score decreased to 2.5 in January 2010 Table .In October 1999, 24 hours after the sudden appearance of the demyelinating brainstem syndrome the patient presented a generalized tonic-clonic epileptic seizure with tongue laceration and urinary incontinence. Post-critical EEG (24 hours after seizure) was normal. No specific treatment for seizures was initiated. Although EDSS score was zero and brain MRI was unchanged, on December 2001 the patient presented a second generalized tonic-clonic epileptic seizure, which was preceded by a simple partial seizure of focal motor activity on the left body part Table . Post-icStarting from March 2002, 3 months before the first MS relapse and initiation of GA treatment, the patient complained of recurrent stereotyped disorders consisting of different types of partial (simple and complex) seizures, lasting less than 3 minutes and sometimes overlapping: i) focal motor activity of the left part of the body, ii) loss of contact with surroundings followed by drowsiness, iii) gestural automatisms. Often, these episodes were followed by secondarily generalized tonic-clonic seizures Figure , Table 1status epilepticus requiring intervention in intensive care department and treatment with intravenous lorazepam, diazepam and/or phenobarbital.Notably, although the non-epileptic MS course was well controlled , the epileptic condition worsened during the period 2005-2007 Figure , Table 1Starting from 2006, seizures were treated with other AEDs  at increasing daily dosage and often in association, as indicated on Table status epilepticus. A month later, an intercritical EEG showed partial epileptic discharges  the patient manifested only 4 short (less than 2 minutes) complex partial seizures (60% reduction compared to 2008). Importantly, during natalizumab treatment we observed no generalized seizures (100% reduction) and the absence of status epilepticus. Intercritical EEG performed in June 2009 was normal Figure . OxcarbaEpilepsy affects between 0.5-1% of world population and, despite medical therapy, about one third of patients develop refractory epilepsy -18. AmonExperimental and clinical studies have shown that inflammation mechanisms are activated in epilepsy . ProinflIn addition to the inflammation mechanisms described above, recent studies performed in experimental animal models with relevance to human disease show a role for vascular inflammatory mechanisms and leukocyte-endothelial adhesion in the induction of BBB leakage and seizure generation ,11. BBB Acute seizure activity induces expression of adhesion molecules on brain endothelium ,30. Impoper se induces vascular inflammation [In support to the results obtained from animal models of epilepsy, it has been shown that BBB disruption in patients with cerebral lymphoma induces focal motor seizures . Vasculaammation , seizureThe generalization of the remarkable effect of natalizumab on refractory epilepsy observed in the present clinical case to other more common types of epilepsy requires further studies. In contrast to the anti-adhesion therapy with natalizumab, a specific and sustained anti-adhesive activity exerted by GA and steroids in the present clinical case seems rather unlikely. In fact, GA treatment causes in vivo changes of the cytokine secretion pattern and effector function of GA-specific T cells but increases the migration rate of Th2 cells and do not affect the migration of Th1 cells ,33, wherCurrent pharmacological treatments for epilepsy do not address the inflammatory component of pathogenesis highlighted by recent studies and most AED drugs aim to depress aberrant neuronal excitation. However, noncompliant and refractory epilepsy cases demand investigation into alternative mechanisms and corresponding treatments, and interfering with the adhesion of immune cells to the cerebral vasculature may potentially open new avenues for epilepsy treatment .Our results indicate treatment with natalizumab as highly effective in patients with MS and epilepsy. As no interactions with AED drugs were described for natalizumab and recent clinical data show that safety may be increased for PML associated with natalizumab therapy , our resTaking into account our current understanding of the pathogenesis of seizures and epilepsy and the emerging key role of inflammation mechanisms in epilepsy, our data suggest a more general application of natalizumab and anti-adhesion therapy in other types of epilepsy.Only public funds from the University of Sassari and the University of Verona were used for the present paper. No conflicts of interest exist with the companies whose products are mentioned and/or discussed in this article. GC is co-author in a patent owned by Stanford University USA and entitled "Anti-leukocyte recruitment therapy for the treatment of seizures and epilepsy" .All authors fulfill the authorship criteria because of their substantial contributions to the conception, design, analysis and interpretation of the data. SS and GC wrote the manuscript, MRM made the clinical follow-up of the patient. All authors gave their final approval of the version to be published.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/10/84/prepub