If the products of these cells are toxic to developing larvae (L3 and L4 stages) of hookworms, this may explain why acquired immune responses are particularly effective against invasive stages, compared with adult worms. Finally,

selleck kinase inhibitor the experiment described in this paper, has made novel contributions to our understanding of the events that occur in the mucosa during both primary and secondary infections with the hookworm, A. ceylanicum in the hamster model. This model is being exploited in the quest to develop a protective vaccine for hookworms and the current data will help to inform those studies, particularly when evaluating the outcome of vaccine trials in the hamster model (35–37). The results reported here raise interesting questions about the role of Paneth cells, and about how Th2-driven intestinal inflammatory responses are controlled by cytokines and regulated by the parasites, ABT888 given the contrasting dynamics of the initial appearance of the different cellular compartments and of their return to baseline levels once the worms have been removed. We thank the Ministry of High Education of the Kingdom of Saudi Arabia for the provision of a postgraduate studentship

for LMMA. We are also grateful to Dr. Catherine Lawrence and Prof. P. Garside for training provided for LMMA in the initial stages of this project. “
“Recently, Sirtuin 1 (SIRT1) has been implicated in the molecular control of ageing and immune response. Although the remodelling of periodontal ligament (PDL) in response to mechanical stress (MS) is mediated by several host factors, including cytokines and chemokines, the transmission of mechanical stimuli into specific cellular activity is still not understood fully. This study aimed to investigate the effects of MS, particularly cyclic strain, on immune response genes, as well as SIRT1 and its signal transduction pathways, in human PDL cells. MS up-regulated the expression Obatoclax Mesylate (GX15-070) of SIRT1 and immune response genes encoding cytokines [tumour necrosis factor

(TNF)-α, interleukin (IL)-1β], chemokines [IL-8, monocyte cheoattractant protein (CCL)-20], defensins [human β-defensin (hBD)-2, hBD-3] and Toll-like receptors (TLR-2 and TLR-4) in a force- and time-dependent manner. The SIRT1 inducers resveratrol and isonicotinamide attenuated MS-induced cytokine and chemokine expression, but enhanced the expression of defensins and TLRs. Blockade of SIRT1 activity by the SIRT1 inhibitors sirtinol and nicotinamide and down-regulation of SIRT1 expression by SIRT1 siRNA reduced the stimulatory effects of MS on defensins and TLRs, but increased its effects on cytokines and chemokines. MS induced activation of protein kinase B (Akt), protein kinase C (PKC), nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK).

The reticular formation is a network of loosely packed, multipolar neurons located inside the brainstem, from the mesencephalon through the pons to the medulla oblongata. The reticular formation provides a transition between ascending Akt inhibitor and descending motor and sensory pathways.[10] The reticular formation helps with automatic regulation of heart rate variability, breathing, and the process of digestion. Reticular formation also helps to regulate the processes of micturition and defecation.[28] In addition, the reticular formation regulates brainstem reflexes, such as blinking, masseter,

and pharyngeal.[10, 11] Pontine reticular formation can be divided into a lateral and a medial tegmental field. The L region is located more ventrally and more laterally in the pontine tegmentum than

the M region, overlapping the AZD6738 cost lateral pontine tegmental reticular formation.[29] The lateral tegmental field also houses the premotor neurons, with long descending axons to the motor neurons of the spinal cord that are involved in micturition.[28] Detrusor relaxation and external sphincter contraction are elicited by electrical stimulation of the lateral tegmental field of the pontine reticular formation.[15, 29, 30] R1 is relayed through an oligosynaptic path, consisting of one or two interneurons;[31] however, R2 are relayed through a polysynaptic path, including neurons in the reticular formation.[32] For the R2, trigeminal sensory impulses are relayed by a pathway that ascends bilaterally to the facial nuclei in the pons. These trigeminofacial

connections pass through the lateral pontine tegmental reticular formation,[27, Liothyronine Sodium 33] from where L region are thought to overlap. In monkeys, microstimulation of the pontine lateral tegmental field suppresses reflex blinks.[34] Another pontine region that is responsible for storage is found in the pontine reticular formation ventral to the M region known as nucleus reticularis pontis oralis.[35] It is suggested that this nucleus takes a role in modulating both micturition and blink reflex. Increased R2 times and urgency have been shown in a patient with lesion of the nucleus reticularis pontis oralis.[36] In the present study, all of the late blink latency times were found to be longer in patients with OAB. The early blink latency times were not significantly different between the groups. Based on these neuroanatomical and possibly functional relationships between blinking and micturition; assessing significant differences in only the late blink reflex latency times suggests that the increase in both the late blink latency times and the storage symptoms may originate from a brainstem structure that can mediate both micturition and the late blink reflex. This structure seems to be the pontine reticular formation. However, it may derive from rostral pontine reticular formation or L region.

In our study the HLA-B*4403/07/13 was present only in the HIV-seronegative couples, while 4402/11/19 and 4405 was the most frequent among HIV-1+ couples. It is important to emphasize that the three B*44

alleles found in discordant HIV+ partner pairs were homozygotic for KIR3DL1. The combination of KIR3DS1/KIR3DL1 with the HLA-B*4403/07/13-Bw4 ligand was MK-8669 solubility dmso not present in HIV-1+ partners. These results would support those of Macdonald et al.,[25] who comment that cytotoxic T lymphocytes discriminate between HLA-B*4402 and B*4403. Polymorphism between HLA-B*4402 and B*4403 modifies both the peptide repertoire and T-cell recognition. Alter et al.[11] performed in vitro tests to examine the functional ability of NK cells to differentially control HIV-1 replication in vitro based on their KIR-HLA types. Functional testing should be performed with AZD9291 ic50 specific HIV-1 peptides to establish the true participation of the alleles B*4403 and A*32 . Herman et al.[26] conclude that the B44 specificity of T cells results mostly from distinct conformations adopted by the same peptides in the two B44 molecules. They found several peptides, different from the three mentioned above, that contain the canonical HLA-B44 binding motif and bind to B*4403 but not to B*4402 molecules. This was consistent with the stronger T-cell alloreactivity toward B*4403 in comparison

with B*4402. Numerous observations suggest that CD8+ T cells play an important role in constraining infection. We can add that there might be selective expression of activating and inhibitory KIR depending on the HLA GNA12 alleles in each individual. If KIR gene evolution were pathogen-driven, some diversity would be expected to correlate with resistance or sensibility to certain infectious diseases. This study observes that KIR3DS1(3DS1/3DL1) could have a greater effect on protection against HIV-1 infection in HESN individuals

when linked to a specific HLA allele, in this case HLA-A*32 and HLA-B44, both Bw4. Besides KIR3DL1/KIR3DL1 homozygosity could be considered as a risk factor in the susceptibility to HIV infection. These results could add epidemiological data to the understanding of complex KIR-HLA interactions that trigger different responses to the disease, depending upon genetic characteristics of studied population. We thank the Director of the ‘Hospital Dr. Julio C. Perrando’ for facilitating this work, and Maria Leonor Santa Cruz, Licenciada en Trabajo Social, for locating the patients and individuals who participated in this study (Servicio de Infectología. Hospital Dr. Julio C. Perrando). We also extend our thanks to Hector Fernandez for technical support (Servicio de Genética Molecular e Histocompatibilidad Hospital Dr. Julio C. Perrando). The authors have declared that no competing interests exist.

4). To confirm the possible role of TCR in the increase in IL-9+ IL-10+ T cells, a group of DO11·10 mice was pretreated with anti-TCR α-chain antibody (500 ng/mouse, daily, intraperitoneally for 1 week. The expression of TCR in T cells was exhausted as examined by flow cytometry; data not shown). The mice were then treated with OVA (1 mg/mouse) daily for 3 days. Indeed, the frequency of IL-9+ IL-10+ T cells was not increased, which was not significantly

different from naive mice (Fig. 4). The results indicate that TCR activation Selleck Roxadustat plays an important role in the induction of IL-9+ IL-10+ T cells; this subset of T cells expresses high levels of MIP1. The results in Fig. 3 showed that abundant Mos were recruited in the intestine during

LPR. Mos consist of several cell types, including lymphocytes, dendritic cells and macrophages (Mϕ). To determine whether Mϕs were recruited in intestinal LPR, in separate experiments we sensitized a group of BALB/c mice to OVA with the procedures in Fig. 1a. Isolated intestinal LPMCs were stained with anti-CD11b and F4/80 antibodies (Mϕ-specific marker), and analysed by flow cytometry. The results showed that the frequency of Mϕ was increased markedly at 48 h, which was abolished in mice pretreated with anti-MIP1 antibody, whereas pretreatment with Hydroxychloroquine mouse control antibody (an isotype-matched IgG) did not have this effect (Fig. 5). The data demonstrate that MIP1 contributes to the Mϕ recruitment to local tissue Immune system in LPR. The finding that abundant neutrophils were noted in the intestine (Fig. 3d) as well as a mild increase in myeloperoxidase (MPO) in local tissue (Fig. 3e) in LPR prompted us to look into the factors which recruited neutrophils to the sites of LPR. As MIP2γ is one of the major chemoattractants of neutrophils, we tried to find the source of MIP2γ. As the number

of Mos was increased in the intestine of mice after antigen challenge, we postulated that Mos might be the putative source of MIP2γ. We thus examined the expression of MIP2γ in isolated intestinal LPMCs by flow cytometry. Indeed, high levels of MIP2γ were detected in isolated LPMCs (Fig. 6). The fact that the MIP2γ+ Mos are also CD11b+ and F4/80+ implies that Mϕs are one of the major sources of MIP2γ in LPR. The data in Fig. 6 imply that MIP2 may play a critical role in intestinal LPR. To test this hypothesis using the same mouse model in Fig. 1a, we treated mice with neutralizing anti-MIP2 antibody 30 min prior to specific antigen challenge that was repeated 24 h later. The results showed that the extravasation of inflammatory cells (Fig. 7a–c) was increased markedly at 2 h after antigen challenge but returned to prechallenge levels at the 48 h time-point.

2d,h). To study the effect of Leishmania virulence on DC differentiation, we tested the ability of the four Lm clones to interfere with the expression of CD1a, HLA-DR, CD80 and CD86 during DC differentiation. We observed that all tested Lm clones were able to down-modulate CD1a expression significantly when compared with DCs differentiated without parasites (P = 0·002) (Fig. 3). Belnacasan manufacturer No significant

differences were observed between HV and LV Lm clones. We also showed a slight decrease in HLA-DR and CD80 expression as well as a slight increase in CD86 expression in the presence of Lm promastigotes when compared to uninfected DCs, but these results were not significant (Fig. 3). To evaluate the impact of virulence on cytokine production by DCs, the

four Lm clones were incubated with immature DCs for 48 h and IL-12p70, IL-10 and TNF-α production was analysed. We did not observe significant differences in IL-12p70, IL-10 or TNF-α production between Lm-infected DCs and uninfected cells for all tested clones. The effect of virulence of Lm parasites was also analysed on cytokine production selleck screening library during IFN-γ-, LPS- or IFN-γ/LPS-induced maturation of DCs. As shown in Fig. 4, highly significant levels of IL-12p70, IL-10 and TNF-α were detected in infected and uninfected LPS or IFN-γ/LPS matured DCs when compared with immature cells. Interestingly, we observed that infected and LPS-matured DCs produced lower levels of IL-12p70 than uninfected LPS-matured DCs, whereas the presence of parasites did not affect IL-12p70 production in IFN-γ/LPS-matured DCs. No IL-12p70 production was detected in infected and IFN-γ-stimulated DCs. These results were observed regardless of Lm clones virulence. We also showed a slight increase of IL-10 production in the presence of all clones except LV and of

TNF-α production in the presence of HVΔlmpdi and LVΔlmpdi clones during LPS-induced maturation of DCs (Fig. 4). In this study, we evaluated correlations between human isothipendyl DC response and Lm clones that were differentially pathogenic in BALB/c mice. The contrasting pathogenicity of these clones was more pronounced than it was for the isolates from which they were derived. Indeed, unlike the LV isolate that induced mild disease, the LV clone was not able to induce lesions in mice (unpublished data). We showed that infection rate and parasite burden were significantly higher in DCs infected with HV than with LV. Previously, using the wild Lm isolates LmHV and LmlV, we showed a significantly higher parasite burden in LmHV-infected human monocytes, suggesting that the high virulent isolate was able to replicate more rapidly inside the phagolysosome [23]. Here, we extend these observations to human DCs. We showed significant differences in uptake and intracellular growth of Lm clones having different levels of virulence. We also observed a significant decrease in infection rate and parasite burden in HVΔlmpdi-infected DCs compared with HV-infected DCs.

PTN and PTPRZ1 are known to control the egress of stem cells from bone marrow. Methods: Livers were harvested from PTN-GFP (PTN reporter) mice, PTN-PTN-knockout (KO) mice, PTPRZ1-KO mice, and wild type (WT) controls (n=6-9 mice/group) 2 weeks after bile duct ligation (BDL). Effects buy Ponatinib on expression of PTN and PTPRZ1, and the DR were evaluated by qRT PCR, quantitative immunohistochemistry (IHC), and hepatic hydroxyproline assay. LPCs and HSC

from WT, PTN-KO, and PTPRZ1-KO mice were also treated directly with PTN to characterize tyrosine-phosphorylated proteins and assess effects on gene expression, migration, and growth. Results: Although freshly-isolated HSC and LPC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was Enzalutamide molecular weight demonstrated in healthy liver. BDL induced strong expression of PTN mRNA and protein in MF-HSC and dramatically increased PTPRZ1 expression in MF-HSC and ductular-appearing LPCs. In WT mice, BDL triggered a DR characterized by periportal accumulation of collagen, Krt19(+) ductular cells, and aSMA(+) MF derived from desmin (+) HSC. All aspects

of this DR were significantly increased in PTN-KO mice and significantly suppressed in PTPRZ1-KO mice. PTN had no effect on the viability or growth of cultured LPCs or MF-HSC but directly inhibited migration of both cell types. The anti-migratory actions of PTN required PTPRZ1 because PTN inhibited migration in HSC that expressed PTPRZ1, but not PTPRZ1-KO HSC. PTN treatment of PTPRZ1(+) liver cells caused accumulation of several phosphoproteins, including an obligatory component of adherens junctions and a protein that regulates podosome function and integrins. Conclusions: The “stemness” factor, pleiotrophin, and its receptor, PTPRZ1, regulate the ductular reaction to liver injury by controlling the migration of resident cells in putative adult liver progenitor niches. Disclosures: John P. Chute – Board Membership: C2Regenerate Anna Mae Diehl – Consulting: Org 27569 Roche; Grant/Research Support: Gilead, Genfit The following people have nothing to disclose: Gregory A. Michelotti,

Anikia Tucker, Mariana V. Machado, Marzena Swiderska-Syn, Steve S. Choi, Leandi Kruger, Katherine S. Garman, Cynthia A. Moylan, Cynthia D. Guy, Heather Himburg MicroRNAs (miRNAs) are small non-coding RNAs that regulate the course of cholestatic liver diseases. miR-125 is a highly conserved family of miRNAs that regulate cellular proliferation during cholestatic liver injury. These miRNAs also regulate the expression of VEGF that potentiates fibrosis during liver injury. miR-125 targets several growth factors (e.g., VEGF) and matrix metalloproteases (MMPs), a dysregulation of which leads to aberrant proliferation, metastasis and cell invasion. Biliary hyperplasia in bile duct ligated (BDL) rats is accompanied by enhanced angiogenesis and fibrosis.

During the several decades after the first description of the disease in 1951, only a limited number of patients have been reported in the literature. Determination of the cDNA and full genomic sequence of FVII in the 1980s allowed the detection of the F7 gene defects leading to the abnormalities of the FVII molecule, and prompted the study of this disease with highly heterogeneous phenotype, ranging from severe to asymptomatic forms, with a poor correlation with FVII plasma levels. Significant advances in the understanding of FVII deficiency were achieved most recently due to

clinical studies carried out in large cohorts of patients from the national and international multicenter registries, Regorafenib clinical trial Tamoxifen supplier aiming at the comprehensive study and analysis of all aspects of the disease including phenotype/genotye relationships and current treatment of FVII deficiency. “
“Summary.  Circumcision is the oldest and most frequent surgical procedure in the world and especially in Turkey as is seen in the other Islamic countries because of religious and traditional pressures. In this study, we aim to report the experience of circumcision at Çukurova University in a total of 76 patients with haemophilia between 1990 and 2011. We retrospectively reviewed medical records of 69

haemophilia patients without inhibitors and seven haemophilia patients with inhibitors who had been circumcised. Before the year 2000, factor concentrates were given before and after circumcision for 6–7 days. Liothyronine Sodium After 2000, we used fibrin glue together with factor concentrates for only 3 days. By-passing agents were used for circumcision in haemophilia patients

with inhibitors. Twelve of 69 patients without inhibitors were referred to our centre with bleeding after the circumcision before diagnosis of haemophilia. Nine of these twelve patients had severe life threatening bleeding and three of them had moderate bleeding. Sixty-four patients with haemophilia were circumcised in our centre under general anaesthesia except for three patients who were given local anaesthesia. Thirteen of 57 haemophilia patients (22.8%) without inhibitors had seven mild and six moderate bleeding complications. A few patients had significant bleeding, despite adequate factor replacement. Five of seven haemophilia patients with inhibitors had two moderate and three mild bleeding complications. Our experience showed that circumcision for patients with haemophilia should be carefully performed by surgeons together with paediatric haematologist, under appropriate conditions in haemophilia centres which has comprehensive coagulation lab. “
“Inhibitor development complicates haemophilia treatment and may impact caregiver burden. Compare overall burden of caregivers of children with/without inhibitors in the United States using a novel disease-specific questionnaire and the previously validated CarerQol.

7, 8 More importantly, DNROL and DOXOL have also been reported to be responsible for the cardiotoxicity of DNR and DOX, respectively.9, find more 10 In humans, the conversion of DNR and DOX to DNROL and DOXOL is mainly catalyzed by carbonyl reductase 1 (CBR1).11 CBR1 belongs to the short-chain dehydrogenase/reductase (SDR) family and is ubiquitously expressed in human tissues with particularly high levels in the liver.12 CBR1 is believed to contribute

significantly to the development of resistance toward DNR and DOX. This is supported by the finding that CBR1 overexpression results in DNR resistance in tumor cells.13, 14 DNR resistance in human stomach carcinoma cells has also been shown to result mainly from an induction of CBR1.15 Furthermore, the role of CBR1 in the severe cardiotoxicity associated with anthracycline treatment has been documented. Mice heterozygous for the null allele of CBR1 have shown reduced sensitivity to anthracycline-induced cardiotoxicity because reduced CBR1 expression produces lower levels of DOXOL.16 Because of CBR1′s role in the resistance to and toxicity of anthracyclines,

it has been speculated that the inhibition of CBR1 to prevent carbonyl reduction may be an effective approach to enhancing the efficiency and reducing the toxicity of anthracyclines.17 Selleck Erismodegib In the SDR family, several enzymes are sensitive to inhibition by flavonoids, a group of natural products of plant origin. Flavonoids were first identified as lens aldose CBR inhibitors

in the 1970s.18, 19 More recently, hydroxy-PP has also been reported old to inhibit CBR1 and increase the sensitivity of cancer cell lines to DNR treatment (Fig. 1A).20 Flavonoids with different chemical structures are widely distributed in plants, vegetables, fruits, and beverages, particularly in tea and red wine. The major flavonoids of green tea extracts are catechins. Among them, (−)-epigallocatechin gallate (EGCG) is most abundant. EGCG has been shown to possess a wide range of pharmacological properties, including chemopreventive, anticarcinogenic, and antioxidant activity.21, 22 We have noticed a structural similarity between catechins and known inhibitors of CBR1, such as quercetin and quercitrin (Fig. 1A). In this report, evidence is presented that EGCG has a previously unknown inhibitory effect on CBR1 and CBR1-mediated tumor resistance to DNR, and this makes EGCG a potential chemotherapeutic agent for HCC.

Karyotype abnormalities, the morphological hallmark of genetic instability, have been consistently described in human HCC, structural chromosomal abnormalities being found predominantly in the pericentromeric region and in advanced tumors.[13] Key cellular functions are inhibited by statins selectively in various karyotypically abnormal cell types (including colorectal and ovarian cancer cells and human embryonic stem cells, which possess neoplastic-like properties) and this is mediated via a suppression of the stemness pathway.[14, 15] Low serum levels of either LDL-[16]

or total-cholesterol[5, 17] are major risk factors for HCC suggesting that HCC itself hi-jacks cholesterol away from the bloodstream because Rapamycin cost its growth is critically cholesterol-dependent.[5] HCC displays perturbed cholesterol metabolism both within mitochondria and in cell membranes.[18] In human HCC, a relatively higher cell membrane cholesterol content contributes to increasing membrane rigidity. This, in turn, alters membrane signal transduction pathways leading to favored cell proliferation.[19] Increased cholesterol levels in mitochondria from either rat or human HCC cells contribute to chemotherapy resistance and cholesterol depletion by inhibition of hydroxymethylglutaryl-CoA reductase enhances sensitivity to chemotherapy.[20] The proto-oncogene myc (c-myc)

codes for a nuclear protein, which controls nucleic acid metabolism and mediates the cellular response to growth factors. The human c-myc gene plays a pivotal role in liver oncogenesis.[21] selleckchem Truncation

of the first exon, which regulates the expression of c-myc, is crucial for tumorigenicity. Given that HMG-CoA reductase is a critical regulator of MYC phosphorylation, activation, and tumorigenic properties, the inhibition of this enzyme by statins may be a useful target for the treatment of MYC-associated HCC. Consistently atorvastatin blocks both MYC phosphorylation and activation and suppresses tumor initiation and growth both in a transgenic model of MYC-induced HCC as well as in cell lines derived from human HCC.[22] The specificity of these findings was proven by showing that the antitumor effects of atorvastatin were blocked by co-administering mevalonate, the product of HMG-CoA reductase.[22] As a gender-dependent risk factor Ixazomib ic50 for HCC explaining why females are less prone to liver cancer than males,[12, 23] IL-6 is a HCC bio-marker and an ideal molecular target to be aimed at.[24] IL-6 activates the transcription factor STAT3 (signal transducer and activator of transcription 3), an acute-phase response factor, which is next phosphorylated by the receptor associated kinases, and then forms homo- or hetero-dimers that translocate to the cell nucleus where it acts as a transcription activator. STAT-3 directly affects cell proliferation, differentiation[25] and angiogenesis.

5 (avoiding the contact). The frequency

of white spots in the progeny depended on the mother’s behavior scores; spotting was more frequent in the progeny of rats tolerant of handling than in the progeny of rats that avoided taking in hands. Our data indicate that not only the selection for elimination Epacadostat of aggressive response to humans is associated with higher frequencies of white spot emergence but also further increase in the degree of tame behavior still affects genetic systems associated with spotting. “
“Functionality of cheek teeth is essential for ruminants to masticate plant materials thoroughly and promote microbial degradation in their rumens. Thus, an excessive rate of tooth wear is expected to lead to premature loss of tooth functionality, and hence to reduced longevity. So far, however, the relationships between food habits, HTS assay molar wear and longevity have not been investigated. We first compared molar wear rates among nine sika deer Cervus nippon populations with different food habits. We then investigated correlations between molar wear rate and two ecological

factors, percentage of graminoids in diet and annual precipitation, relating to intrinsic and extrinsic abrasiveness of the ingested food, respectively. Secondly, we estimated ‘retained molar durability’ (molar height at a given age divided by wear rate) at successive ages for each population, and tested for correlation between molar durability and life expectancy among populations. The M1 and M3 wear rates differed among the populations and showed a positive correlation with graminoid consumption and a negative correlation

with precipitation, suggesting that both Glycogen branching enzyme ecological factors influence molar wear rates in the Japanese sika deer. M3 durability had a stronger correlation with life expectancy than M1 durability, especially at the older age stages. This implies that the influence of M3 durability on life expectancy becomes stronger at the time when the M1 is severely worn and loses its functionality, and is therefore more important for life span elongation than the M1. These results are concordant with the fact that the M3 is the most hypsodont molar in many ungulates. In the Japanese sika deer, microevolutionary acquisition of hypsodonty appears to be the case in a northern population (the Kinkazan Island), whose molar wear rates are extremely rapid due to their food habits. “
“The considerable impact of beavers on the species’ composition and structure of plant communities has led to intensive research on their feeding habits. To date, most of the available data originate from monitoring gnawing on woody plants but they rarely include feeding on non-woody plants. This study presents data on the dietary composition of beavers during the vegetation season based on macro- and micro-histological analysis of 97 faeces. The study was carried out during 2004–2008 at four sites in the Czech Republic.