The ability to subdue relatively large targets without the assistance of webs implies the necessity of physical strength and effective venoms that should be able to act rapidly upon the victim. Theraphosids are distributed across the globe, occurring in various habitats. It has been previously postulated that the variety of ecological niches and predatory behaviors correlate with the pharmacological and molecular diversity of their venoms (Escoubas and Rash, 2004). The theraphosid venom is a complex mixture of salts, nucleotides, free aminoacids, neurotransmitters,

polyamines, peptides, Baf-A1 datasheet proteins and enzymes (Escoubas et al., 2000; Escoubas and Rash, 2004; Rash and Hodgson, 2002; Savel-Niemann, 1989). The peptide mass mapping by MALDI-TOF MS of the venom of 55 tarantula species revealed a bimodal distribution of peptide molecular masses: 57.8% of the detected peptides have masses ranging from 3500 to 4500 Da, and 6.9% have masses ranging from 6500 to 7000 Da (Escoubas and Rash, 2004). Galunisertib in vitro Due to the potential biotechnological application of theraphosid venom components, intense toxinological investigations have been performed. By contrast, studies dedicated to the identification and structural and pharmacological characterization of peptides and other components of the venom of the theraphosid genus Acanthoscurria

are still lacking. The genus Acanthoscurria possesses 34 described species broadly distributed across the Neotropical region, especially in South America ( Lucas et al., 2011). Studies dedicated to the characterization of biologically active molecules from Acanthoscurria have been restricted to the investigation of antimicrobial peptides and polyamines present in the hemolymph of Acanthoscurria gomesiana ( Barbosa et al., 2007; Fazio et al., 2007, 2006; Lorenzini

et al., 2003a, b; Miranda et al., 2009; Moreira et al., 2007; Pereira et al., 2007; Remuzgo et al., 2008; Silva et al., 2000). The present study reports the purification, primary structure determination and electrophysiological effects on cockroach dorsal unpaired median (DUM) neurons of an anti-insect toxin, named μ-theraphotoxin-An1a (μ-TRTX-An1a, according to the IMP dehydrogenase nomenclature proposed by King et al. 2008),1 from the venom of Acanthoscurria natalensis – a tarantula species occurring in the Brazilian biomes caatinga and cerrado. Adult female A. natalensis specimens, collected in the vicinities of Feira de Santana (Bahia, Brazil), were kept in captivity and subjected to multiple venom extraction procedures. Venom samples were extracted by electrical stimulation on the base of the chelicerae and were collected with a micropipette tip. The venom samples were then transferred to chilled acidified water (0.1% aqueous trifluoroacetic acid) and centrifuged to remove cellular debris and mucus. The supernatant fractions was lyophilized, pooled and stored at −20 °C until required. μ-TRTX-An1a was purified by two- or one-dimensional chromatography, as described below.

A.F.U., D.O-S., G.E.W., A.S-G., J.A.M., P.B-S, have acquired all data and interpreted the results, C.B-F. and C.R.C. have conceived and supervised this study. C.R.C. wrote the manuscript whose final version was approved by all authors. The authors declare that: a) the material has not been published

in whole or in part elsewhere, except in the form of an abstract or part of academic thesis; b) the work is not currently being considered for publication elsewhere; c) all authors have agreed upon the content and form of the manuscript; d) all relevant ethical safeguards have been met regarding animal experimentation. This work was supported by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq; Coordenação this website Ibrutinib ic50 de Aperfeiçoamento de Pessoal do Ensino Superior – CAPES; Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul – FAPERGS and Fundação de Amparo

à Pesquisa do Estado do Rio de Janeiro – FAPERJ. “
“The scorpion represents a deadly and serious public health problem in some countries due to the high incidence and/or severity of cases, and difficulty of management by public health services (Chippaux and Goyffon, 2008). In Brazil, approximately 10,000 human cases of scorpion sting are treated at hospital centers and recorded annually, and 50% of these cases occur in the states of Minas Gerais and São Paulo (Ribeiro et al., 2010 and Soares et al., 2002). Several species of scorpions are known to cause accidents which can lead to death in Brazil, most of them belonging to the genus Tityus ( Fialho et al.,

2011). The scorpion species Tityus serrulatus is the most prevalent (95%) and accounts for fatal stings, especially among children ( Ribeiro et al., 2010 and Soares et al., 2002). The main problems involved with use of immunoadjuvants for vaccines and serums, includes the toxicity and side effects of these formulations (Gupta and Siber, 1995). Aluminum hydroxide is the only immunoadjuvant licensed for human use. However, recent studies bring concerns about toxicity involved with the cumulative effect Idelalisib manufacturer of aluminum (Gherardi et al., 2001, Petrovsky and Aguilar, 2004 and Zaharoff et al., 2007). Its secondary effects involved with subcutaneous administration include delayed hypersensitivity (Bergfors et al., 2003), severe granulomatous inflammation (Gupta and Siber, 1995 and Zaharoff et al., 2007) and pruritic subcutaneous nodules (Bergfors et al., 2003 and Thierry-Carstensen and Stellfeld, 2004). Thus, technologies or devices able to encapsulate and release recombinant or native proteins and able to induce the antibody production without side effects, represent a promising advance in the serum therapy. Some aspects are important to ensure the optimal efficacy of vaccines, particular attention is needed during their storage, distribution or handling.

All patients had magnetic resonance imaging (MRI) including diffusion-weighted imaging BTK inhibitor mw (DWI) and MRA before tPA administration. Follow-up MRA was performed immediately after the end of tPA infusion, if possible. We could monitor residual flow in 5 patients who had good echo windows (4 male, mean age; 60.8 ± 6.4 years). Two patients had proximal occlusion of the middle cerebral artery (MCA), one patient had distal occlusion of the MCA, one patient had a M2 occlusion and one patient had a distal occlusion of the unilateral vertebral artery.

One patient with proximal MCA occlusion had an insufficient acoustic window, but we could monitor residual flow at M2. Four patients had early complete recanalization within 60 min after the t-PA bolus – two patients at 60 min and other two patients at 30 min. In the patient who could be monitored at M2, one of M2 (M2a) was partial at 30 min, another M2 (M2b) www.selleckchem.com/products/Bortezomib.html was complete at 30 min. On the other hand, the occlusion persisted during 120 min monitoring in one patient with proximal occlusion of MCA. NIH Stroke Scale of two patients with very early recanalization (within 30 min) was 0 at the end of the treatment (dramatic clinical recovery). In

three patients a follow-up MRA could be performed after the end of tPA infusion. Follow-up MRA showed early recanalization in two patients and no recanalization in one patient. These findings of MRA were consistent with diagnosis of TCCS. There was no symptomatic and asymptomatic intracranial hemorrhage in 4 patients except for the patients without recanalization. Table 1 shows clinical detail data of 5 patients, and Fig.

1 shows the information of TCCS und MRA in patients with very early recanalization (within 30 min). The present study showed that patients with early recanalization had a favorable outcome after tPA therapy. In these studies, recanalization after tPA was evaluated by MRA [6] and [7] or TCD [2] and [3]. There are different benefits and limitations between MRA and TCD/TCCS in their diagnostic ability and characteristics as a diagnostic device. MRI is the standard device for the detection of vessel 17-DMAG (Alvespimycin) HCl occlusion or stenosis, however, it cannot be monitored during tPA infusion because patients who get a MRI have to be transferred to the MRI laboratory. On the other hand, TCD/TCCS is useful for real-time evaluation of intracranial hemodynamics at patient’s bedside. Several cases, however, had an insufficient acoustic window especially in Asian elderly female. In TCD study (2), 25% patients recanalized within the first 30 min, 50% recanalized within 30–60 min, 11% recanalized 61–120 min, and 14% recanalized after first 2 h after tPA bolus administration.

, 2004) seem to play essential roles in the edematogenic, hypotensive and nociceptive effects. Additionally, studies using molecular and biochemical approaches have reported the biological effect of the venom fractions on different cell types Autophagy signaling pathway inhibitors in vitro. Lopap and Losac, for example, besides their significant effects

on hemostasis, have been shown direct effects on endothelial cells, upregulating the expression of pró-inflammatory molecules as IL-8, ICAM-1 and E-selectin, inducing NO production ( Chudzinski-Tavassi et al., 2001). Most of studies using L. obliqua crude venom, in vivo or in vitro, have been done treating animals with doses that strongly affect the coagulation and fibrinolitic systems, causing MS-275 datasheet extensive hemorrhage. Under this situation, the mechanisms underlying the onset of inflammatory events, especially those related to alteration of endothelial

cells physiology and the involved intracellular signaling cascades could be masked. In our study, using intravital microscopy, we show that low (1–3 μg/ml), non-hemorrhagic doses of L. obliqua venom induced remarkable affects in the micro-vascular circulation of hamster’s cheek pouch. At those concentrations, although no significant vasodilatation was seen, occurred a marked slowing in blood flow, increasing in leukocyte rolling and adhesion on endothelial wall that were dose-dependent and more evident through time ( Supl. Fig. 7). The activation of vascular endothelial cells by inflammatory stimuli increases adhesion molecules expression and endothelial interactions with circulating blood leukocytes at post-capillary venules ( Granger and Kubes, 1994). Consistently, the analysis of those tissues of hamster cheek pouches by confocal find more microscopy showed that the venom activates the vascular endothelium, increasing the expression

of pro-inflammatory adhesion molecules E-selectin and VCAM-1 and inducing leukocyte adhesion and extravasation to neighboring inflamed tissue. This clear and typical inflammatory response occurred without the hemorrhage usually described for L. obliqua envenomation, although an intravascular effect on coagulation system cannot be discharge. For example, the potent pro-coagulant components of the venom may act triggering intravascular platelet aggregation and blood clotting ( Berger et al., 2010), and then contribute to slow down blood flow, to the activation of endothelial vascular cells and the overall inflammatory panel. Increase in vascular permeability requires cytoskeleton reorganization, a necessary prerequisite to inter-endothelial cell gap formation. The actin reorganization from its cortical distribution into stress fibers is an important component of the endothelial response to inflammation. We show, for the first time, that L. obliqua venom promoted a marked effect in the cytoskeleton reorganization in endothelial cells.

The catchment area had 328,542 inhabitants in 2007. The Danish National Health Service provides tax-supported health care for all inhabitants, guaranteeing free access to general practitioners and hospitals. All acute medical conditions including TIA are exclusively treated at public hospitals, either as in or as outpatients. We established an acute TIA-team, which served TIA-patients LDK378 both on the stroke unit and the TIA-clinic. Patients with TIA symptoms during the preceding 48 h or crescendo TIA

were admitted directly to the stroke unit and monitored for 1–2 days. All other patients were seen as outpatients 1–3 days after received referral. TIA was defined as a sudden focal neurologic deficit of presumed vascular origin lasting less than 24 h. Inclusion criteria were: TIA according to definition, residence in the Aarhus area, TIA during the last six months, and date of referral 1 March 2007–28 February 2008. Patients with a modified Rankin Score (mRS) >2 were excluded. Informed consent was obtained from all participants. All patients fulfilling the inclusion criteria for TIA were registered prospectively, including those admitted for suspected stroke but ending up as TIA. The TIA diagnosis

was made by a specialist. Patients underwent a neurological examination (more than 95% of the TIA patients were examined by the first author), CT or MR of the brain, ECG, laboratory tests and ankle brachial index. Furthermore, we performed Sotrastaurin in vitro duplex sonography of the extra- and intracranial vessels (TCCS). All ultrasound examinations were done by one experienced neurologist, performing at least 500 examinations per year and certified by the European Society of Neurosonoly and Cerebral Haemodynamics (ESNCH). Atherosclerosis of the carotid arteries was considered significant if a stenoses ≥50% was found (NASCET criteria). Intracranial stenoses were defined according else to the criteria established by Baumgartner: stenoses in the anterior (ACA), middle (MCA)

and posterior (PCA) cerebral artery was defined by peak systolic velocity of ≥120 cm/s, ≥155 cm/s, and ≥100 cm/s respectively, Stenoses in the VA and BA was defined by peak systolic velocity of ≥90 cm/s, and ≥100 cm/s respectively [7]. Additionally to these criteria, stenoses in ICA, and the extracranial VA was defined by systolic peak velocity ≥120 cm/s. All intracranial velocities were measured with an insonation angle of 0° without angle correction. A stenosis was considered symptomatic if a patient had TIA symptoms during the last six months before inclusion, related to the supply area of a carotid artery with a significant stenosis, or an extracranial vertebral or an intracranial stenosis according to the criteria above. Patients with combined extra- and intracranial stenoses e.g. ICA and MCA-stenoses on the symptomatic side were counted both as symptomatic ICA- and MCA-stenoses.

Water temperatures followed the expected annual dynamics with winter 2012 minima (16.68 ± 0.24°C) and summer maxima (29.19 ± 0.16°C). No spatial variation in water temperature could be detected. Salinity exhibited seasonal fluctuations and reached maximum values (38.22 ± 0.26 PSU; 38.30 ± 0.59 PSU) in winter and autumn 2012, respectively, whereas the lowest values (27.73 ± 3.64 PSU) were measured in spring. Lowest

values were observed at stations 1 and 2 due click here to the freshwater discharged. Minimum pH value (8.11) was recorded in winter 2012, 2013, while the highest value (8.40) was measured in autumn. The harbour’s water was always well-oxygenated and reached maximum values in spring (17.04 ± 3.23 mg l−1) and minimum values in summer (9.44 ± 3.16 mg l−1). The concentration of DIN, SRP, and RS varied widely and showed excess nutrient during autumn and high concentrations in summer with an apparent excess of SRP. Seasonal and spatial variation of nutrient concentrations showed that highest values of DIN were observed in summer (41.50 ± 10.13 μM) and lowest registered UK-371804 in spring (5.52 ± 5.20 μM). Stations 1, 9 and 10 usually presented peaks of DIN. Nitrate was the most dominant nitrogen form in winter and summer 2012 (55.89%; 52.97%, respectively) with maximum values observed at stations 1, 9 and 10. Ammonium was the dominant

nitrogen form in spring and winter 2013 (57.40; 83.20%, respectively) with maximum values registered at stations 1, 7 and 9. Nitrite was the dominant during autumn (69.22%) with maximum values recorded at stations

6, 7 and 8. The highest SRP concentrations were measured in summer (5.93 ± 2.07 μM) and lowest in spring (0.85 ± 0.47 μM). Station 6 reached maximum values, 9.75 μM in summer and 9.60 μM in autumn. Highest values of RS were observed during summer (28.95 ± 14.13 μM) with maximum values at stations 1 and 2. The DIN/SRP ratio changed both seasonally and spatially. In general, DIN/SRP were lower than the algal N/P (Redfield ratio) throughout DOK2 most of the harbour stations, increasing to >16:1 only at station 1 (summer and autumn) and stations 9 and 10 (summer). Low DIN:SRP ratios (<5) during spring and winter 2013 suggested that nitrogen could be the principal limiting nutrient. The RS/SRP ratio underwent more complex seasonal changes. Except in winter 2012, the ratio RS/SRP was <16:1 all the year round. Higher ratios were observed in winter 2012, suggesting less demand for RS relative to SRP. This is consistent with high proportions of Si-requiring diatoms in the phytoplankton community during spring-winter 2013 and primarily non-siliceous forms in spring. From the analysed data, a visible change in phytoplankton community with regard to numerical abundance and species composition was evident among stations and in the seasonal cycle.

8 °C one individual of six (17%) did not survive ABT-263 mw past 9 h, at Ta = 39.7 °C three of four wasps (75%) died within 9–12.5 h. At Ta = 42.4 °C all four individuals (100%) died within 1.7 to 2.5 h. In Fig. 4 the percentage of mortality at the tested Ta is indicated. Fig. 5 displays the CO2 production and the thoracic temperature excess (Tth − Tab) of a wasp that did not survive the experiment. After cease of cyclic respiration the individual showed a characteristic pattern of CO2 release. This was accompanied by a distinct endothermic phase. The thermograms show that it was induced by thoracic heating activity. In these experiments solely V. vulgaris foragers were investigated. Fig. 6 shows a representative

thermolimit experiment. With increasing temperature the wasps were more agitated, they ran around looking for an exit from the measurement chamber, gnawed

into the chamber’s fittings and showed self-grooming as well as cooling behavior. Coordinated bodily activity ceased with mortal fall ( Fig. 6, stage 4). The averaged values of mortal fall provided the knockdown temperature ( Klok et al., 2004 and Stevens et al., 2010) or activity CTmax of 44.9 °C ( Table 1). However, spasms as well as occasional abdominal movements (which might evade automated activity detection because of diminutive appearance) could be observed in the IR recordings of some individuals until the postmortal peak. CO2 production followed the stages of response to rising ambient temperature first described by Lighton and Turner (2004) (Fig. 6). The respiratory CTmax was determined via the inflection point of the rADS residual values 10 min before and after the mortal fall.

Averaged values were considered Ruxolitinib mouse as the respiratory CTmax amounting to 45.3 °C. Activity CTmax and respiratory CTmax did not differ significantly (P = 0.357507, t-test, Table 1). For comparison, we determined both the activity and also the respiratory Docetaxel molecular weight CTmax in honeybee foragers (A. mellifera carnica). Their activity CTmax of 49.0 °C was nearly identical with their respiratory CTmax of 48.9 °C (P = 0.899966, t-test, Table 1). The honeybees’ activity CTmax was 4.1 °C and their respiratory CTmax was 3.6 °C higher than that of the wasps. Values differed significantly between both species (P < 0.001, t-test, see Table 1). Vespula showed a characteristic CO2 release pattern before the postmortal valley ( Fig. 6A, dotted arrow) which could not be found in other hymenopteran CO2 curves evaluated from thermolimit respirometry (e.g. A. mellifera, Käfer et al., 2011; Pogonomyrmex rugosus, Lighton and Turner, 2004). Fig. 6B also shows the typical thermal reaction (Tth–Tab) of the same wasp, following failure of respiration at the respiratory CTmax. The postmortal peak of CO2 release was accompanied by a heating bout in the thorax (compare also Fig. 5). The mean increase of the thoracic temperature excess over the abdomen at the peak of this bout was as high as 2.5 °C (SD = 0.7 °C, n = 8, maximum = 3.6 °C).

neuwiedi and B. moojeni showed significantly higher LAAO activities, followed by that of B. jararaca and B. jararacussu. B. alternatus venom showed significantly lower LAAO activity. In order to compare the various Bothrops venoms, in terms of their protein profiles, the venoms were submitted to electrophoresis under non-reducing conditions. The results of the SDS-PAGE analysis are shown in Fig. 4. Despite the fact that several venoms had some bands in common, their overall profiles showed substantial differences, except in the case of B. moojeni

versus B. neuwiedi. The presence of PLA2 in the venoms was analyzed by an egg yolk zymogram. All venoms displayed a clear zone at approximately 15 kDa, which corresponds to PLA2 activity against lecithin on the gel (Fig. 5). Although equal amounts of venom were used, different patterns of clear zones were observed. This observation can be explained by differences in the activity level of each enzyme and its concentration

PD0325901 mouse in the venom. These findings are in accordance with the results obtained in the hemolytic assay. The presence of proteinases in the venoms was confirmed by the appearance of clear zones against the blue background on the casein zymogram (Fig. 6). B. jararaca venom showed intense casein degradation in the 25–28 kDa range, while B. neuwiedi venom showed intense degradation at 28 to 30 kDA. The venoms of B. jararacussu and B. moojeni showed a lower degradation profile at approximately 30 kDa, while no clear Selleckchem Epacadostat zone was observed for B. alternatus venom. Although all of the venoms showed proteinase activity, as indicated in Fig. 2, only the venoms of B. jararacussu and B. moojeni were similar in their patterns of casein degradation. The venoms also showed LAAO activity, as confirmed by the presence of yellowish bands in the OPD zymogram (Fig. 7),

nevertheless, their molecular mass was variable. B. jararaca venom showed the most intense yellowish band, around 97 kDa. While B. jararacussu and B. moojeni venoms showed similar band profiles at approximately 84 and 82 kDa, respectively. B. neuwiedi venom DOK2 was unique in that it displayed two yellow bands. One intense band of 75 kDa and another, less intense band of 119 kDa were detected. B. alternatus venom displayed the enzyme at approximately 107 kDa. Proteinases and PLA2s are considered the major toxic compounds in almost all snake venoms, although other enzymes also contribute to the toxicity (Correa-Netto et al., 2010 and Serrano et al., 2005). LAAO is also an important enzyme present in the venom of pit vipers, however, it accounts for about 0.5% of the total toxin transcripts from the venom glands, a small percentage when compared with the 53.1% and 28.5% reported for metalloproteinases and serine proteinases, respectively (Cidade et al., 2006). In the present study, we evaluated the PLA2, proteolytic, and LAAO activities of the venoms of five different Bothrops species: B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi, and B.

The estimate of total GDP for the fisheries sector updates the 2005-estimate of the fisheries contribution

to GDP of US$0.6B [5], and indeed increases the estimate with a factor of five. It also learn more exceeds the 2008-gross value of the fisheries exports of US$2.4B, (which does not consider costs), [5]. The increased estimate of contribution to GDP was higher than the previous estimate, partly because it was for 2009 rather than 2005, and partly because of the much more comprehensive description of the fisheries sector that was derived here. Fisheries have always been important in Peru for providing livelihood and food, and this is still the case. The total employment in the fisheries sector was here estimated to 232,000 jobs (full time), which exceeds

the previous estimate from FAO of 145,000 (full and part time) with more than 50%. Yet, the estimate should be considered conservative, as the study did not account for all parts of the fisheries sector. The estimate of the total sector employment was lower than that of Teh and Sumaila [28], who estimated the employment to 440,000±200,000 jobs. As the estimate for primary sector employment (79,500 jobs) derived here was close to the estimate (72,000 jobs) of Teh and Sumaila [28] the difference was in the higher multiplier used in their study (6.1 vs. 2.9 in the present study). Among enterprise types, the anchoveta-based industry Trichostatin A cell line is not the leading employer – fishmeal plants only provided 5% of the jobs in the industry (Table 1). Instead, fish restaurants dominated with 35% of the employment, followed by freezing and canning plants with 8% and 7%, respectively. By categories, the retailer section was dominating with 45% of the jobs, followed by the primary sector (producers) responsible for 32% of the total employment, and processing with 20% (Table 2). There were only males employed in the primary sector;

the 10 women that were estimated to be working in the sector work with guano processing, (which we have lumped with guano extraction) Non-specific serine/threonine protein kinase (Table 2). In the processing sector there was a 50–50 split between males and females, and in the retailer section there was a small dominance of females with 57% of the total. Overall, however, the total fisheries sector was male-dominated with 64% of the total employment. The study followed the fish products from the primary sector through to the consumers and could therefore be used to evaluate for each fishing fleet how much it contributed to the economy and the employment. This is illustrated in Table 3, from which it is clear that more than half of the GDP contribution came from the steel (34%), artisanal (15%), and wooden (5%) purse seiners. There are numerous species that contribute to this, with anchoveta being the most important. The economic multipliers from the primary sector to the entire fisheries sector varied around an average of 2.

RPMI 1648 medium (Gibco, Karlsruhe) was supplemented with 25 mM HEPES buffer 1 mM l-glutamine (Gibco, Karlsruhe), 1× Penicillin/Streptomycin (Cölbe, selleck chemical PAA) and 10% heat-inactivated fetal calf serum. 8 ml were aliquoted into 50 ml polypropylene tubes (Sarstedt, Nürnbrecht) and warmed to 37 °C. Upon removal from liquid nitrogen storage, no more

than two cryovials at a time were thawed in a 37 °C water bath until the cell suspension was melting and a little ice remained. One ml of warmed media was slowly added to the thawed PBMC and the cell suspension had been transferred to a corresponding polypropylene tube (final volume 10 ml). The tubes were centrifuged at 400 g for 5 min at room temperature. The PBMC were resuspended in 10 ml medium per 1 × 107 cells and transferred in a cell incubator (5% CO2, 37 °C) overnight with the cap of the tube loose. The effect of the 3 different storage conditions on cell recovery was evaluated using the ViCell cell analyser (Beckman Coulter,

Krefeld). Five samples per donor Mitomycin C order per storage condition were thawed and cell recovery and viability measured immediately post-thaw and again after overnight culture using the trypan blue dye exclusion test. Each sample was measured three times. Recovery (%) (after thawing): %recovery=(number of viable PBMC after thawing/number of frozen viable PBMC)×100 Recovery (%) (after overnight culture): %recovery=[number of viable PBMC after overnight rest/(number of frozen viable PBMC-number of viable PBMC removed for measurement directly after thawing)]×100 Viability: %viability=(number of viable PBMC/number of total PBMC)×100 PBMC were assayed for IFN-γ production in the presence of CMV pp65 peptide pool (BD Bioscience,

Heidelberg), CEF peptide pool (CTL, Bonn), PHA (Sigma–Aldrich, Taufkirchen) and background control (culture Progesterone media containing 0.4% DMSO) in triplicates. 96 well plate anti-human-IFN-γ mAb 1-D1k precoated (Mabtech, Hamburg) were washed four times with PBS (Gibco, Karlsruhe) and blocked with culture medium, RPMI 1648 medium (Gibco, Karlsruhe) containing 25 mM HEPES buffer 1 mM l-glutamine (Gibco, Karlsruhe), 1× Penicillin/Streptomycin (Cölbe, PAA) and 10% heat-inactivated fetal calf serum, for 30 min. Cryopreserved PBMC were thawed as described above and used the next day. Approximately 1 × 105 PBMC were added to the CEF, CMV and background wells and 0.5 × 105 PBMCs to the PHA wells. CEF peptides and CMV peptides were added to a final concentration of 2 μg/ml/peptide and 1.75 μg/ml/peptide, respectively. The final PHA concentration was 4 μg/ml. The final DMSO concentration was between 0.1% and 0.25%. The plates were incubated at 37 °C, 5% CO2 for 20–22 h.