The spectrum of presentation of intracranial hypotension is clinically perplexing. We report a case of 31-year-old post-partum woman who underwent an uneventful caesarean section under spinal anesthesia. From the second day of surgery she developed postural headache, the headache lost its postural character after few days. She then developed seizures and ataxic hemiparesis. Magnetic resonance imaging showed features of severe intracranial hypotension in the brain and the spinal cord, and magnetic resonance venography showed cortical vein and partial superior sagittal sinus thrombosis. Prothrombotic  work-up showed Protein C and S deficiency. She responded to anticoagulation therapy and recovered completely. On review of literature two distinct groups could be identified obstetric and non-obstetric. The non-obstetric group included patients who underwent diagnostic lumbar puncture, intrathecal injection of medications and epidural anesthesia for non-obstetric surgeries. Poor outcome and mortality was noted in non-obstetric group, while obstetric group had an excellent recovery. Post-lumbar puncture intracranial hypotension syndrome (PL-IHS) with persistent headache and neurological deficits is rare. PL-IHS is observed in multiple settings post-diagnostic lumbar puncture2, post-spinal anesthesia/analgesia for abdominal and lower-limb surgeries3, post-epidural anesthesia/analgesia during labor for pain and caesarean section4, and post-intrathecal injection for chemotherapy and post-myelography5. Usually it has a benign course, with most patients making complete recovery with or without an epidural blood patch (EBP). Rarely complications may develop in the form of subdural hygroma6 and neurological deficits due to brainstem compression7. We present a case with PL headache in a post-partum female progressing to cerebral venous thrombosis (CVT). We reviewed the literature to identify predictors of outcome.Post-lumbar puncture (PL) headache is common, occurring in 10–30% of patients due to persistent cerebrospinal fluid (CSF) leakage after a lumbar puncture performed for anesthesia or diagnostic purposesth post-partum day she had a generalized tonic clonic seizure and had persistent drowsiness. She was taken to local hospital for seizure; there she received lorazepam 4mg slow IV bolus followed by phenytoin 900mg IV infusion over 60 minutes. After stabilization of vitals she was transferred to our comprehensive stroke care center. Prior to this episode she had been detected to have PIH in two earlier pregnancies and the first pregnancy had resulted in intrauterine death. During the present pregnancy she received dalteparin 5000 IU/day subcutaneous injection from 6 weeks pregnancy from local maternity centre with suspicion of anti-phospholipid syndrome; it was continued until the 8th post-partum day. There was no past history of abortions or deep vein thrombosis.A 31-yr-old female with pregnancy-induced hypertension (PIH) underwent caesarean section (CS) at 35 weeks of pregnancy . Spinal anesthesia was used for the procedure. On the second day post-partum, she noticed diffuse occipital headache and neck pain when she tried to get off the bed, which improved on lying down. This postural headache was persistent over next 8 days following which it changed character and became continuous. A diagnosis of PL headache was made and she was discharged on Ibuprofen 400mg thrice daily. On 12On examination in the emergency room after 12 hours of seizure she was conscious and oriented. She had no papilledema and her visual acuity and visual field were normal. She had weakness and appendicular ataxia of the left upper limb and mild dysarthria. Her National Institute of Health Stroke Scale on admission was 3.She was investigated with a CT scan of the head  after 8 Unfractionated heparin was started (16 hours after time of onset) at 800 units/hour to maintain plasma thromboplastin time between 75–90 seconds along with bed rest and caffeine immediately after the MRI venography. She responded early and was discharged after 6 days on warfarin 4mg once daily and optimized international normalized ratio (INR) of 2.1. On discharge she did not have any headache and neurological deficit had subsided, with NIHSS of 0 and modified Rankin Scale (mRS) of 0. On follow-up after 3 months, she had no further recurrences or new symptoms.et al.3; of their five PL-IHS patients, three had a predisposing prothrombotic condition.We describe a case of PL-IHS as suggested by the typical postural headache of the patient and the change of pattern of headache, which could have been the time of transition when cortical venous thrombosis developed. The brain and spine features seen on MR imaging are also supportive of this diagnosis, showing classical features of intracranial hypotension and cortical venous thrombosis. Our patient had multiple predisposing conditions, such as the early post-partum period, a history of previous intrauterine death and reduced activity of Protein C and Protein S. These predisposing conditions increased the likelihood of occurrence of thrombosis in our patient with PL-IHS. Similar findings have been also described by Wilder-Smithet al.8 who showed reduction in the velocity of blood flow by approximately 50% in the straight sinus in patients after lumbar puncture.The pathogenesis of the PL-IHS-induced CVT can be explained by the Monro-Kellie-Abercrombie doctrine. This suggests that the skull is a rigid structure, and the brain volume, venous blood and CSF are in state of equilibrium, so reduction or increase of either element leads alteration in the volume of the other two. In IHS, the CSF volume and pressure are reduced significantly. Two changes occur as a result of this: first in the venous compartment, there is increase in the intracranial venous volume, and second there is descent of the brain and brainstem structures. The venous volume expansion is due to the venous stasis and dilation of the sinuses, cortical and spinal veins. This change occurs first in the meninges, both pachymeninges and leptomeninges. The pachymeninges do not have a blood-brain barrier; this leads to contrast extravasation and hence the post-contrast enhancement. Further, due to the descent of the brain there is distortion and stretching of the veins. All the above changes are further aggravated in erect posture when there is acute dilation of the veins and further stretch on the venous walls, which leads to postural headache. This along with venous stasis may lead to CVT in some patients. This hypothesis has been further bolstered by the study by CanhaoPubMed on September 2013 (Data Set 1). These patients could be divided into two major groups  in September 2013. Details include obstetric associated PL-IHS patients and non-obstetric associated patients, subjected to post-diagnostic lumbar puncture spinal or epidural anesthesia for non-obstretic surgeries, lumbar intrathecal injection for chemotherapy or myelography, and insertion lumbar drain.Click here for additional data file.11, heterozygous factor V Leiden mutation12 and prothrombin mutation 20210A13. Patients in this group had uniformly good response with complete resolution of symptoms in less than 4 weeks. Neither prior epidural blood patch12 nor prophylactic heparin were able to prevent occurrence of CVT in post-partum PL-IHS.The CVT symptoms developed after 4–12 days of the lumbar puncture. Approximately three quarters of the patients had postural headache at the onset and a change in the pattern of headache was observed in approximately half of the patients with the onset of CVT symptoms. This was the headache becoming continuous, becoming diffuse or losing its postural character. These patients had prothrombotic predisposition in 35% of cases or prior history of oral contraceptive pill intake as additional pathogenetic factors. The prothrombotic conditions were Protein C and S deficiencya) Post-diagnostic lumbar puncture (n=17)2, recurrent optic neuritis3 and probable Neuromyelitis optica14. None of these patients had any prothrombotic condition as observed in the Obstetric associated Group. All patients uniformly had postural headache at the onset and this was followed by a change in the pattern of headache as described above in 77.8% of patients with the development of CVT. Superior sagittal sinus was involved in 70% of the cases. One-quarter of the patients in this group were reported to have had a poor outcome15. The underlying disease and sagittal sinus involvement may have been responsible for the poor outcome.The majority of the patients (82.5%) in this group had demyelinating disorder and had received intravenous high dose corticosteroids (IVCS) within a few days, such as multiple sclerosis2 but the exact pathogenesis remains to be elucidated. The concomitant use of IVCS has been proposed to be causative factor for the CVT. This can partly be explained by the role of high dose steroids on CSF dynamics and vascular endothelium: steroid treatment has a paradoxical effect on CSF absorption and can either impair or facilitate its absorption. Steroids have a vasodilatory effect on the vessel; this could further aggravate the venous stasis present in the PL-IHS. We hypothesize that the combined changes in CSF absorption and increased venous stasis in patients with lower intracranial hypotension may increase the likelihood of the venous thrombosis inpatient receiving IVCS.Reports of CVT in demyelinating disorders have increased in last decadeb) Other procedures group17, post-intrathecal methotrexate5, post-myelography3, post-epidural anesthesia3, post-spinal anesthesia18 and post-lumbar drain19. Underlying prothrombotic conditions were Factor V Leiden mutation3, Protein C deficiency17 and lymphocytic lymphoma5. In other cases either etiological work-up was non-revealing or was not done (Data Set 1). Poor outcome was noted in 27.2% (3/11) patients and one patient died during the illness3. Apart from the age, post-partum state and outcome this group had similar characteristics and risk factors as obstetric associated group.The average age of at presentation was 35.5±10.76 years. This group was heterogeneous, including patients that had experienced post-epidural steroid injection18, there has been the ongoing debate about whether they are both mere associations and whether their occurrence is only coincidence. But over the last two decades there is formidable evidence suggesting causality8, that is, of PL-IHS causing CVT, though the exact pathogenetic mechanism remains to be conclusively demonstrated. Further, the various settings in which the phenomenon has been observed suggest multiple risk factors. Over the last 25 years of its description, the clinical characteristics have consistently demonstrated the headache pattern, starting as postural headache and then with a change in the headache character as it loses its orthostatic character; this should alert the treating clinician that the patient may have developed CVT. Though the phenomenon is very rare compared to the number of lumbar punctures being performed, the morbid outcome in one-quarter of patients reported should caution us. The propensity for PL-IHS is increased in patients with prothrombotic conditions; prophylactic heparin may prevent the occurrence of the CVT in this group of patients. This generates questions regarding the dose and duration of the heparin, type of heparin to be used, assessment measures etc, which further studies can answer. Patients with known prothrombotic conditions who undergo lumbar puncture for various procedures should be aggressively managed, including hydration and supine posture, and they may benefit from heparin prophylaxis.Since its initial description by Schou and Scherb in 1986 of PL-IHS and CVThttp://dx.doi.org/10.6084/m9.figshare.92019220Figshare: Review of literature of cerebral venous thrombosis associated with post-lumbar puncture intracranial hypotension,Written informed consent for publication of clinical details and clinical images was obtained from the patient on Institutional format. This case report and review of the literature may be helpful to the patients of physicians who are not aware of this syndrome.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. An interesting case with a well conducted review of similar cases in the literature. It is a good clinical teaching point to consider dural sinus thrombosis if there is a change in pattern of post-lumbar puncture headache.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Here we report mesocosm experiments designed to test the influence of plant density and species composition (wheat monoculture or wheat and faba bean polyculture) on the emergence of multi-predator effects between Adalia bipunctata and Chrysoperla carnea, in their suppression of populations of the aphid Metopolophium dirhodum. The mesocosm experiments were followed by a series of behavioural observations designed to identify how interactions among predators are modified by plant species composition and whether these effects are consistent with the observed influence of plant species composition on aphid population suppression. Although plant density was shown to have no influence on the multi-predator effect on aphid population growth, plant composition had a marked effect. In wheat monoculture, Adalia and Chrysoperla mixed treatments caused greater suppression of M. dirhodum populations than expected. However this positive emergent effect was reversed to a negative multi-predator effect in wheat and faba bean polyculture. The behavioural observations revealed that although dominant individuals did not respond to the presence of faba bean plants, the behaviour of sub-dominants was affected markedly, consistent with their foraging for extra-floral nectar produced by the faba bean. This interaction between plant composition and predator community composition on the foraging behaviour of sub-dominants is thought to underlie the observed effect of plant composition on the multi-predator effect. Thus, the emergence of multi-predator effects is shown to be strongly influenced by plant species composition, mediated, in this case, by the provision of extra-floral nectar by one of the plant species.The prediction of pest-control functioning by multi-predator communities is hindered by the non-additive nature of species functioning. Such non-additivity, commonly termed an  In recent years, there has been considerable interest in the relationship between community attributes and the provision of ecosystem services, such as pest population suppression. Although numerous studies have focussed on the role of natural enemy species richness or composition in determining prey population suppression, predicting the impact of natural enemy community change on ecosystem functioning is often hindered by emergent multi-predator effects; the functional impact of a species assemblage is not a straight-forward linear combination of the constituent species impacts Emergent multi-predator effects on prey population suppression are classically thought to arise when there is a trait-mediated or density-mediated interaction Aside from interactions among species, general biodiversity – ecosystem functioning theory tells us that emergent multi-species effects also arise when there is niche-differentiation between species, such that multispecies communities occupy a greater proportion of total niche space It is clear that multi-predator assemblages can exhibit a range of emergent multi-predator effects depending on the relative strengths of a range of potential mechanisms. There is also evidence that the same multi-predator assemblage can exhibit different emergent effects in different environmental contexts; multi-predator effects are not determined solely by fixed traits of the predator species concerned, but vary depending on how the environment affects the underlying mechanisms. For example, prey life-history Here we conduct experimental tests to determine whether plant density and plant composition (monoculture vs biculture) affect the emergent multi-predator effects occurring between two aphidophagous predators. Our plant species include one plant that produces extra-floral nectar as a supplementary/alternative food source, which is predicted to affect foraging activity. We formally test the hypotheses: 1) that increased plant density reduces the influence of negative intraguild interactions leading to more positive emergent effects on prey suppression; 2) that the presence of extra-floral nectar provides an alternative food source leading to reduced prey consumption; and 3) that foraging responses of predators to changing resource environments result in modification of emergent multi-predator effects.Adalia bipunctata (Coccinellidae) and second instar larvae of Chrysoperla carnea (Chrysopidae), and a common prey species, the cereal feeding aphid Metopolophium dirhodum. This combination of predator life stages was chosen to generate an asymmetric negative interspecific interaction in the multi-predator treatment, with the larger Adalia adults being the potential intraguild predator and dominant competitor of Chrysoperla larvae. This combination facilitated a direct test of the hypothesised moderating effect of plant density on negative intraguild interactions.A combination of mesocosm experiments and independent behavioural observations were undertaken to test the effect of plant density and species composition on the emergence of multi-predator effects, and to identify the behavioural mechanisms underlying these effects. The experimental system included two commonly co-occurring aphidophagous predators, adult Adalia bipunctata and second instar larvae of Chrysoperla carnea on population growth of M. dirhodum. Four predator treatments were used in the experiment: Adalia alone, Chrysoperla alone, the two species in combination, and a predator-free control. A substitutive design was used, such that four individual predators were introduced into each cage, with two of each species in the mixed predator treatment. These predator treatments were fully crossed in a factorial combination with three vegetation treatments comprising low-density wheat monoculture , high-density wheat monoculture, and a mixed culture of wheat and faba bean (Vicia faba var Hobbit). Plants were sown in compost in seed trays (35×21×6.5 cm) separated into 5 strips (each 6.5×21×6.5 cm). The low-density wheat treatment had three plants in each of three strips, the high-density wheat had three plants in each of five strips, and the wheat/bean polyculture had three wheat plants in three strips plus three bean plants in two strips. All plants were reared from seed in a glasshouse in compost (John Innes #2 compost) and were approximately five weeks old at the start of the experiment. Faba bean was used as the second species because it is commonly inter-cropped with cereals and because it produces extra-floral nectar and is, therefore, a source of supplementary/alternative resources for predators.Experimental mesocosms  housed in an unheated glasshouse were used to measure the impact of adult M. dirhodum (in three groups of ten) were introduced into each cage. M. dirhodum were obtained from a single-clone culture housed at Lancaster University. After 24 h the appropriate number of predators was introduced to each cage. All predators were purchased immediately prior to each experimental run  and were maintained on an aphid-free diet (buckwheat seeds) at 5°C for approximately 24 h before use. 48 h after introduction of the predators, each cage was destructively harvested and the number of surviving M. dirhodum counted. For logistical reasons, the experiment was replicated across seven temporal blocks, with each block comprising the full set of treatment combinations.At the start of each experimental run, 30 third or fourth instar M. dirhodum was calculated as c,t/Np,t)/4m = ln in the same block. The variable m represents the extent to which the intrinsic population growth rate r is reduced by each predator assuming dN/dt = rNm values, expected prey population suppression was calculated as the average of the m values for the constituent single species treatments under the same vegetation treatment in the same block. The emergent multi-predator effect (MPE) was defined as the deviation from expectation: = m/Em)ln, values <0 indicate lower prey suppression than expected (risk reduction).The per capita impact of predators on the population growth of plant with a contrast representing the effect of plant richness (high and low-density wheat treatments vs. wheat/bean mix) followed by a contrast testing the effect of wheat density Analysis of per capita impact and MPE were done by ANOVA in R Triticum aestivum var Tybalt) or one wheat and one faba bean (Vicia faba var Hobbit). In all cases, ten individual M. dirhodum were placed on an upper leaf of the wheat plant in wheat/bean combinations, or on one of the pair of wheat plants in the wheat/wheat combinations. M. dirhodum all came from a single-clone culture maintained at Lancaster University. Predator foraging observations were made of Adalia adults and second-instar Chrysoperla larvae alone, in combination with a conspecific, and in combination with a heterospecific individual. Approximately eight independent trials of each predator combination were made in each of the plant composition treatments. All predators were purchased from a biological control supplier  prior to the experiments and were maintained on an aphid-free diet at 5°C before use. All observations were made in a controlled-environment room at 20°C and all predators were allowed to acclimatise for 1 h prior to observations. At the start of each observation period (approx. 30 min), a clean transparent bridge (7×3 cm) was positioned to join the plant pots such that the each end of the bridge was touching the stem of the plant. The appropriate predator combination was then released at the centre point on the bridge (equidistant from the two plants) and the following data were recorded for each predator: total time spent on each plant; time spent on the bridge, soil surface or other areas in the cage; time spent feeding on aphids; and time spent feeding at extra-floral nectaries (wheat/bean treatment only).In order to provide mechanistic explanations for the population responses observed in the mesocosm experiments, a series of observations of predator interactions were undertaken in an experimental arena , modified to have one transparent side to facilitate observation. The experiment was designed to test the effect of interpredator interactions (conspecific and heterospecific) on foraging behaviour and how these were modified by plant composition. Two plant pots (7-cm diameter), each containing a single plant, were placed in the arena in one of two arrangements: two wheat  that was spent on the infested or alternative plant was analysed. This gives an indication of how foraging pressure on the two plants was influenced by the predator treatments and the plant treatments. Second, the foraging behaviour of individual predators was analysed. In predator treatments with two conspecific individuals, the individual spending most time on the aphid-infested plant was deemed to be the dominant and the individual spending least time on the aphid-infested plant the sub-dominant. This revealed whether similar dominance hierarchies existed within and between species, and whether these were affected by the identity of the alternative (aphid-free) plant offered to the predators. As with the mesocosm experiment, all analyses were performed by ANOVA in R m) varied significantly between both the predator treatments  and plant treatments , but there was no significant interaction between plant and predator treatments. Suppression of the prey population was lowest in the wheat and bean polyculture treatment, significantly lower than in the low-density wheat treatment  and marginally lower than the high-density wheat treatment . The contrast comparing aphid population growth suppression (m) in treatments with both wheat and bean plants compared with the wheat-only treatments was significant  or the Adalia treatment . The Adalia treatment did not result in significantly greater suppression than the Adalia plus Chrysoperla treatment .All predator treatments caused significant suppression of aphid population growth but suppression was highest in the reatment . The Chr1,16 = 6.06; P = 0.026). This analysis also revealed that there was a change in the nature of the MPE from significantly positive in the wheat treatments (greater than expected prey suppression) to negative (lower than expected prey suppression) in the wheat plus faba bean polyculture treatment , but not by plant treatment, and there was no significant interaction between plant and predator treatments. The predator effect arose from significantly less foraging time on the aphid-infested plant in the Adalia plus Chrysoperla combination treatment than in the Adalia treatment .The percentage of total observation time spent foraging on the plant infested with aphids was significantly affected by predator treatment  and there was a highly significant effect of the predator combination within species . When heterospecific individuals were paired, Adalia tended to be dominant, spending a greater proportion of time foraging on the aphid-infested plant than Chrysoperla .The time budgets of individual insects showed that 1, 104 = 9.79; P = 0.002; 7, 104 = 3.55; P = 0.002), and these two factors had a statistically significant interaction . Generally, more time was spent by predators on bean plants than aphid-free wheat plants , and the plant species identity had a significant impact on foraging time allocated to the aphid-free plant by sub-dominant individuals in conspecific and heterospecific pairs  of their foraging time at a nectary compared with 21.3% (±3.5 s.e.) for Chrysoperla. Of the 24 trials in which two predators were present, only in three trials were both predators observed to feed on nectar .Feeding at the extra-floral nectaries on the bean plant was recorded in 10 The data reported here show that plant composition, but not plant density, can have a strong impact on the sign and strength of emergent multi-predator effects on prey suppression. Previous studies of the effect of vegetation on the functioning of multi-predator communities have shown variable results, leading to the suggestion that structural complexity of vegetation or habitat may be more important than plant identity in determining the outcome and magnitude of emergent multiple predator effects Several studies have shown an effect of habitat complexity on emergent multi-predator effects caused, for example, by lower rates of intraguild predation in more complex environments Although the experiments revealed little effect of plant density on multi-predator effects, they did reveal strong effects of plant composition on emergent multi-predator effects, characterised by a switch from positive (increased prey suppression) multi-predator effects in wheat monocultures to negative (deceased prey suppression) multi-predator effects in wheat and bean polyculture. Although some previous experiments have failed to show an effect of plant identity on diversity effects among aphidophagous predators Adalia individuals in the conspecific pairings, and Chysoperla individuals in heterospecific pairings, increased markedly when bean was present compared with wheat. There was no such effect with sub-dominant Chrysoperla in conspecific pairings. We propose that the interactive effect of predator and plant treatments on the foraging behaviour of sub-dominant Chrysoperla individuals may underlie the observed impact of plant identity on the multi-predator effect in the mesocosm experiment.Under a substitutive experimental design, emergent multi-predator effects reflect a difference in impact of interspecific interactions compared with intraspecific interactions on prey suppression M. dirhodumChrysoperla individuals, in particular, behaved differently depending on whether they were paired with conspecifics or heterospecifics. In the trials where the aphid-free plant was wheat, Chryspoperla individuals spent a much higher proportion of time off the plants when paired with Adalia than when paired with a conspecific, which may have promoted facilitation between Adalia and Chrysoperla in the wheat monoculture treatments, mediated by aphid dropping behaviour. However, where the aphid-free plant was bean, foraging allocation to the aphid-free plant by Chrysoperla was high and, consequently, their aphid consumption was unlikely to have been affected by aphid dropping behaviour.Why should changes in the behaviour of sub-dominant individuals, which generally did not forage on the aphid infested plant, affect the multi-predator effect on prey suppression? Positive multi-predator effects can arise from resource-use complementarity among the predators, such that a greater proportion of the aphid population is susceptible to predation, or from facilitation among predators. For example, facilitation between foliar and ground foraging aphidophagous predators has been commonly reported Medicago sativa and Trifolium pratense, can cause a marked reduction in aphid consumption resulting in a negative impact of plant species richness on aphid consumption Whatever the precise mechanism for the change from positive to negative multi-predator effects on prey suppression as vegetation composition changed from wheat monoculture to wheat and bean polyculture, it is likely that this was mediated by the change of foraging behaviour of individual predators in the presence of extra-floral nectar. Previous studies have shown that the presence of faba bean, compared with the other legumes Chrysoperla may not persist over longer time scales, when feeding on more protein-rich food may be required to maintain growth and development. Similarly, increase in spatial scale would likely reduce encounter rates and the strength of inter-predator interactions. Increase in spatial scale has also been predicted to strengthen richness effects on functioning due to the influence of habitat heterogeneity on species functional complementarity There is an open debate about the value of increased natural enemy diversity on pest control in agricultural systems; increasing biodiversity improves pest control in most cases, but in a substantial minority of cases the opposite is true


Schistosoma haematobium. In high transmission areas, children acquire schistosome infection early in life with infection levels peaking in early childhood and subsequently declining in late childhood. This age-related infection profile is thought to result from the gradual development of protective acquired immunity. Age-related differences in schistosome-specific humoral and cellular responses have been reported from several field studies. However there has not yet been a systematic study of the age-related changes in human dendritic cells, the drivers of T cell polarisation.Urogenital schistosomiasis is caused by the helminth parasite S. haematobium prevalence of 47.5%. Two subsets of dendritic cells, myeloid and plasmacytoid dentritic cells (mDCs and pDCs), were analyzed by flow cytometry.Peripheral blood mononuclear cells were obtained from a cohort of 61 Zimbabwean aged 5–45 years with a In this population, schistosome infection levels peaked in the youngest age group (5–9 years), and declined in late childhood and adulthood (10+ years). The proportions of both mDCs and pDCs varied with age. However, for mDCs the age profile depended on host infection status. In the youngest age group infected people had enhanced proportions of mDCs as well as lower levels of HLA-DR on mDCs than un-infected people. In the older age groups (10–13 and 14–45 years) infected people had lower proportions of mDCs compared to un-infected individuals, but no infection status-related differences were observed in their levels of HLA-DR. Moreover mDC proportions correlated with levels of schistosome-specific IgG, which can be associated with protective immunity. In contrast proportions of pDCs varied with host age, but not with infection status.Our results show that dendritic cell proportions and activation in a human population living in schistosome-endemic areas vary with host age reflecting differences in cumulative history of exposure to schistosome infection. Schistosoma haematobium infection. We found an age-related pattern in the proportions of myeloid dendritic cells (a subset of dendritic cells) in this population. Furthermore, in the case myeloid dendritic cells, the age profile differed between schistosome infected and un-infected people. Thus our study suggests that activation and migration of myeloid dendritic cells also develop in an age-related pattern consistent with the cumulative history of exposure to schistosome parasites.A characteristic feature of most helminth infections is the convex age infection profile, where infection levels rise to peak in early childhood and decline in adulthood, a pattern thought to result from the development of protective acquired immunity. Thus, several investigations characterizing protective responses to inform vaccine research have focused on responses present in older people, who despite continued exposure to infection carrylittle or no infection. To date, such studies have identified key responses which are correlates of resistance. However, there is a paucity of information on cell types that are mediators rather than effectors of the immune responses. One such group where there are limited studies in human schistosome infections is dendritic cells which are important for the polarizations of CD4+ T cell responses. Therefore, we characterized the age profile of dendritic cells in Zimbabweans exposed to  Schistosoma haematobium helminth parasites cause urogenital schistosomiasis which affects about 112 million people mainly in rural areas of subtropical countries S. haematobium are most common in school-age children. Populations in endemic areas show a characteristic age-infection profile with infection levels increasing to peak in early to late childhood, typically around 9–15 years and then declining in adulthood versus egg negative adults) can differ significantly in their immune response against the parasite and thus their levels or resistance to re-infection. Several studies characterizing human immune responses to schistosome infections have shown age related differences in antibody levels H1, TH2 and regulatory responses H2 response is important in the immune response against schistosomes H2 immune responses in the context of schistosome infections has been recently highlighted in an experimental mouse model H2 responses during schistosome infections supporting other studies showing the importance of DCs in the context of TH2 induction As investigations of the nature and development of protective acquired immunity progress, there has been a move to decipher the mechanisms and pathways behind these observed age-related patterns. Human field studies show that the immune response against schistosomes is characterized by a very complex interaction of TH2-promoing activities can be diminished by TLR-3 It has been shown that schistosome derived compounds can activate dendritic cells by toll-like receptors (TLR)-2 and 3 S. haematobium) In particular, studies analysing DCs directly from humans exposed to schistosomes are limited. The first study addressing this question, published by Everts and colleagues in 2010, elegantly showed changes in DCs during chronic schistosomiasis , which elicit immune responses that cross-react with those against S. haematobiumS. mansoni prevalence is consistent with earlier surveys in this area of Zimbabwe S. haematobium  should not have received anti-helminthic treatment prior to this study, (3) should have provided at least two urine and two stool samples on consecutive days for parasitological diagnosis, (4) should have tested negative for intestinal helminths including S. mansoni to focus on single infections with S. haematobium, (5) should be negative for HIV and malaria  and (6) have provided a sufficient volume of blood to isolate peripheral blood mononuclear cells (PBMC). The selected cohort comprised 61 individuals and details to the cohort are provided in This study was performed in Chipinda village which is located in the Mashonaland East Province in Zimbabwe . The village was selected because health surveys regularly conducted in the region showed little or no infection with soil-transmitted helminths (STH) and a low Permission to conduct the study in the region was obtained from the Provincial Medical Director and institutional and ethical approval was received from the University of Zimbabwe's Institute Review Board and the Medical Research Council of Zimbabwe respectively. Only compliant participants were recruited and they were free to drop out at any point during the study. At the beginning of the study, participants and their parents/guardians (in case of children) had the aims and procedures of the project explained fully in the local language, Shona, and written consent was obtained from participants and parents/guardian before parasitology and blood samples were obtained. After collection of all samples, all participants were offered anti-helminthic treatment with the recommended dose of praziquantel (40 mg/kg of body weight).Depending on age of the participants up to 25 ml of venous blood was collected in heparinized tubes of which approximately 5 ml was used for serological assays as well as microscopic detection of malaria parasites. The remaining blood was used for the isolation of peripheral blood mononuclear cells (PBMC) through density gradient centrifugation using Lymphoprep . These PBMC were subsequently enumerated, cryo-preserved in 10% DMSO, 90% fetal calf serum and stored in liquid nitrogen in Zimbabwe prior to shipping to Edinburgh in dry shippers for assaying.Thawing of cryo-preserved PBMC was performed by rotating cryovials in a 37°C water bath until a small crystal was remaining in the cell suspension. Cells were then slowly re-suspended in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine and 100 U Penicillin/Streptomycin . Cells were washed twice with media, counted and viability assessed using trypan blue . The median viability of the PBMCs was 71.4% which is within the range of published values Serum antibody levels were measured by enzyme-linked immunosorbent assays (ELISA) following established protocols 10 (mean egg count+1) transformed) and proportions of mDCs was conducted after allowing for the effect of sex. The correlation coefficients were then tested for homogeneity using the Fisher's r-to-z transformation The proportions of DC cell subsets were square root arcsine transformed whereas expression levels of HLA-DR and CD86 were square root transformed to allow the use of parametric tests in subsequent analyses S. haematobium. The northeast part of Zimbabwe is endemic for S. haematobium with many regions having a moderate to high prevalence of S. haematobium, but low prevalence of S. mansoni and soil-transmitted helminths S. haematobium in the selected study population (N = 61) was 47.5% which is higher but not significantly different from  the village prevalence of 39% and still within the WHO definition of moderate transmission. The difference in infection prevalence between the youngest age group (35.3%) compared to more than 50% in the older age groups was not significant  from pDCs , neither mDCs (negHLA-DR+ (data not shown). Myeloid DCs showed a significant association with sex  between un-infected and infected people in any of the three age groups as shown in In contrast, to the clear picture in the case of mDCs, there was no significant difference in the proportion of pDCs  and mDCs proportions were considered. In this case both parameters were positively correlated in the youngest age group and became a negative correlation with increasing age. This pattern is consistent with that predicted by quantitative studies for protective immune responses and has already been demonstrated for antibody responses associated with protection to re/infection in human hookworm et alet al (17–39 years). Since cellular immune responses might depend on many different parameters such as transmission dynamics or genetic background of the investigated study population the consistency in DC proportions in schistosome-exposed human between our study and that by Everts et al, by itself is an important finding. A major difference between the two studies is that we did not observe differences in pDC percentages in regards to infection status, although we had high variation in pDC levels in the oldest age group. In deed a slight tendency of lower pDC proportions (expressed as percentage of PBMC) was observed in this age group. Differences in co-infections may also account for the differences between our observations and those of Everts et alOur data confirm some of the major findings of a previous study of DC populations in human schistosomiasis conducted by Everts et alH2 induction has been recently shown Both our study and that of Everts Changes with age have been also observed for serum cytokines in other study populations with moderate/high schistosome transmission et alH2 priming DCs has been reported S. haematobium infected people et al reported a more general impairment of DCs affecting both TH1 and TH2 cytokines as well as IL-10 in older individuals (17–39 years). The mechanisms involved are still unclear since despite this impairment of DC function, older individuals carrying schistosome infection can show enhanced immunopathology Other reasons for reduced relative numbers of mDCs in infected individuals in older age groups remain to be determined, e.g. enhanced apoptosis, reduced bone marrow output or increased migration to inflamed tissues or lymphoid organs were proposed by Everts In contrast, our data show that in the younger individuals (5–9 years) mDCs proportions were higher in peripheral blood of infected individuals in comparison to un-infected. Potential reasons for this include enhanced bone marrow output or reduced migration of DCs in the context of blood-born antigens et alDendritic cell activation/maturation is characterised by up-regulation of cell-surface markers such as CD86 and MHC class II molecules. As previously reported by Everts et al, we did not observe a significant difference of HLA-DR levels in the older age groups. Again more significant changes of HLA-DR in these age groups might be due to cumulative effect of co-infections, not present in our study population.In contrast to the study by Everts Inhibition of up-regulation of activation markers such as CD86 has been suggested to be involved in induction of tolerogenic DCs Functional studies, will be very informative and will clarify some of these areas, but were beyond the scope of this study.S. haematobium infection as is typical for many already investigated humoral and cellular responses. Furthermore, in the case of mDCs, this pattern differs between schistosome infected and un-infected people and also follows the age profile with infection of protective responses, which is supported by the correlation between schistosome-specific IgG and proportions of mDCs.In summary this study clearly shows there is an age-related pattern in the proportions mDCs in people exposed to The mechanism and consequences  behind the association between mDCs and infection remain to be determined. Understanding the nature and dynamics of immunological parameters in natural helminth infections and, in particular, changes of DCs involved in polarising the immune responses has an important impact on the development of vaccines against schistosomes.Figure S1Proportions of mDCs and pDCs expressed as percentage of CD14neg-HLA-DR+ cells. The study population was divided into three age groups: 5–9 years (A), 10–13 years (B) and 14+ years (C). Proportions of mDCs (A) or pDC (B) were compared between un-infected (white box) and infected (gray box) individuals and expressed as % percentage of CD14neg-HLA-DR+. Differences between un-infected and infected groups in the different age groups were analysed by univariate analysis after accounting for the effect of sex.(TIF)Click here for additional data file.Table S1Interaction between WWH-IgE and mDC or pDC.(TIF)Click here for additional data file.Table S2Correlation between WWH-IgE and mDC or pDC.(TIF)Click here for additional data file.


Therefore, at physiological pH, histamine exists as an equilibrium mixture of tautomeric cations: the monocation making up 96%, the dication only 3% and the rest being nonprotonated histamine. As a protonated molecule histamine most probably uses a carrier protein in order to cross the cell membrane. In the present work we wanted to determine the kinetic properties of histamine uptake and the influence of other biogenic amines on its transport.Histamine is a double-protonated molecule with corresponding pKt-test was used for statistical analysis of uptake data.We performed histamine uptake assays in the model system of cultured neonatal rat astrocytes. The mRNA expression of organic cation transporters (OCTs) was determined by qPCR. Student’s +, but not Cl−-dependent, with low affinity (Km 116 µM) and high capacity (158 pmol/mg protein). The uptake was inhibited by millimolar concentrations of other biogenic amines . The histamine metabolite tele-methylhistamine affected both directions of histamine uptake. In spite of the presence of OCT2 in neonatal astrocytes, the OCT inhibitors decynium-22 and corticosterone had no affect on histamine clearance.Histamine uptake in neonatal rat astrocytes is a bidirectional process, which was found to be dependent on pH and NaHistamine is taken up into astrocytes by low-affinity high-capacity uptake, which involves transporter(s) other than OCT.


Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes.fbxoo) was obtained by assembling sequences from an EST clone and a 5′RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening.Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene  were identified as potential Fbxoo protein binding partners.Results suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout. Figla), growth differentiation factor 9 (Gdf9) and newborn ovary homeobox (Nobox). Deficiency of these essential genes in mice leads to the failure of primordial follicle formation or the disruption of early follicular development  and a predicted hypothetical protein [GenBank: XP_002932171.1] in Xenopus tropicalis , an open reading frame (ORF) of 1,545 bp and a 3′untranslated region (3′UTR) of 438 bp  and early embryos (day 4 and day 7), but at much lower levels relative to the developing ovaries.To determine the expression profile of Using a polyclonal antibody raised in rabbit against a synthetic peptide of Fbxoo protein, the expression of Fbxoo protein in ovaries of different developmental stages was determined by Western blot analysis. As shown in Figure In situ hybridization was performed on rainbow trout ovarian sections to determine the localization of fbxoo transcript. Strong hybridization signals (blue staining) were detected in the cytoplasm of small oocytes at early pre-vitellogenesis and early vitellogenesis stages . Three positive clones were finally identified as they showed y Figure A. The ply Figure B, Ak2  particles ,19. Duri protein . Sumoyla protein  and sumo protein .fbxoo mRNA and protein is detected in both cytoplasm and nucleus of oocyte at early developmental stages. While many F-box proteins are ubiquitously expressed [Many F-box proteins are located in both cytoplasm and nucleus . Our resxpressed , tissue xpressed -28. In pxpressed ,29. As aTimps has been detected in unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts as well as in granulosa and theca cells [F-box proteins bind to their specific substrates for ubiquitin-mediated proteolysis. They often have additional carboxy-terminal motifs (e.g. WD repeats and leucine-rich repeats) capable of protein-protein interaction. As an FBXO protein, Fbxoo does not have the common secondary motifs but may contain potential protein-protein interaction domains not yet identified. As an attempt to understand the function of the novel protein, we screened for Fbxoo-interacting proteins using the yeast two-hybrid system and identified three proteins including Timp2 as potential substrates of Fxboo protein. TIMP2 is a member of the TIMP family proteins known to play an important role in regulating the activity of matrix metalloproteinases (MMPs), which are the key metal-dependent enzymes in the extracellular matrix remodeling of follicular tissue. TIMPs are highly abundant in reproductive tissues and are well known to be involved in follicular development and early embryogenesis in a number of mammalian and fish species -32. The ca cells . The funca cells . As a TIWe have cloned a novel oocyte-specific gene encoding an F-box only protein in rainbow trout and demonstrated that it is abundantly expressed in early pre-vitellogenic oocytes. Our results suggest that Fbxoo may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.The authors declare that they have no competing interests.LW, SKT and WW performed the experiments. LW drafted the manuscript. CER and JY designed the study, supervised the experimental work and revised the manuscript. All authors read and approved the final manuscript.Complete cDNA and deduced amino acid sequences of rainbow trout Fbxoo. The F-box domain is shaded in grey and the predicted Sumo site is shaded in blue. The sequence of the peptide used to produce anti-Fbxoo antibody is boxed. The start codon (ATG) and stop codon (TAG) are highlighted in yellow. The polyadenylation signal (AATAAA) and the cytoplasmic polyadenylation element (TTTTTAA) are both underlined and bolded. The cDNA sequence has been deposited in the NCBI database with the accession number: HQ201417.Click here for file


Based on our theory, main triplets of amino acid residues have been discovered in cell-adhesion receptors (integrins) of marine sponges, which participate as homologies in the interface between two major immune molecules, MHC class I (MHCI) and CD8αβ. They appear as homologies also in several human neural receptor heteromers and subunits. The obtained results probably mean that neural and immune receptors also utilize these structural integrin triplets to form heteromers and ion channels, which are required for a tuned and integrated intracellular and intercellular communication and a communication between cells and the extracellular matrix with an origin in sponges, the oldest multicellular animals. Based on a mathematical approach, Tarakanov and Fuxe , 2011 ha20102012a2012 abcIn the current paper, many of such triplets have been found in integrins of marine sponges together with human alpha and beta integrins. This means that such triplet homologies may play a role in alpha-beta heterodimeric complexes forming integrin receptors and interact with extracellular matrix proteins  and the Universal Protein Resource (http://www.uniprot.org). Table http://www.clustal.org/), AGGRESCAN (http://bioinf.uab.es/aggrescan/), accelrys (http://accelrys.com/), and so on seems to be able to deal with so specific and complicated combinatorial puzzle. Our original software has been developed to determine such basic set of triplet homologies from two given sets of protein-protein pairs (which bind and do not bind). The core of this software is the computing of all triplet homologies between two given words . The method consists in forming the binary matrix of all one-letter homologies (which element is 1 if there is homology and 0 otherwise) and then filtering this matrix using rather specific rules of so called cellular automata .Amino acid codes of receptors and other proteins have been obtained from the National Center for Biotechnology Information , RPA (Arg-Pro-Ala), DGR (Asp-Gly-Arg), LLG (Leu-Leu-Gly), and GLL (Gly-Leu-Leu) of the integrin receptors of marine sponges appear as homologies in murine and human MHCI, GABAB1, and human integrin receptor heteromers (see Tables The triplet DGR (Asp-Gly-Arg) is in fact the inverse triplet of RGD (Arg-Gly-Asp) that provides the binding site for integrin RGD-binding receptors (see Table Tetraodon) and then continues to man, while it is missing in zebrafish (Danio rerio). Thus, the usefulness of the ITL triplet in recognition is rediscovered in the fish GABAB1 receptor.The evolution of the ITL triplet in the GABAB1 receptor subunit is displayed in Figure Furthermore, the RPA triplet homology in the beta and delta interacting nicotinic subunits of the neuromuscular nicotinic receptor  found in integrins of marine sponges is presented as a homology in the interface between MHC Class I and CD8αβ heterodimer (coreceptor in T cells). It is postulated that this triplet homology can contribute to the formation of the MHCI-CD8 heteromeric complex which leads to a strong activation of the T cell by guiding the T-cell receptor into relevant self-MHC recognition (see Wang et al. Integrin triplets of marine sponges found in the interface of human receptor heteromers and even in the interface between two major immune molecules MHCI-CD8 seem to confirm once more our theory. This triplet puzzle arose as a surprising merger of pure mathematics and most recent biochemical studies of receptor-receptor interactions. As a result, it appears that neural and immune receptor heteromers in humans may also utilize these structural elements originating in sponges, the oldest multicellular animals. Thus, the triplet puzzle may be an ancient and general mechanism for protein-protein recognition.


Foot-and-mouth disease virus (FMDV) targets specific tissues for primary infection, secondary high-titer replication  and long-term persistence at some primary replication sites. Although integrin αVβ6 receptor has been identified as primary FMDV receptors in animals, their tissue distribution alone fails to explain these highly selective tropism-driven events. Thus, other molecular mechanisms must play roles in determining this tissue specificity. We hypothesized that differences in certain biological activities due to differential gene expression determine FMDV tropism and applied whole genome gene expression profiling to identify genes differentially expressed between FMDV-targeted and non-targeted tissues in terms of supporting primary infection, secondary replication including vesicular lesions, and persistence. Using statistical and bioinformatic tools to analyze the differential gene expression, we identified mechanisms that could explain FMDV tissue tropism based on its association with differential expression of integrin αVβ6 heterodimeric receptor (FMDV receptor), fibronectin (ligand of the receptor), IL-1 cytokines, death receptors and the ligands, and multiple genes in the biological pathways involved in extracellular matrix turnover and interferon signaling found in this study. Our results together with reported findings indicate that differences in (1) FMDV receptor availability and accessibility, (2) type I interferon-inducible immune response, and (3) ability to clear virus infected cells via death receptor signaling play roles in determining FMDV tissue tropism and the additional increase of high extracellular matrix turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, play a key role in the pathogenesis of vesicular lesions. Aphtovirus). Susceptible hosts include several domesticated  and wild  cloven-hoofed animals. The morbidity is very high but the mortality is generally low in adult hosts. The infection in cattle commonly occurs via the respiratory route by aerosolized virus Foot and mouth disease (FMD) is one of the most contagious and economically devastating viral animal diseases. It is caused by FMD virus (FMDV), a positive-sense, single stranded RNA virus member of the family Picornaviridae  amino acid sequence motif present on the FMDV VP1 Like other pathogenic viruses, the FMDV genome encodes several proteins that facilitate evasion of the innate immune response of infected cells. For example, the leader protease (Lpro) can inhibit host cap-dependent translation by cleaving translation initiator factor eIF4G but has no effects on viral cap-independent translation Because cells differentiate by alternating their gene expression, differential tissue gene expression may provide valuable insights into the molecular mechanisms involved in determining FMDV tissue tropism. We designed a custom bovine whole genome expression microarray and used microarray analysis to identify genes differentially transcribed among tissues targeted and not targeted by FMDV for primary, secondary, and persistent infection in infected and non-infected cattle. The bioinformatic analyses of the differentially expressed genes lead to the identification of several biological processes/pathways which activity could be affected by the differential expression. Based on the expression of the genes involved in these biological processes and on relevant findings from the literature, we propose molecular bases that help explain the mechanisms of FMDV tissue tropism.All animal studies were conducted under an approved Institutional Animal Care and Use Committee protocol. Five Holstein steers weighing 225 to 300 kg were obtained from an accredited experimental-livestock provider . Two of the steers were aerosol-inoculated as previously described Selected tissues were collected based on pathogenesis studies as previously described http://genome.ucsc.edu/cgi-bin/hgGateway). The DNA probes for all available FMDV genome sequences were also designed and included in the array to detect FMDV RNA levels.A bovine whole genome expression microarray was designed based on all bovine expressed sequence tags and RNA sequences from the NCBI database and assembled into unique sequences with the CAP3 program The custom bovine microarrays used in this study were manufactured by Agilent technologies. Agilent Quick Amp RNA labeling kit  was used for the sample labeling. Total RNA prepared from a metacarpal skin sample was labeled with Cy5 and used as a universal control for cross-array data normalization, whereas all samples including 8 tissues from each animal (40 samples) were labeled individually with Cy3. Each Cy3 labeled sample together with the control was hybrided to a microarray. The entire procedure from RNA quality check to microarray hybridization and washing was conducted using reagents and protocols provided by Agilent Technologies. The microarrays were scanned at 5 µm resolution with Cy3:Cy5 signal ratio of between 0.9 to 1 using a GenePix 4000B scanner and GenePix Pro 6.0 program .http://www.genomine.org/edge/edgehelp.pdf) to avoid statistical analysis involved with division by zero. The normalized signal data were square-root-transformed and analyzed using t-test. Differentially expressed genes (DEG) were defined as having a difference of ≥75% or 1.75 fold with a P value of 0.05 or smaller in all pair-wise two tissue comparisons between tissue groups. The significant differences obtained from bovine sequences without known human homologous genes were not included in the gene sets and were excluded from further analysis. Eight gene sets including up- and down-regulated in non-targeted sites (NTS) vs targeted sites (SRS+PRS), up- and down-regulated in secondary replication sites (SRS) vs other tissues (NTS+PRS), up- and down-regulated in persistent infection sites (PIS) vs others (NTS+SRS+LNG), and up- and down-regulated in primary replication sites (PRS) vs nasal turbinate epithelium comparisons were created from data obtained from three not infected animals and identified as NTS-up, NTS-down, SRS-up, SRS-down, PIS-up, PIS-down, PRS-up, and PRS-down, respectively . The data of extracted gene expression signals were background-corrected with the average signals of nearby negative control features. All negative intensity readings were adjusted as 0. The expression data were normalized with the signal of Cy5-labeled universal control to have equal averaged signal intensity. After normalization, all expression data were added with an arbitrary small value of 1 as suggested in the EDGE program user manual  using a subscribed account to identify the pathways/networks that can regulate the differential expression by analyzing over-represented transcription factor binding sites in the promoter sequences of the identified DEG and can be significantly affected by the differential gene expression using ExPlain™ program . To identify over-represented transcription factor binding sites, all bovine gene sequences used to design the microarray probes were mapped to human genes based on the result of BLAST or annotation from the bovine genome sequence displayed in the UCSC genome browser. The promoter sequences in −500 and +100 bases of transcription starting sites of human genes homologous to the identified bovine DEG were compared to a human house-keeping gene set provided by BIOBASE as a control. The over-represented transcription factor binding sites not shared in compared gene sets were considered as the sites of interests, and signal transduction pathways that can activate transcription factors to bind to these sites are considered to regulate the differential expression. To detect biological networks that may be significantly affected by the differential gene expressions, the gene sets were analyzed to identify those with significant more DEG than that may occur randomly  using statistical tests included in the ExPlain™ program. Both up- and down-stream networks of key nodes were analysis to identify DEG regulating and regulated by the key nodes, respectively.The gene sets were submitted to a BIOBASE web site (P = or <0.05) were analyzed with t-test. Biological impact of differential gene expression was evaluated based on known biological functions of the genes with an assumption that higher transcription levels are associated with higher biological activity of the genes. The tissue activity of indentified pathways/networks was determined based on the expression levels and functions of the genes in the pathways. Biological inferences were made according to the impact of predicted differences in the activity of the pathways on FMDV infection. The data from three non-infected and two infected animals were used to make the inferences.Once candidate pathways/networks were detected, all genes known to be involved in these biological processes were identified and their expression levels were examined in terms of up- or down-regulation and the magnitudes of the differences between tissue groups. Significant differences  at 2–3 days post infection and vesicles in the mouth and/or feet detected at 3 days post infection indicating that both animals were at the start of generalized FMDV infection when euthanized for tissue collection. Steer #1 had vesicles in the interdigital cleft of one foot and in the tongue, whereas steer #2 had vesicles on all 4 interdigital clefts. No other lesions were identified. Both animals were subsequently confirmed to have FMDV and viral RNA  in sera (viremia) and in nasal secretions at the time of euthanasia.Varying signal intensities of FMDV microarray features were observed in the infected tissues , whereasWhole genome gene expression profiles of the tissues from the three naïve animals were compared across four tissue categories selected according to their classification as not targeted sites, primary infection, secondary replication, or persistence sites. Tissue categories were based upon previously published studies describing tissue-specific FMDV loads as stated earlier. Eight gene sets were created from the tissue comparisons based on the statistical analysis described earlier . In the Integrin β subunits may compete with each other for ITGAV if the expression of ITGAV is a limiting factor. Our results show that the signal intensity of ITGAV was significantly lower than the total of all RGD-binding integrins β . TherefoAlthough the expression of ITGAV was not significantly different among tissues, the SRS expressed higher ITGB6 and lower FN1 than other tissues in infected and non-infected animals . The expBecause there were less than 5 genes in the NTS-down and PIS-down gene sets, only other six gene sets were used in the bioinformatic analysis to infer biological mechanisms of FMDV tissue tropism. Analysis of the promoter sequences of the DEG identified 33 over-represented transcription factor binding sites . Six of Using the ExPlain™ program to identify networks that may be significantly affected by the differential expression, we detected several networks relevant to those identified using the promoter sequences of the DEG and to FMDV infection. Several network key nodes associated with type I interferon signaling were detected in the analysis using the SRS-down , PRS-down (IFNα), and PRS-up  gene sets, whereas some involved in IL-1 signaling were found using the SRS-down , PRS-down (p38α), and PRS-up  gene sets . In the Cell attachment involves two types of interactions; cell-ECM and cell-cell adhesion. Cell-ECM interaction is mediated by RGD-binding integrins and regulated by urokinase plasminogen activator receptor (PLAUR or uPAR)-integrin pathways When the expression of these individual genes was normalized to be equally weighted, the SRS expressed significantly higher activators and lower inhibitors of ECM degradation than the negative control tissue, whereas the PRS had significantly lower expression of the inhibitors than their negative control tissue in the not infected animals and the activators were expressed significantly higher in the lung than the control in the infected and non-infected animals. These results indicate that tissues susceptible to primary and secondary infection have gene expression profiles promoting higher ECM turnover.In order to evaluate the impact of differential IL-1 cytokine expression, we examined the expression ratios of agonist(s) vs their antagonist(s) instead of the individual genes according to Johnston et al. We examined the expression of genes in the interferon JAK-STAT and PI3K-AKT pathways  according to Kroczynska et al. http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) based on the functions discussed by Saddler and Williams To compare the status of interferon signaling pathways among tissues, 48 interferon-inducible antiviral effector genes were identified from the Gene Database , has been reported to play critical roles in the clearance of viruses by specifically targeting infected cells to induce apoptosis and/or necrosis and by facilitating cell-mediated cytotoxic immune response In conclusion, we have identified differential gene expression at a genomic scale between tissues with different FMDV tropism. FMDV tropism could not be explained by the differential expression of a single gene alone. The bioinformatic analysis of the genes differentially expressed led to identify a few biological processes or pathways. Further examination of genes in those relevant to FMDV infection according to reported findings provide the molecular basis that could explain, at least in part, the mechanisms of why FMDV targets specific tissues for primary, high-titer secondary and persistent infection and causes vesicular lesions. Based on these results, we hypothesize that multiple contributory factors including increased FMDV receptor availability and accessibility resulted from high ECM turnover and differential FMDV receptor and the ligand expression, reduced antiviral innate immunity due to low type I interferon response and/or type I interferon production, and decreased ability to clear infected cells via death receptor signaling play roles in determining FMDV tropism. We also hypothesize that additional increase in already high tissue ECM turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, plays a key role in the pathogenesis of vesicular lesions. This work provides guidance for future research to rationally test these factors and further elucidate the molecular mechanisms of tissue tropism for FMDV. The approaches used in this study may also be applied to understanding tropism of other viral agents.


Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed.Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF-SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF-SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes  was not effective. By using a recently developed technique of a two step  Nano-sized particles are finding increasing practical applications and commercial use. Consequently, the possibility of occupational exposure to nano-particles has increased. Furthermore, as the use of nanoparticles increases, they may diffuse and accumulate in environment increasing the risk of environmental exposure. Health effects of exposure to nanoparticles are now under investigation by many researchers in vitro as well as in vivo. AF-SWCNTs were found to bind with erythrocytes and cause significant membrane damage as well as externalization of phosphatidylserine. Administration of AF-SWCNTs intravenously induced a significant though transient anemia in mice. Older as compared to the younger subpopulation of erythrocyte in blood circulation, was more susceptible to the toxic effect of AF-SWCNTs. Taken together our results show that non-agglomerated form of SWCNTs may exert significant and rapid toxic effects on blood erythrocytes.Since AF-SWCNTs were toxic to some types of nucleated cells that are capable of self repair, we hypothesized that the AF-SWCNTs may have a more aggravated toxic effect on erythrocytes that have no nucleus and lack substantial cellular repair mechanisms. This proposition was examined in the present study. Our results indicate that unlike pristine SWCNTs, AF-SWCNTs are significantly toxic to mouse blood derived erythrocytes ad libidum. All the experimental protocols were approved by JNU Institutional Animal Ethics Committee (IAEC Project Code: 5/2010) and performed accordingly.Inbred Swiss and C57BL/6 female mice  were used throughout this study. Animals were bred and maintained in the animal house facility at JNU, New Delhi or obtained from the National Institute of Nutrition, Hyderabad. The animals were housed in positive-pressure air conditioned units  and kept on a 12 h light/dark cycle. Water and mouse chow were provided For in vitro studies, erythrocytes derived from mouse blood were suspended in RPMI complete medium (RPMI-CM) containing glutamine (2 mM), HEPES buffer pH 7.2 (25 mM), gentamycin (20 µg/ml) obtained from Sigma-Aldrich (India) and fetal bovine serum, obtained from Hyclone . ANS  was obtained from Sigma-Aldrich (India). Alexa fluor 488/633 hydrazide was obtained from Molecular Probes . Biotin-X-NHS Ester (BXN) was from Calbiochem  and Streptavidin Allophycocyanin (SAv-APC) and Annexin-V PE were obtained from BD biosciences . Single-wall carbon nanotubes (SWCNT's) were purchased from Sigma . Similar results were obtained for SWCNTs preparations procured from  and their derivatives.3:H2SO4 in 100 ml high-pressure vessels in a microwave digester as described previously 2O and dialyzed four times against 5 liter water over a two day period. Dialyzed suspensions were freeze dried, weighed and resuspended in 5 ml water. Particle size distribution and surface charge on AF-SWCNTs were as reported before Acid functionalized SWCNTs (AF-SWCNTs) were produced by suspending 20 mg of powder SWCNTs in 20 ml of 1∶1 concentrated HNOFor covalent attachment of fluorescent probes to the nanotube surface, -COOH groups on AF-SWCNTs were exploited. AF-SWCNTs were suspended in water and treated with 1-ethyl 3-(3-dimethyl aminopropyl) carbodiimide (EDAC) and N-hydroxy succinimide (NHS) in order to get a succinimidyl intermediate. The mixture was continually shaken for 2 hrs and dialysed thereafter to remove excess NHS, EDAC and urea by-product. AF-SWCNTs thus activated were incubated with Alexa Fluor 488/633 hydrazide  in dark with continuous mixing, followed by dialysis to remove free dye Attachment or internalisation of AF-SWCNTs was confirmed by incubating erythrocytes with alexa fluor 488/633 tagged AF-SWCNTs, as described previously max = 365 nm) using a Fluorimeter (Spectramax M2e). Fluorescence spectra were scanned from 400 to 600 nm. The excitation and emission band passes were 5.0 nm in width.Hydrophobicity of the erythrocytes was determined by staining with 8-anilino-1-naphthalenesulfonic acid (ANS) as described before in vivo studies, mice were given 100 µg AF-SWCNTs or saline control through intravenous, intraperitoneal, gavage and intratracheal routes and blood samples were taken at several time points thereafter. Blood erythrocyte count was determined by automated hematology analyser .Erythrocytes were suspended in RPMI 1640 supplemented with 10% Fetal bovine serum and incubated with control and AF-SWCNT's at different concentrations over rotisserie (17 RPM) at 37°C for different time intervals. For In vivo biotinylation of circulating erythrocytes was done as described previously high population, age>40 days), intermediate , and young  populations of erythrocytes. Biotin high, low and negative erythrocyte populations could be identified and enumerated by staining with streptavidin-APC followed by flow cytometry as described before 6 erythrocytes were stained with streptavidin allophycocyanin (APC) as described before 2) and annexin-V PE for 20 min at 25°C in dark. Stained erythrocytes were immediately analysed on FACS Calibre flow cytometer  using Cell Quest software for acquisition and analysis. A minimum of 10,000 events were recorded for each sample.To enumerate erythrocytes with high, low or no biotinylation, 1×10Each experiment was repeated at least three times. Statistical analysis by two-way ANOVA and student's t-test were done using Sigma plot and Sigma stat software. Data are presented as means ± SEM.The effect of control and acid-functionalized SWCNTs was examined on murine erythrocytes. For this purpose, blood erythrocytes derived from female Swiss or C57BL/6 mice were cultured with control and acid-functionalized SWCNTs. To facilitate the interactions between carbon nanotubes and erythrocytes, the culture tubes were gently rotated (17 RPM) throughout the culture duration. Erythrocyte recoveries at different time points are shown in in vitro for 1 h and analysed on flow cytometer before and after washing the erythrocyte preparations. Results in For examining the interactions between AF-SWCNTs and erythrocytes, AF-SWCNT preparations tagged with fluorescence probe were used. Erythrocytes freshly derived from female C57BL/6 mice were incubated with fluorescence tagged AF-SWCNT particles at 50 µg/ml Exposure of hydrophobic regions in erythrocyte membrane is associated with cell damage Phosphatidylserine (PS) externalisation on cell membrane is an early marker of apoptosis and cell death in vivo too, mice were administered control or acid functionalized SWCNTs through different routes and the erythrocyte count in blood was monitored. Results in In order to determine if AF-SWCNTs exerted toxic effect on erythrocytes Results above show that treatment with AF-SWCNTs resulted in a transient anemia in mice. A second dose of AF-SWCNTs given 24 h after the first dose resulted in a more sustained anemia that was not alleviated even at 72 h time point . Adminisin vivo biotinylation of circulating erythrocytes, erythrocytes of different age groups can be identified as a biotin negative population , a biotin high population  and a biotin low population (erythrocytes of age between 10 to 40 days). Results in Results so far indicate that the intravenous administration of AF-SWCNTs caused a marked and transient anemia in mice. It was of interest to determine if the toxic effect of AF-SWCNTs was generalize or was selective for erythrocytes of specific age groups. To determine the susceptibility of erythrocytes of different age groups, we used a technique that we recently developed to enumerate circulating erythrocyte cohorts of different age groups in vitro. The effect was dose and time dependent and after one day of incubation with AF-SWCNT, 70–90% of the erythrocytes were lost at all the doses  of AF-SWCNT. It was also interesting to observe that the damaging effect is seen earlier with the highest dose of 50 µg/ml leading to 70–90% decline in recovery after just 10–12 h while interaction ensures the similar damaging effect on erythrocytes treated with lower doses also (10 and 25 µg/ml) when kept for a longer duration (24 h). It should be noted that the process to generate AF-SWCNTs involved extensive dialysis for several days and it is therefore unlikely that some material leaching from AF-SWCNTs was responsible for the toxic effect on erythrocytes. To further test this possibility, we separated AF-SWCNTs from erythrocytes by a dialysis membrane  during incubation and found that no loss of erythrocytes occurred (results not shown). Interestingly control SWCNTs had no significant effect on survival of erythrocytes. The reason for this lack of effect of control SWCNTs could be that the control SWCNTs preparations are highly agglomerated and do not interact effectively with cells. These results corroborate our previous findings on the effects of control and acid functionalized SWCNTs on LA4 lung epithelial cells lines where too the AF-SWCNTs induced a marked cellular damage but control SWCNTs lacks a significant cytopathic effect Our first set of experiments indicated that the AF-SWCNTs had a marked toxic effect on erythrocytes Using fluorescence tagged AF-SWCNTs, we could demonstrate a significant binding of AF-SWCNTs with erythrocytes. Flow cytometric analysis indicated that almost 70% of erythrocytes incubated with fluorescence tagged AF-SWCNTs bound nanotubes. Further confocal microscopic studies suggested that some AF-SWCNTs could enter the erythrocyte also. Since an active uptake of AF-SWCNTs by erythrocytes appear unlikely, the observed association of AF-SWCNTs could reflect loose binding of AF-SWCNTs with erythrocyte membrane or a stronger binding perhaps reflecting embedding of nanotubes in erythrocyte membrane or even some passive entry of the nanotubes into the cells. Our results showing that 18% of the erythrocytes retained the association with fluorescence tagged AF-SWCNTs even after three rigorous washings suggest that AF-SWCNTs may interact effectively with erythrocytes.Membrane damage as well as PS externalization seen in response to treatment with AF-SWCNTs could be a direct consequence of interactions of the AF-SWCNTs with erythrocytes. Erythrocytes treated with AF-SWCNTs were found to have a marked increase in ANS binding within one hour of treatment indicating that the membrane damage in erythrocytes may be early consequence of interaction with AF-SWCNTs. Erythrocytes treated with AF-SWCNTs also displayed increasing externalization of PS in the membrane that was related to the dose and time period of treatment. While these results clearly show a response similar to apoptotic response in nucleated cells, it is not clear from our results if PS externalization alone lead to the death/removal of erythrocytes. We also examined the AF-SWCNT induced erythrolysis by time lapse micrography  and founin vitro studies established the toxic effect of AF-SWCNTs on erythrocytes, which was further confirmed by in vivo studies. Intratracheal and intravenous administration of AF-SWCNTs induced transient anemia in mice. As observed in vitro, control SWCNT preparations did not result in decrease in erythrocyte count. Interestingly, intra-peritoneal and intra-gastric administration of AF-SWCNTs didn't reduce the RBC count too. The drop in erythrocyte count in blood seen in case of intravenous administration of AF-SWCNTs could be due to an efficient interaction of the nanotubes with erythrocytes in circulation. In all other forms of in vivo treatments, nanotubes were not deposited into blood circulation directly. As a result, the immediate physical contact of nanotubes with erythrocytes that was possible in case of intravenous administration only, would not have taken place. Significant anemia in response to intratarcheal administration of AF-SWCNTs could be a result of the relatively easy diffusion of nanotubes through thin alveolar membranes as reported earlier also Results of Acute anemia is known to stimulate the release of erythrocytes from spleen and bone marrow in order to compensate for the loss of erythrocytes in vitro experiments. It was important to understand whether the cytotoxic effect of nanotubes was exerted uniformly on all erythrocytes in circulation or it was related to the age of erythrocytes in circulation. This question could be addressed by help of a technique involving two steps of in vivo biotinylation of circulating erythrocytes that has recently been developed in our laboratory. By using this technique it became clear that the oldest subpopulation of erythrocytes (age > 40 days) was specifically sensitive to nanotubes. Blood count of young erythrocytes (<10 days) actually increased after administration of AF-SWCNTs. This increase could be a consequence of release of fresh erythrocytes into circulation from spleen and bonemarrow. Interestingly the erythrocytes population of intermediate age group (10–40days) didn't change in response to AF-SWCNTs. These results suggest that the older erythrocytes in circulation that have become weakened by oxidative damage may be easier targets for the nanotubes. It should however be stressed that even though greater susceptibility of older erythrocytes to AF-SWCNTs is indicated by the in vivo experiments, a more generalized erythrolysis observed in vitro  and were placed on the observation platform of a live cell imaging (Nikon Eclipse Ti) microscope in a chamber maintained at 37C in 5% CO2 atmosphere. Cells were allowed to settle for 30 min before recording was started.(AVI)Click here for additional data file.


Baseline offset errors on phase-contrast velocity images can be corrected using stationary tissue, for example subtracting fitted corrections from the image (1). Although corrections are often curved over the FOV, 1st order (linear) fitting is typical. This may partly be due to low SNR of static tissue making higher-order fitting unreliable (2). Aim: To evaluate a new method acquiring additional high SNR velocity images specifically to improve offset correction.A retrospectively-gated cine phase-contrast sequence was edited so that the RF pulse was 25% of its normal duration, with all other pulse timings and amplitudes unchanged. This excites a thicker slice in the patient, increasing the SNR but without changing the offset error. The modified sequence used an extra breath-hold after the original clinical flow sequence, on 18 vessels in 10 patients. The high-SNR image was not for vessel flow measurement, only baseline correction (Figure Compared to the clinical scan Figure  left, thDiscussion & Conclusions: The new high-SNR method improved fitting of higher order surfaces for offset correction, but with marginal improvement of accuracy. For some protocols and scanners with larger uncorrected offsets and/or lower SNR in the clinical flow study, this method might have more impact. The reduced stdev implies that this new method may improve reliability. However, with this data the improved accuracy of correction was insufficient to justify the extra breath-hold with each phase-contrast scan.Cardiovascular Biomedical Research Unit funding from the National Institute for Health Research.


Basilar artery dolichoectasia, ectasia of internal carotids and intracranial aneurysm have been described in late-onset glycogenosis II (GSD II). Incidence of these anomalies and their relationship to the genotype are unknown. Also, because studying cerebral vessels is not recommended for diagnosis of late-onset GSDII, the frequency of these anomalies may be underestimated. Therefore, we investigated the occurrence of vascular anomalies in eight siblings  of an Italian family affected by late-onset GSDII due to a compound mutation (c.118C>T in exon 2 and c.2647-7G>A in intron 18) of the acid α-glucosidase (GAA) gene (17q21-23).The clinical picture in these siblings is typical of late-onset GSDII: Patients never complained of symptoms of cerebro-vascular disease and none had cardiac involvement. Blood pressure and blood glucose were normal. Brain Magnetic Resonance Angiography  was performed in 7  out of 8 patients , and in 10 controls. Vessel total square diameter was measured at the apex and middle third of the basilar artery and at the middle portion of both right and left internal carotid artery. Blind measurements were carried out by three investigators. Basilar artery dolichoectasia  was found in 3M and 2F, and a slight dilatation of internal carotids compared to controls was found in all patients.These data confirm that anomalies of main cerebral vessels are relatively frequent in late-onset GSDII patients. The role of genotype in determining these anomalies is uncertain because studies on informative families are rare. Particularly intriguing is the relation of these vascular anomalies with the risk of stroke in these patients. Even though all have ventilatory insufficiency with reduced blood oxygen saturation, nocturnal apnea, and impaired glycogen metabolism, a minority suffer TIA or stroke. Therefore, an imaging study of the cerebral circulation is recommended in all patients with late-onset GSDII. In those with anomalies, further research is needed to evaluate cerebral metabolism and to search for specific vascular risk factors.


Cortical changes associated with cognitive decline in Parkinson's disease (PD) are not fully explored and require investigations with established diagnostic classification criteria.We used MRI source-based morphometry to evaluate specific differences in grey matter volume patterns across 4 groups of subjects: healthy controls (HC), PD with normal cognition (PD-NC), PD with mild cognitive impairment (MCI-PD) and PD with dementia (PDD).We examined 151 consecutive subjects: 25 HC, 75 PD-NC, 29 MCI-PD, and 22 PDD at an Italian and Czech movement disorder centre. Operational diagnostic criteria were applied to classify MCI-PD and PDD. All structural MRI images were processed together in the Czech centre. The spatial independent component analysis was used to assess group differences of local grey matter volume.We identified two independent patterns of grey matter volume deviations: a) Reductions in the hippocampus and temporal lobes; b) Decreases in fronto-parietal regions and increases in the midbrain/cerebellum. Both patterns differentiated PDD from all other groups and correlated with visuospatial deficits and letter verbal fluency, respectively. Only the second pattern additionally differentiated PD-NC from HC.Grey matter changes in PDD involve areas associated with Alzheimer-like pathology while fronto-parietal abnormalities are possibly an early marker of PD cognitive decline. These findings are consistent with a non-linear cognitive progression in PD. Dementia is highly prevalent in the advanced stages of Parkinson's disease (PD) with important consequences for quality of life of affected patients and caregivers. Imaging evidence suggests an association with discrete regional grey matter abnormalities, but the extent and topographical distribution of such changes at initial stage of cognitive decline are still unknown. Understanding the underlying mechanism of the early cognitive changes in PD is important for diagnosis and prognosis, and ultimately for developing drug treatment to slow progression to dementia MRI analysis methods may also contribute to the results inconsistency. The source based morphometry (SBM) is a newly established multivariate technique that has been shown to be superior to mass-univariate methods using either voxel or cluster level of inference in the identification of specific between-group grey matter changes In order to shed further light on the specific grey matter volume (GMV) changes in well-defined cognitively impaired subjects with PD, we used the SBM analysis and examined a relatively large sample of 126 PD patients including PD with normal cognition (PD-NC), MCI-PD and PD with dementia (PDD) groups using recent MDS criteria. Findings were compared with cognitive functioning based on a comprehensive battery of tests.We examined altogether 151 participants, including 25 HC, 75 PD-NC, 29 MCI-PD, and 22 PDD at two movement disorder centres: an Italian centre  and one Czech centre . A series of 89 consecutive PD patients  and 10 HC were recruited in the Italian Centre. In the Czech Centre, 37 PD patients  and 15 HC were consecutively enrolled. All PD patients were diagnosed with idiopathic PD according to UK Brain Bank criteria The study was approved by the ethics committee of the IRCCS San Camillo, Venice, Italy and the local ethics committee in Brno, Czech Republic. Written informed consent was obtained from all participants.At the Italian centre, all participants performed a comprehensive neuropsychological evaluation including assessment of attention/working memory, executive functions, memory, language and visuo-spatial functions  PDD was diagnosed according to the Movement Disorder Society criteria Italian partners utilized the Level II (comprehensive assessment) operational schema according to MDS criteria Whole head scans were acquired at the Czech centre  and at the Italian centre .http://www.fil.ion.ucl.ac.uk/spm) with its internal toolbox DARTEL. Data were segmented into grey and white matter segments and registered using DARTEL to the study specific template. Resulting grey matter volume images were resampled to the resolution of 1.5×1.5×1.5 mm and smoothed with 8 mm FWHM Gaussian kernel.All images were combined into one cohort and processed in the Czech centre (by RM) using SPM8 software (3) were superimposed on the study-specific grey matter images to visualize regions with strong inter-group differences.The method uses spatial Independent Component Analysis (ICA) to reveal naturally grouping spatially independent sources of local grey matter volume variability with common co-variation among subjects. Initially the grey matter images were concatenated and reshaped to form 2D matrix with number of voxels and number of subjects as matrix dimension. Prior to ICA, Principal Components Analysis (PCA) was done to reduce the data dimensionality with the Minimum Description Length algorithm in order to estimate the optimal number of components Pearson correlation analyses were used in order to assess relationship between components-of-interest expressions and cognitive tests Z-scores in the Italian cohorts. The multiple testing problem was handled by FDR and the significance level was set to p<0.05. = 16.2, p = 0.001]) and education  = 14.3, p = 0.003]) between cognitive groups, but not for gender . Detailed cognitive tests z-scores  and ACE-R domains z-scores (Czech centre) for all groups are displayed in Subjects' characteristics are presented in  = 12.4, p = 0.0063) represented areas of GMV reduction particularly in the bilateral temporal lobes including the inferior temporal gyrus, hippocampus, parahippocampal gyrus, amygdala, fusiform gyrus, and medial and superior temporal gyri, as well as areas of GMV increases in the posterior lobes of both cerebellar hemispheres  = 13.2, p = 0.0042) represented areas of bilateral grey matter reduction in the medial frontal and inferior parietal regions, as well as areas of GMV increases in the brainstem and cerebellum  copy  involved particularly the GMV reductions in the hippocampus, amygdala and neocortical temporal regions, while the second (C2) involved GMV major deviations in the fronto-parietal regions and the pons/cerebellum. The temporal lobe atrophy pattern was related to impairment in visuo-spatial abilities in PDD, which was in fact the most affected cognitive domain in this group. The inferior and medial temporal cortex is the final route of the ventral visual pathway Our C2 pattern consists of both GMV decreases in the middle frontal gyrus and inferior parietal lobules including the supramarginal gyrus  The C2 pattern differentiated PDD from all other groups and was associated with impairment in letter verbal fluency task. In addition, the C2 pattern differentiated PD-NC from HC. Of note, HC participants were significantly better educated than the PC-MCI subjects which might indicate a higher level of cognitive reserve in this patient group To our knowledge this is the first time that metabolic changes assessed by the FDG-PET are replicated in a structural MRI study. In that study the authors differentiated multiple-domain PD-MCI from PD-NC but not single-domain PD-MCI from PD-NC While it is well accepted that regional metabolic decreases and increases can coexist in PD, it may be more difficult to explain the GMV increases observed in the infratentorial regions. Unfortunately, underlying mechanisms for these changes cannot be directly answered and resolved by structural MRI.Using fMRI, several studies have shown that L-dopa may modify (increase) connectivity of specific brain networks Our aim was to investigate GMV deviations associated with cognitive decline in PD and in this regard results of a recent study by Biundo et al. Here we show for the first time that both deficits and compensatory mechanisms may be involved in PD patients who do not yet meet the criteria for PD-MCI. Interestingly, these patterns become more pronounced at later stages of cognitive decline – when dementia develops. Although this is a novel morphological result in PD population it accords well also with functional results described in MCI-AD and AD-dementia patients Finally, it has to be acknowledged that both grey matter and white matter tracts are present in the pons as well as in the cerebellum. Therefore, our result of increased GMV in these regions should be considered with care, as different microanatomical factors could be related to described changes We have to acknowledge our study has limitations. Even if both centres followed operational guidelines to diagnose MCI-PD we applied different instruments for cognitive testing. The respective groups of participants  differed with respect to their age and years of education. We controlled for these variables in the second-level analysis when comparing the groups. Nevertheless, the possible effects of age and length of education cannot be fully excluded. We also have to acknowledge an unbalance in the number of participants in specific subgroups . This discrepancy in sample sizes could have lowered the power of the Kruskal-Wallis test.We report correlations between the MRI component loadings and specific cognitive outcomes in the Italian PDD groups which are small in size. Although the correlations support our hypothesis the results cannot be generalized. The strength of our study include comparison of a fairly large number of controls and PD patients at well-defined stages of cognitive decline, application of the novel consensus criteria for PD-MCI along with a comprehensive neuropsychological battery for the largest centre. Finally, our SBM analysis is a novel method allowing identification of patterns of grey-matter loss which may more closely mirror the actual changes occurring in PD compared to traditional univariate voxelwise analyses.Future prospective studies will be required to investigate whether these structural patterns can be used as biomarker of cognitive progression.