PARK JI HYEON, PARK KYUNG SUN, JANG HYE RYOUN, LEE JUNG EUN, HUH WOOSEONG, KIM YOON-GOO, OH HA YOUNG, KIM DAE JOONG Division of Nephrology, Department of Internal Medicine, Samsung Medical center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Introduction: Living-unrelated donors (LURD) have been widely click here used for kidney transplantation (KT). We compared clinical outcomes of

KT from LURD and from concurrent living-related donors (LRD), and identified risk factors associated with acute rejection, graft and patient survival in living KT. Methods: We retrospectively reviewed 779 patients who underwent living donor KT (264 from LURD, 515 from LRD) at Samsung Medical Center from January 2000 to December 2012. Results: Median follow-up was 67 months. Mean age (43.2 vs. Protease Inhibitor Library supplier 38.4 years, P < 0.001), mean number of total HLA mismatches (4.1 vs. 2.5, P < 0.001) and HLA-DR mismatches (1.2 vs. 0.8, P < 0.001) were higher, and mean estimated glomerular filtration rate (eGFR) was lower (87.6 vs. 90.9 ml/min, P = 0.007) in LURD. Acute rejection-free survival (64.9% vs. 72.7% at 5 years, P = 0.018) and graft survival (92.9% vs. 96.5% at 5 years, P = 0.025) were lower for LURD than LRD whereas patient survival rate was comparable (97.9% vs. 98.7% at 5 years, P = 0.957). Cox-regression analysis showed that HLA-DR mismatches (OR 1.77, 95% CI 1.20–2.62, P = 0.004

for 1 mismatches; OR 2.63, 95% CI 1.64–4.20, P. Conclusion: Our data suggest that HLA-DR mismatches and donor eGFR are independent risk

factors for clinical outcomes of living KT. In living KT, these factors should be considered to prevent acute rejection and improve graft survival. ADACHI HIROKI, MUKAI KIYOTAKA, OKUSHI YUKI, OKINO KAZUAKI, SHOJIMA KIYO, MATSUI YUKI, FUJIMOTO KEIJI, ATSUMI HIROKATSU, OKUYAMA HIROSHI, YAMAYA HIDEKI, YOKOYAMA HITOSHI Division of Nephrology, Kanazawa Medical University Amisulpride School of Medicine Introduction: Although the risk for morbidity and mortality is studied in subjects with renal transplantation, there are very limited data to access the reno-protective effects of lipid and adiponectin. Methods: We studied 105 adult subjects (age 19- to 72-year old; 66 males, 21 cadaveric donors) between January 2004 and December 2012, with at least three years of allograft survival in our hospital. We examined clinical backgrounds, donor types (living or cadaver), treated drugs, blood pressure (BP, mmHg), body mass index (BMI), blood chemistry including cholesterol (total, LDL-C, HDL-C), glucose, glycated hemoglobin (HbA1c), and total, high- and low-molecular adiponectin (ADPN) levels. Results: The initial 4 years alteration of eGFR was at −2.2(−14.3∼7.0) ml/min/1.73 m2/year in 105 subjects. In single analyses, both LDL-C/HDL-C ratio below 2.0 and statin treatment also decreased the reduction rate of eGFR at −1.4 and −1.0 ml/min/1.73 m2/year (p = 0.01, p = 0.02), respectively, but not by total ADPN levels.

Interestingly, Ehirchiou et al. [44] found that TH17 cells in lymph nodes may negatively interfere with tolerance induction to fed allergens, which suggests that IL-17A could be involved in the allergic airway sensitization

in our i.n. model. Apparently, the youngest mice had augmented airway responses compared with older mice. In both the i.p. (10 μg) NSC 683864 research buy and i.n. model, the youngest mice had higher BALF eosinophil influx and higher cytokine secretion than older mice. In the i.n. model, the OVA-only immunized 1-week-old mice also presented with increased OVA-specific IgG1 levels accompanied by a neutrophil inflammation in BALF. It may be argued that endotoxin contamination of the OVA could have an inflammatory effect particularly in the youngest mice. However, acute lung responses to endotoxin did not differ between newborn and adult mice [45], which argue against endotoxin as an explanation for the observed age differences. Allergen doses that

induce tolerance in adult rodents may, when applied mucosally in newborns, induce IgE sensitization [46, 47]. However, we did not observe effects on OVA-specific IgE after i.n. exposure to OVA alone. If the inflammation in mice sensitized at 1 week of age may be ascribed to an IgG-immune-complex-induced reaction cannot be defined from this study, but would explain the neutrophil-dominated buy Ruxolitinib inflammation [48, 49]. Whether the general propensity to elevated inflammation in very young mice may be

Rho linked to early onset of allergy and asthma in children remains to be determined. Further, half of children with early-onset asthma outgrow their disease [50]. It could be speculated that this is because of the maturation of the immune system, because bronchial hyperreactivity and airway inflammation persisted for a shorter time in mice sensitized when newborn compared to when sensitized as 8 weeks old [34]. Although ‘new’ allergy can occur throughout life, generally, allergy prevalence and severity tend to decrease after young adult life [51], and TH2-type responses may weaken with age [52]. Immunological ageing studies have included mice of much higher age than the present study. However, our study clearly demonstrates that age may exert a pronounced effect on experimental allergy even in mice up to 5–6 months of age. Further, allergy responses in female and male mice may be affected differently by age and allergen doses. The study also indicates that to develop appropriate models of allergy in children, adults and aged humans, good knowledge of age-related effects in human allergic diseases is required. The data presented here demonstrated that age, sex and immunization dose interact to be significant determinants of experimental allergy. Therefore, optimal modelling must be performed to mimic human disease. The study was financed by The Norwegian Research Council.

Results of studies will also allow health professionals to more accurately describe the benefits and harms of dialysis therapy on quality of life

and outcomes for patients. Assumptions are made that dialysis is appropriate for all individuals; however this may not be a valid assumption for everybody. Dialysis by the nature of the intervention has a large potential to influence the quality of life of the individual and immediate family. Dialysis may prolong life, however it also ‘remains an aggressive tertiary intervention FK506 that may challenge the priorities and attitudes of older patients in particular’.[8] Dialysis also has hazards, and in some patients it will shorten life. This is a particularly critical issue in the older age group. The patient’s preference and quality of life are central issues.[8] It has also been found that both dialysis patients and their partners are overwhelmed by the impact of dialysis on their lives.[4] In a patient survey conducted by Davison and colleagues,[9] 60.7% of patients regretted the decision to start dialysis. However, if patients opt for conservative therapy (no dialysis) it is unknown how much life expectancy, as well as the quality of life, is actually altered. It is possible Depsipeptide datasheet that the intervention

of dialysis may actually make the quality of life worse, particularly in the presence of significant comorbidity. Currently, there is a small amount of retrospective data only,[5] but no prospective scientific data to support either point of view to help clinicians, their patients and family/whanau to make a decision. A study from a large London dialysis centre looked at outcomes between two groups of older patients, one group that opted for dialysis therapy and the other that chose maximal conservative care. Those opting for conservative care were older (mean age 82 years vs 76 years). Although the dialysis group survived for a longer period (mean 2 Non-specific serine/threonine protein kinase years), the majority in the conservative group survived for over 13 months with substantially lower hospital days (16 days per patient per year) and the majority in

this group died at home.[10] The dialysis patients were dialysed in a hospital centre that meant they averaged 173 days per patient per year at the hospital. This study did not record any quality of life assessment, data related to patient satisfaction, cost-effectiveness or the socioeconomic impact of the hospital-based treatment.[10] 1. In a thematic analysis of the literature Morton and colleagues demonstrated that awareness of factors associated with decision-making related to the management of chronic kidney disease (CKD) can provide health professionals with evidence on how best to deliver education programmes for patients and their family, as well as enhancing the patient and their family’s capacity to share in that decision-making process.

This IFNAR/STAT1 signalling up-regulates Axl, which may feed forward SOCS protein production. SOCS1 promotes INCB018424 the degradation of the TLR4 adaptor protein MAL,28 and SOCS3 inhibits TRAF6 ubiquitylation.29 In the present study, we demonstrate that TLR ligands reduce Gas6/ProS expression via NF-κB activation. NF-κB activation results in the induction of various cytokines. Whether NF-κB activation-driven

cytokines are involved in the reduction of Gas6/ProS should be investigated. Evidence that autocrine Gas6 and ProS synergistically inhibit inflammatory cytokine expression by macrophages in baseline conditions suggests that these two cytokines play important roles in the maintenance of immune homeostasis in normal physiological conditions. Several observations are consistent with this speculation. Regarding autoimmunity, patients with systemic lupus erythematosus have low circulating levels of ProS.30,31 Because ProS is a TAM ligand, a low ProS level selleck compound may lead to reduced TAM signalling, which consequently leads to immune hyperactivation. At the same time, patients with systemic lupus erythematosus are prone to thrombosis,32 which corresponds to a role of ProS as a blood anticoagulant.24 On the other hand, increased TAM signalling may also result

in diseases. A clinical report showed that circulating Gas6 levels were elevated in patients with severe sepsis, and that Gas6 elevation was correlated with a patient’s clinical score and the occurrence of septic shock,33 suggesting that hyperactive TAM signalling might play a role in sepsis. The treatment of macrophages with TLR ligands rapidly up-regulated the production of IL-6, TNF-α and IL-1β, why which declined

to low levels 24 hr after the treatment. Thereafter a secondary mild up-regulation of the inflammatory cytokines was observed, the mechanism of which has yet to be determined. Evidence that reduced Gas6 and ProS levels are responsible for the secondary up-regulation of inflammatory cytokines after 24 hr of LPS stimulation is provided. The results provide novel insights into inflammatory regulations. In recent years, increasing research on the Gas6/ProS-TAM system function has been observed, which has provided essential clues on the biological implications of this system. Gas6 and ProS have exhibited crucial roles in the clearance of apoptotic cells and autoimmune diseases.12,13 Interestingly, links between the two phenomena have been known for many years.34 However, the regulation of Gas6 and ProS expression remains largely unknown. In the current study, evidence that Gas6 and ProS can be down-regulated by TLR activation in macrophages is provided, which may feed forward the inflammatory responses against infectious pathogens. Appropriate TAM signalling is critical in the homeostatic regulation of the immune system and resolution of inflammation.

Multiparity induces transferable-specific hypo-responsiveness or even true tolerance to either HY or paternal alloantigens.53,54

Placental products, be them placental learn more extracts or water-soluble material obtained from these, co-injected with alloantigenic cells, induce systemic antigen-specific LyT2+ Ts.81 These were traced in the first pregnancy in mice and in rats by Baines and Liburd. Similarly, antigen-specific MHC-restricted Ts were found in humans.82 Controversies about in vitro assays can still be traced in proceedings of the Gusberg meeting.83 In the 1980s, we studied, in detail, the in vitro properties and mode of action of these suppressor cells (specificity, mediation by a soluble factor). A part of these studies was carried out with anti I–J antisera, as many other labs working on suppression did at the time. Lee Hood’s demonstration that the I–J region does not exist while properties of the suppressor Opaganib price factor(s) of

Gershon and Cantor were more and more improbable doomed Ts. For an excellent revision of the history of Ts, see references.84,85 We nevertheless still tested/published the role of Ts in CBA × DBA/2 matings.51 As reviewed, in,86 the CD25 and Foxp3 markers again boosted Ts on the forefront. Yet the I–J trauma lead to a more benign denomination of ‘regulatory T cells’ (Tregs), rather than ‘CD4+ Ts’, which we first saw in 1981, but termed ‘inducers’ .87 CD8+ cells are still important partners, as shown in studies by Arck, Clark and coworkers.88 Aluvihare and Darasse convincingly demonstrated that CD4+ CD25+ elimination causes foetal deaths in allopregnancy by transfer or direct in vivo experiments.89,90 Saito traced/ quantified Foxp3 cells DCLK1 (T regs) in human decidua as well as regulatory NK/T cells.91 Robertson and coworkers92 showed that the Foxp3

marker decreases in unexplained infertility endometrial biopsies. These, and Fainbolm, detected periodic T reg modulation during the menstrual cycle, peaking in the late follicular phase.93,94 For Fainbolm, T regs from patients with RSA are ‘functionally deficient’,93 and T reg decidual recruitment correlates with expression levels of CCL3, CCL4, CCL5, CCL22, and CX3CL1.90 Finally, placenta-dependent CD8+ T regs have been demonstrated by Shao et al.,95 and this is reminiscent of earlier data in mice about LyT2 Ts.81 Could the placenta escape immune attack by resisting effector cell lysis? We have discussed the Fas/Fas ligand interaction. Membrane and soluble HLA-G (sHLA-G) also play a role, including sHLA-G secretion by the MHC-syncytiotrophoblast. Moreover, trophoblasts (and choriocarcinomas) are resistant intrinsically to cell-mediated lysis.96–98 This resistance is independent of HLA-G.99,100 The once debated soluble factors96,101,102 had properties which fits with what is now known of soluble HLA-G, be it sHLAG1/ G2 characteristics.

RNA was

isolated from CD4+ T cells by using the RNeasy Mini kit (Qiagen, Courtaboeuf, France). cDNA synthesis involved Enhanced Avian HS RT-PCR (Sigma-Aldrich). CD40L and β-actin cDNA levels were determined Selleck PD0325901 using Light Cycler-based kinetic quantitative PCR (Roche Diagnostics), and PCR product detection involved Light-Cycler FastStart DNA Master SYBR Green I (Roche Diagnostics). CD40L expression was normalised to that of β-actin. Amplification primer sequences were for CD40L (forward) 5′-CACCCCCTGTTAACTGCCTA-3 and (reverse) 5′- CTGGATGTCTGCATCAGTGG-3′; and β-actin (forward) 5′-GCT GTG CTA CGT CGC CCT-3′ and (reverse) 5′-AAG GTA GTT TGG TGG ATG CC-3′. Each sample was analysed in duplicate. After CD4+ T cell isolation, DNA was isolated using the QIAamp DNA Mini Kit (Qiagen), bisulphite treated with the EpiTect Bisulfite Kit (Qiagen) and then stored at −20 °C. Pyrosequencing was used for quantitative assessment of the methylation level at each studied CpG dinucleotide [9]. Briefly, methylation data were analysed using pyro q-cpg software (Qiagen). The degree of methylation at each CpG was expressed as proportion of methylated cytosines to total LBH589 cell line methylated

and unmethylated cytosines at the respective CpG. Non-CpG cytosines were used as a control to verify completeness of bisulphite conversion. Each sample was processed in duplicate. Eight CpG dinucleotides were analysed within the promoter region and four CpG dinucleotides within the downstream enhancer. CD40L promoter and downstream

enhancer methylation patterns in CD4+ T cells were compared for patients with pSS and controls. CpG positions were the same as those found differentially methylated in SLE [2]. Data are presented as mean percentage methylation. Statistical analyses involved use of GraphPad Prism 5. Differences between patients and controls were analysed by the nonparametric Mann–Whitney U-test. Relative mean fluorescence intensity (MFI) and 95% confidence intervals (95% CI) were calculated. To adjust for age between patients and controls, we used ANCOVA. P < 0.05 was considered statistically significant. Characteristics of women with pSS and controls are in Table 1. Median ESSDAI was 2 [0–18]; patients and controls differed by age (56 ± 15.4 versus Progesterone 41 ± 14.6, P < 0.05). We used flow cytometry to investigate CD40L expression on CD4+ T cells ex vivo and after 4 days of culture followed by PMA/ionomycin stimulation for 4 h. Ex vivo expression of CD40L was not detectable among both patients with pSS and controls. After 4 h of PMA and ionomycin stimulation, membrane-bound CD40L expression was higher on CD4+ T cells from patients with pSS than controls (n = 20): the mean MFI was 3,758 (95%CI: 2,636–4,879) versus 2,344 (1,512–3,177), respectively (P = 0.0167) (Fig. 1). Conversely, CD40L mRNA level in CD4+ T cells did not differ between patients and controls, either ex vivo or after 4-day culture with 4-h PMA and ionomycin stimulation (Fig. 2).

25 Renal hL-FABP binds to lipid peroxidation MLN0128 chemical structure products generated by oxidative stress, and redistributes them into the tubular lumen, thereby preventing tubulointerstitial damage. Cisplatin is a platinum-based chemotherapy drug used to treat various types of cancers, including sarcomas and some carcinomas. However, acute kidney injury is a serious side effect of cisplatin, thus, strategies to reduce acute kidney injury is important for continuation of cisplatin as a cancer treatment modality. This model is used to evaluate the pathophysiology of AKI after platinum-based chemotherapy. Intraperitoneal

injection of cisplatin in mice induces acute tubular necrosis and apoptosis, which are similar to the phenotypes observed in human cisplatin induced nephropathy. In the proximal tubules of this model, it was reported that metabolism of intracellular FFA was suppressed and nonesterified fatty acid and triglycerides accumulated

in kidney tissue. When the metabolism of FFA is activated by activation of the peroxisome proliferator-activated receptor (PPAR), the degree of cisplatin induced nephropathy is attenuated, therefore, increased intracellular FFA is considered to be closely associated with the generation and progression of nephropathy. Since there is a PPAR response element (PPRE) in the promoter region of hL-FABP, the presence of PPAR ligand, which activates PPAR, upregulates the expression of hL-FABP as well.30 In the cisplatin-induced nephropathy model, gene and protein expressions DAPT of hL-FABP are upregulated and urinary excretion of hL-FABP is also increased.26,27 Although the degree of tubulointerstitial

damage in the Tg mice is similar to those in the WT mice, accumulation of FFA in the kidney of Tg Lepirudin mice is significantly inhibited and acute kidney injury in the Tg mice is significantly reduced by administration of PPAR ligand, which further upregulates the expression of renal hL-FABP. Further, urinary hL-FABP levels are decreased in the Tg mice administered both cisplatin and PPAR as compared to the Tg mice with cisplatin administration alone. From these results, it is concluded that more upregulation of renal hL-FABP by PPAR activation is protective of acute kidney injury in this model. Adenine is one of the two purine bases used in the formation of DNA and RNA. Adenine injected into the body is oxidized to 2,8-dihydroxyadenine (DHA) by xanthine dehydrogenase (XDH). Since DHA has low solubility in body fluid, injection of a large amount of adenine causes DHA to be filtered through the glomeruli and to accumulate in the tubular lumen, thereby leading to tubulointerstitial inflammation and subsequent tubulointerstitial fibrosis. XDH inhibitors, such as allopurinol, inhibit the production of DHA derived from adenine and attenuate adenine-induced nephropathy.

16 In the current study, AFLP was found to be useful for discrimination between inter- and intrapatient isolates. Moreover, beta-catenin inhibitor all isolates could be identified down to the species

level according to the current taxonomic status. A majority of patients were exclusively colonised by one AFLP genotype. Only one genotype was shared between two patients. The colonisation of CF patients by multiple AFLP genotypes was already reported previously [37] but this study was performed in 2002, well before the recent taxonomical changes. Therefore, from the present perspective, we cannot appraise if intra- or interspecific variations were detected. Defontaine et al.37 state multiple colonisations with up to three different genotypes, comprising one predominant genotype associated with up to two accompanying genotypes. Exceptionally, in our study, we found patients colonised with up to five different genotypes over a period of up to 5 years, with re-appearing genotypes. Therefore,

it is very likely that those patients are colonised with multiple S. prolificans genotypes. Our data mirror that CF patients can be chronically colonised with a specific genotype or multiple genotypes for prolonged periods of time (several years). Co-colonisation by multiple genotypes, also in non-CF patients, has been recognised before for other fungal species, such as A. fumigatus and A. flavus.36,38,39 If multiple colonisation turns into multiple infections by different genotypes of one species, this might have an impact on disease outcome, this website as we found that different Scedosporium isolates from the same patient (Table 1) can vary considerably in their AFSP. In particular, when patients are colonised by two or more Obatoclax Mesylate (GX15-070) isolates with different susceptibility patterns, this may result in an overestimation of MIC values. This situation is exemplified in this study for instance in patient 13 where one clinical sample contained an MICA-susceptible, as well as MICA-resistant isolate of the same species. Apparently,

also patient 1 was colonised at the same time with two isolates of the same species, but with different AFSPs. For this reason, clinical specimens should be carefully analysed for the possible presence of multiple strains expressing variable antifungal susceptibilities. Overseeing such mixed infections due to S. prolificans may in part explain the therapy refractive nature of S. prolificans. In conclusion, we found that S. prolificans represents the most prevalent Scedosporium species in the respiratory tract of CF patients and immunocompromised patients in Northern Spain. In CF patients, P. boydii or S. prolificans were exclusively found as respiratory colonisers. All patients were colonised over years exclusively with isolates affiliated to one Scedosporium species, but to multiple AFLP genotypes carrying variable AFSP.

Hypoxia can regulate the degree of inflammation and the anti/pro-tumoral functions of immune cells in the tumor microenvironment, thus tilting selleckchem the balance between cancer progression and regression [43-45]. Furthermore, both pro- and antiapoptotic consequences of hypoxia have been documented depending on the cellular context [42],

resulting in cell death [46], or survival [47] of distinct immune cell populations. Recent evidences indicate that low pO2 can affect NK-cell differentiation from hematopoietic stem cells in vitro [48]. Limited information, however, is currently available on the impact of hypoxia on mature, ready to kill, NK cells. In this study, we investigated this issue and we show that NK cells can adapt to the hypoxic environment by upregulating HIF-1α. This response is associated with inhibition of the NK-cell LY294002 cytolytic activity against tumor or virally infected target cells, without significantly affecting ADCC. We analyzed whether hypoxia affected NK-cell viability. To this end, NK

cells were isolated from PB of healthy donor, cultured with IL-2 under hypoxic (1% O2) or normoxic (20% O2) conditions. Cells were then harvested after 96 h and analyzed for Annexin V (AV)/ propidium iodide (PI) staining to detect apoptotic/necrotic cells. As shown in Figure 1A, there was no loss of cell viability under hypoxia, as indicated by a similar high percentage of viable nonapoptotic NK cells in both normoxic and hypoxic cultures. The response of NK cells to hypoxia was assessed by evaluating the expression of HIF-1α. HIF-1α protein levels were measured by Western blot analysis of cell lysates from NK cells either freshly isolated or cultured under

normoxic or hypoxic conditions (either in the absence or in the presence of IL-2). As shown in Figure 1B, HIF-1α expression was not detectable in fresh cells or in cells cultured under normoxia but was rapidly induced at 3 h and maintained up to at least 48 h in NK cells cultured under hypoxic conditions. Interestingly, HIF-1α was inducible by hypoxia in both resting and IL-2-treated NK cells. We next assessed whether hypoxia could HSP90 modulate NK-cell function. First, we evaluated the effects of hypoxia on the expression of the main receptors capable of triggering cytolytic activity in short-term cultures. Surface expression of NCRs (NKp46, NKp30, and NKp44), NKG2D, and CD16 was assessed by flow cytometry on freshly isolated PB NK cells and after culture under normoxic or hypoxic conditions. As shown in Supporting Information Fig. 1, hypoxia downregulated NKp46, NKp30, NKG2D, and, minimally, CD16 expression on resting NK cells (i.e. on NK cells cultured without IL-2). More importantly, hypoxia was effective also on activated NK cells.

The donors recognized four peptides of the 23 20-mer peptides in DENV-1, five peptides of the 35 20-mer peptides of DENV-2, five peptides of the 35 peptides of the DENV-3 and five peptides of the 28 20-mer peptides of DENV-4 (Table 2). All dengue immune donors responded to the peptides of at least two DENV serotypes. Two donors responded to peptides of all four DENV serotypes. The number of healthy donors responding to at least two peptides of the four DENV serotypes in the cultured ELISPOT assays is shown in Table 3. Eight of 20 (40%) of the individuals responded

to at least two peptides of DENV-4 and responses to at least two peptides of other serotypes ranged from 30 to 50% (Table 3). The frequency RAD001 cost of cultured ELISPOT responses to each of these peptides is shown in Fig. 1. These peptides had <15% homology between the four DENV serotypes except for 30% homology for four peptides (DENV-1 peptide with DENV-1 pep-11, DENV-2 pep-33, DENV-4 pep-12, DENV-2 pep-11, DENV-3 pep-11. DENV-2 peptide 17 with DENV-3 pep-21, DENV-3 pep-11 with DENV-4 pep-19). Of the 19 conserved and non-cross-reactive regions identified from the four DENV serotypes, two peptides were from the envelope region,

one peptide from the DENV-2 was from the NS1 region, six peptides were from the NS2A region, two peptides from the NS2B region, one peptide of learn more DENV-1 was from the NS3 region, four peptides were from the NS4A region and three peptides were from the NS5 region (Table 2). Of the six peptides identified which were from the NS2A Tenofovir region, one peptide each was from DENV-2 and DENV-3, two peptides from DENV-4 and two of the peptides were from DENV-1. Three of six of these peptides were from the region represented by amino acids (aa) 99–133, and two of six peptides were from the region represented by

aa 184–216. One peptide from DENV-4 was from the aa 135–148. Variants of all the peptides are shown in supplementary Table S1 and are based on NCBI Virus Variation website data. In the current study we have used the most common sequence, which accounted for >90% of the detected variation in the majority of cases. The three peptides, from aa 99 to 133, were again found to be highly conserved. Of these three peptides, peptide 28 of DENV-3 (RENLLLGVGLAMATTLQLPE), which was the most frequently recognized peptide among all donors (nine of 20), had two changes in the amino acids in only two sequences. In these two variants, threonine in position 14 is replaced by alanine and arginine in position 17 was replaced by methionine. Peptide 10 of DENV-4 (AMTTTLSIPHDLMELIDGIS) had the amino acid leucine in position 6 replaced by isoleucine in some sequences. Although we also used this sequence in our peptide matrix, we did not detect any responses to the sequence with the altered amino acid.