The ureter was ligated at approximately 1 cm below the renal hilum with 3-0 silk suture. The abdominal wound was closed, and rats were returned to the cages. Control rats underwent abdominal learn more incision and approximation with no ligation of the ureter.19,20 Six rats of the two groups were killed at 14 days and 28 days after surgery, respectively, and their renal tissues were collected for histological and molecular biology determination. After

10% neutral formaldehyde fixation, the renal tissues were dehydrated through a graded ethanol series and embedded in paraffin. Sections were prepared on a microtome and stained with masson’s trichrome staining. Renal pathology was observed by light microscope, the severity of the renal lesion was presented by the RIF index. Blue granular or linear deposits were

interpreted as positive areas for collagen staining. Semi-quantitative evaluation was performed by computer-assisted image analysis (DMR + Q550, Leica Co., Germany). The area of positive staining for fibrosis was measured at 400-fold original magnification in 50 fields (ignoring the fields containing glomerular parts) and expressed as a percentage of the total area.21 The extent of interstitial fibrosis was scored as absent (0), involving less than 25% of the click here area (1), involving 26–50% of the area (2), and involving greater than 50% of the area (3).22 RIF index was obtained by the formula as follow: GSI = (0 × n0 + 1 × n1 + 2 × n2 + 3 × n3)/(n0 + n1 + n2 + n3) = (0 × n0 + 1 × n1 + 2 × n2 + 3 × n3)/50. All the fields were selected from coded sections for each rat at random and the scores obtained by two investigators were averaged. Cell apoptosis was examined by the TdT mediated dUTP nick end labelling (TUNEL) assay (Roche Inc., Basel, Switzerland) as described

previously.23,24 Six slides from each kidney were evaluated for percentage of apoptotic cells by using the TUNEL assay. Then 10 watch fields, which didn’t include the glomerular parts, were chosen at random under a microscope on each section. Brown staining not of cell nuclei was considered as apoptotic cells. Positive brown cells and total cells were counted. The formula for apoptosis index as the indicator of apoptosis was as follows:23 cell apoptosis index = positive cells/total cells × 100%. The scores obtained by two investigators were averaged. Renal tissue fixed with 4% buffered paraformaldehyde was embedded in paraffin, and 4 µm thick sections were stained. The positive area was measured quantitatively using a computer-aided manipulator (DMR + Q550, Leica Co., Germany). For immunohistochemical analysis of PHB, Caspase-3, TGF-β1, collagen-IV (Col-IV) and fibronectin (FN), the sections were deparaffinized, washed with phosphate-buffered saline (PBS), and treated with 3% H2O2 in methanol for 10 min.

Therefore, for amplifying the O157-9 locus of the O26 and O111 serogroups, we designed a new reverse primer to equate the size of the offset sequence from the O26/O111 isolates with that from O157. By using this new reverse primer, we found that the O157-9 locus of the O26 and O111 isolates exhibited high allele numbers (11 and 12, respectively) and high D values (0.81 and 0.87,

respectively) (Fig. 1a). Two loci (O157-19 and O157-25) were also present in the genome sequences of O26 and O111, but showed no repeat copy number variation between the O26 and O111 isolates. There were some problems associated with the O157-34 locus. Re-inspection of the sequence of the O157-34 locus revealed that O157 contained two repeats in this locus in addition to those described PARP inhibitor PS-341 cell line in a previous study (15) (Fig. 2). Furthermore, although the sequenced O26 and O111 strains contain one and three repeats, yielding PCR products of 153 bp and 195 bp, respectively, a sequence variation, including a 6-bp deletion, was found in the O157-34 locus-flanking region of the O26 genome sequence. Therefore, we set the offset size for O157 and O111 at 141 bp and

that for O26 at 135 bp. To summarize, of the nine loci that are currently used for analyzing the O157 isolates, eight were not suitable for analyzing the O26 and O111 isolates when the original primers were used (Fig. 1a). Only the O157-37 locus could be used for the O26 and O111 isolates, which exhibited D values of 0.25 and 0.93, respectively. When a new O157-9 reverse primer was used for the O26/O111 isolates, the O157-9 locus in both the O26 and O111 isolates exhibited high D values. Among the nine additional genomic loci that we used in the present study, three were previously used for O157 analysis (EH157-12, EHC-1, and EHC-2, designated as O157-13, O157-11, and O157-2, respectively, in the previous report (15))

and six were newly developed Ribonucleotide reductase (EH26-7, EH111-8, EH111-11, EH111-14, EHC-5, and EHC-6). Of these nine loci, EHC-1 was very useful for genotyping all the serogroups: the D values were 0.83, 0.91, and 0.85 for the O26, O111, and O157 isolates, respectively. EHC-2 was also useful for all the serogroups, especially for the O26 isolates that exhibited an extremely high D value (0.92). EH157-12 was suitable mainly for O157 and exhibited moderate D values for the O26 and O111 isolates, despite the low allele numbers in these two serogroups. EHC-5 and EHC-6 also yielded high or moderate D values for all the serogroups. Although these five loci are not included in the current MLVA system for O157, they can be used for analyzing the O157 isolates, as well as the O26 and O111 isolates.

However, the negative results obtained by daily injections of TNF-α and the fact that anti-TNF-α or soluble TNF-α receptors (etanercept) did not modify the tolerance induced by LPS in vitro indicated clearly that, in our hands, TNF-α is not a cytokine responsible for the establishment of tolerance. Our results are in agreement with those of Medvedev et al. [48], but not with other authors, who suggested that TNF-α was capable

of inducing LPS tolerance [49,50]. This discrepancy could be the result of using a different animal model (rat) and/or the fact that these experiments were carried out using a non-physiological dose of TNF-α (200 µg/kg/day for 5 consecutive days) [49] or from a different species [50]. However, as GC and Dex inhibit BTK pathway inhibitors the production of a set of proinflammatory cytokines such as TNF-α, IL-1-α, IL-1β, IL-12, IFN-γ, IL-6 and IL-8 [28,51,52], this suggests that inflammatory agent(s) other than TNF-α would be necessary for the establishment of LPS tolerance. In line with this, we have found previously Rucaparib purchase that IL-1β was capable of inducing the establishment of endotoxin tolerance, an effect determined through protection against LPS, increasing the level of GC and by down-regulation of Toll-like receptor 4 (TLR-4) and up-regulation of GC receptors, both indicators of endotoxin tolerance

[53]. Considering that RU486 can overcome the tolerant state, and taking into account all the previously described data, a central role for GC in the maintenance of endotoxin tolerance is suggested. Similarly to GC, IL-10 has been recognized as an important cytokine in tolerance, although its mode of action is also controversial. In fact, some authors consider IL-10 to be a central cytokine

for the establishment of tolerance [25], while others consider that IL-10 is critical for the maintenance but not for the establishment of endotoxin tolerance [54,55]. The fact that we found a low level of IL-10 in tolerant animals and high values in RU486-treated tolerant mice suggests that this cytokine is not Tideglusib crucial in the maintenance of tolerance. This is in line with Baykal et al. [56] and with those authors who show that IL-10 knock-out mice (IL-10–/–) can be tolerized by LPS [54]. However, we cannot discard a possible role for IL-10, as redundant mechanisms in the regulation of endotoxin tolerance could be possible, although it has been shown that this anti-inflammatory cytokine regulates GC synthesis in a negative manner through the inhibition of adrenocorticotrophic hormone (ACTH) effects [57,58]. During recent years, endotoxin tolerance has been reported as one of the causes of immunosuppression in Gram-negative infections and considered to be one of the principal causes of mortality in late sepsis [23,32].

Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized BMS-354825 chemical structure integrin β1 to lysosomal compartments with

a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. “
“The transcobalamin II (TCN2)

INK 128 ic50 776C>G polymorphism has been reported to be a genetic risk factor for idiopathic recurrent

spontaneous abortion (RSA). However, the sample size in previous studies was small, and other TCN2 polymorphisms have not been studied. Moreover, the TCN2 67A>G and 776C>G polymorphisms, and the transcobalamin II receptor (TCblR/CD320) Rebamipide 1104C>T polymorphism, have demonstrated associations with immune responses. Three hundred and seventy-eight RSA patients who had at least two consecutive spontaneous abortions were enrolled. Two hundred and seven control subjects were collected from a convenience sample. Polymerase chain reaction and restriction fragment length polymorphism analysis were performed to identify the TCN2 67A>G and 776C>G polymorphisms, and the TCblR 1104C>T polymorphism. RSA patients showed significantly different frequencies of the TCN2 67AG+GG genotypes compared with control subjects. The TCN2 67G allele is a possible risk factor for idiopathic RSA. “
“Infection with murine gammaherpesvirus 68 has become an accepted model for studying the virus/host interactions with regard to gammaherpesvirus infections. Previous studies using gene-deficient mice have revealed that neither IFNγ nor perforin is essential in controlling the outcome of infection or the virus load during chronic infection in C57BL/6 mice. However, pronounced multiorgan fibrosis and splenic atrophy are observed in mice lacking IFNγ or the IFNγ receptor.

As shown in Fig. 2B, CCL25 levels were increased in supernatants

of IL-4-stimulated meso-thelial cells within 12 h, suggesting that those cells may be an important source of CCL25 during allergic pleurisy. IL-4 stimulation did not induce CCL25 production by mesothelial cells recovered from unsensitized mice. OVA challenge find protocol also induced the accumulation of γδ T cells expressing CCR9 and α4β7 integrin in previously sensitized mice within 48 h (Fig2.C). Interestingly, the majority (65%) of CCR9+ γδ T cells recovered from OVA-challenged mice coexpressed α4β7 integrin. The representative histograms (Fig. 2D) show the increased expression of CCR9 on pleural γδ T cells recovered from OVA-stimulated mice as compared Quizartinib concentration with those from nonstimulated mice (SAL 223.9 ± 36.5

versus OVA 336.1 ± 41.9 mean fluorescence intensity (MFI)). No increase in the expression of α4 integrin chain (SAL 42.6 ± 1.3 versus OVA 37.5 ± 0.7 MFI) and α4β7 integrin (SAL 168.8 ± 6.9 versus OVA 105.5 ± 8.3 MFI) by γδ T cells were observed between groups (Fig. 2D). The involvement of CCL25 in γδ T-cell migration during an allergic response was assessed. The anti-CCL25 monoclonal antibody (mAb) treatment failed to inhibit γδ or αβ T-cell migration to pleura 48 h after OVA challenge (Fig. 2E and F). However, the in vivo neutralization of CCL25 specifically impaired the accumulation of γδ T cells expressing α4β7 integrin (Fig. 2G). Since CCL25 induced the migration of α4β7+ γδ T lymphocytes, we further evaluated the role of α4 integrins on γδ T-lymphocyte migration induced by this chemokine. The in vitro blockade of α4 integrin chain and α4β7 integrin by mAbs inhibited γδ T-cell transmigration across endothelial monolayers prestimulated with the Th2 cytokine IL-4 toward CCL25 and cell-free pleural wash recovered from OVA-challenged mice (OPW; which contains CCL25) (Fig. 3A and B). Cell-free pleural wash recovered from saline-injected mice (SPW) was used as negative control. To confirm that α4β7 integrin mediates γδ T-cell transmigration from blood into inflamed pleura during

allergic response, Etomidate 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled splenocytes recovered from OVA-challenged mice, ex vivo treated (or not) with anti-α4 integrin mAb, were adoptively transferred into recipient mice 24 h after OVA i.pl. challenge. Adoptively transferred γδ T cells migrated into challenged mouse pleura in a higher extent than into saline-injected recipient mice, a phenomenon which was reduced by α4 integrin blockade (Fig. 3C and D). Moreover, the in vivo blockade of α4β7 integrin inhibited the migration of γδ T lymphocytes into mouse pleural cavities after OVA challenge (Fig. 3E). By contrast, the pretreatment with anti-α4β7 integrin failed to inhibit the migration of αβ T lymphocyte (Fig. 3F).

A further issue relates to whether or not nephrectomy increases

the risk of developing hypertension in the long term. An increase in BP is commonly observed following nephrectomy, however, an increase in BP into the hypertensive range in previously normotensive individuals, remains to be determined.8,9 Studies examining this possibility are varied and have often used different control groups. Most commonly, the general population is used, and this may not be the most appropriate group to compare with healthy donors. A number of studies report an incidence of hypertension following nephrectomy ranging from 9% to 48%.9–19 It is important to note that the definition of hypertension varies between these studies. Additionally, there are no studies that compare age- and gender-matched individuals in a prospective manner for individuals who either undergo nephrectomy or are followed without Vadimezan datasheet a nephrectomy. Torres et al.10 followed patients post-nephrectomy for 10 years

and defined hypertension as a systolic/diastolic BP of ≥160/95 mmHg. Ten of 66 patients (15%) who were previously normotensive became hypertensive and 9/24 (38%) of patients who had borderline hypertension developed hypertension according to the study definition. Clearly, the level of BP used to define hypertension here, is much higher than is generally used selleck kinase inhibitor now and the relevance of the data from this study remains unclear. Another study of 250 patients followed long-term for up to 10 years or more, demonstrated that ‘borderline hypertension’ (defined as 150–159/90–94 mmHg) developed in 8.8% and definite hypertension old (160/95 mmHg or greater) developed in 5.6% of patients. The investigators compared the incidence of hypertension with the general population and concluded that this was lower than that seen in age-matched individuals.16 Some small studies comparing BP in donors to control groups have suggested an increase in the risk

of developing hypertension.19–21 However, most of the larger studies have not confirmed this. Goldfarb et al.22 studied 70 donors followed for a mean time of 25 years and found no increase in the risk of developing hypertension compared with age-matched individuals. Two larger studies, one of 402 donors with a mean follow up of 12 years23 and another of 733 donors with a follow up of up to 30 years or more,24 showed that the age-matched incidence of hypertension was not increased. Grossman et al.25 followed 152 donors with a mean time after uninephrectomy of 11 ± 7 (range: 1–28) years with a 93% retrieval rate. BP increased from 125 ± 15/79 ± 11 to 134 ± 19/81 ± 9 mmHg (P < 0.01) but remained in the normotensive range. A large meta-analysis by Kasiske et al.26 of the long-term effects of reduced renal mass in humans examined mostly nephrectomy for renal donation, however, the group of patients was not uniform.

This demonstrates a STAT4-dependent, IL-12/IL-23-independent pathway of parasite control. Type I IFNs

are important in viral and other infections and can activate STAT4. We found that IFN-α/βR-deficient mice have a nonpersistent, early IFN-γ defect, and a persistent, early IL-10 defect, without changes in serum IL-12 or LN-derived nitric oxide. We found less IL-10 per cell in CD25+CD4+ T cells and possibly fewer IL-10-producing cells in the draining LN of IFN-α/βR-deficient vs. wild-type mice. IFN-α/βR-deficient mice have chronic, nonprogressive disease, like wild-type mice, suggesting that IL-10 and IFN-γ defects may balance each other. Our data indicate that although type I IFNs help promote early Th1 responses, they are not the missing activators of STAT4 responsible for partial control SAHA HDAC cell line of L. mexicana. Also, the lack of lesion resolution in IFN-α/βR-deficient mice despite lower IL-10 levels indicates that other pathways independent of T cell IL-10 help prevent an IL-12-driven clearance of parasites. Leishmania (L.) mexicana, a New World intracellular protozoan parasite, causes chronic cutaneous infection in mice and humans. The immunology and outcome of infection

of L. mexicana are quite different from those of the better-studied Old World relative, L. major. In C57BL/6 (B6) this website mice, L. major induces a strong Th1 response with interleukin (IL)-12-driven interferon (IFN)-γ from CD4+ T cells. This IFN-γ leads to an upregulation of inducible nitric oxide synthase (iNOS) in infected macrophages, which in turn generates nitric oxide, leading to killing of the intracellular L. major. The outcome is that rapidly growing lesions develop but then heal,

with very low persistent parasite burdens (typically <100 per lesion). In L. mexicana infection of B6 mice, a very weak IFN-γ response occurs and parasites induce chronic, but nonprogressive, lesions that plateau in size at around 10–12 weeks post-infection, with higher parasite burdens (generally 107–108 per lesion). Understanding the similarities and differences between these two related parasite infections is instructive in dissecting the immunological mechanisms. We have found that despite these learn more differing outcomes, many of the pathways involved in control of L. mexicana parallel those seen in L. major infection. B6 mice lacking IFN-γ or iNOS have progressive disease with lesions that do not plateau in size and parasite burdens much higher than in wild-type (WT) mice (1). Interestingly, the Th2 response, as determined by IL-4 production, is not increased in the IFN-γ- and iNOS-deficient mice, showing that susceptibility is because of a lack of an adequate Th1 response rather than an increased Th2 response. We also found that mice lacking STAT4, an important signalling molecule, had progressive disease (1) similar to the increased susceptibility of STAT4 knockout (KO) mice to L. major (2).

CD122 was expressed at only marginal levels by both induced and natural CD8+Foxp3+ T cells (Fig. 4C), consistent with the finding that CD8+CD122+ Tregs lack Foxp3 expression 8. In contrast, all T-cell populations were predominantly CD28+ (Fig. 4C). IL-6 was recently suggested to positively regulate the expansion of CD8+Foxp3+

T cells in vitro and in vivo 17. We, therefore, compared IL-6Rα (CD126) expression among the different subsets to judge their potential sensitivity towards IL-6. Interestingly, CD126 expression was absent from both induced CD8+Foxp3+ and CD8+Foxp3− T-cell populations, whereas CD126 https://www.selleckchem.com/products/apo866-fk866.html expression was noted on all T-cell populations ex vivo (Fig. 4C). Notably, naturally occurring CD8+Foxp3+ T cells expressed a CD8-αβ heterodimer, TCR-αβ, CD3-ε (data not shown) and partially CD4 (Supporting Information Fig. 4); the latter consistent

with previous reports 2, 25. In summary, CD8+Foxp3+ T cells express classical CD4+Foxp3+ Treg markers in a pattern distinct from activated CD8+Foxp3− T cells and previously described CD8+ Tregs. Since Foxp3 is expressed by certain effector T-cell populations in humans 26 and IFN-γ is an important effector molecule of CD8+ T cells, we next asked whether CD8+Foxp3+ and CD8+Foxp3− T-cell populations differ in IFN-γ expression. CD8+Foxp3+ and CD8+Foxp3− T cells were generated from Rag1−/−×OTI selleck products mice. Additionally, WT splenocytes were obtained and all populations were restimulated with PMA/ionomycin. Importantly, the majority (75.8%) of activated CD8+Foxp3− T cells produced IFN-γ, whereas almost no IFN-γ production (5.5%) was observed in induced CD8+Foxp3+ cells (Fig. 5A),

consistent with a previous study 27. Similarly, fewer CD8+Foxp3+ T cells produced IFN-γ in comparison to their Foxp3− counterpart ex vivo (Fig. 5A). IFN-γ production LY294002 by CD8+ T cells activated under Foxp3-inducing conditions could be partially restored when Foxp3 was mutated (Supporting Information Fig. 3D), yet Foxp3-independent mechanisms also seem to be involved in the repression of IFN-γ. Since suppressive function is a hallmark of Tregs, we finally tested induced CD8+Foxp3+ T cells in in vitro suppression assays. Suppressive activity was compared with activated CD8+Foxp3− T cells, CD4+Foxp3+ nTregs and induced CD4+Foxp3+ Tregs, all isolated based on eGFP reporter expression. Interestingly, not only CD8+GFP+ T cells but also activated CD8+GFP− T cells showed a mild suppressive effect on CD4+ (Fig. 5B) and CD8+ (Supporting Information Fig. 5) T-cell proliferation and on IFN-γ production by CD8+ T cells (Fig. 5C), which was however inferior to that of CD4+GFP+ natural and induced Tregs (Fig. 5B and C). In conclusion, CD8+Foxp3+ T cells are actively restricted in pool size and not enriched in suppressive function, although they share certain developmental and phenotypic characteristics with CD4+Foxp3+ Tregs.

Despite this, the lactobacilli inhibited IL-13 induction, regardless of donor, either allergic or not. In the long-term cultures and the αCD3/αCD28-stimulated cultures, the increased IFN-γ and IL-12 MLN0128 mw secretion in hPBMC cultures exposed to the lactobacilli could mediate the Th2-suppressive effect, as observed previously (Pochard et al., 2002; Bickert et al., 2009). However, the Th2 cytokine inhibition could be dependent on several parameters depending on the strains used (Pochard et al., 2002; de Roock et al., 2010; Lopez et

al., 2010). The exact mechanism by which probiotic lactic acid bacteria modulate the host immune response is largely unknown. Bacterial cell surface macromolecules (such as long surface appendages, extracellular polysaccharides and teichoic acids) are in direct contact with several immune cell types by binding various pattern learn more recognition receptors of the host. The structure

of the main cell wall macromolecules is strongly conserved, but various modifications, such as glycosylation and also quantitative differences, can contribute to the strain-specific properties of probiotics. As little information is available regarding the specific bacterial components that for example induce the expression and production of cytokines, advances can be made in this area through the sequencing of genomes and transcriptomes that can be correlated to measured effects and enable testing which bacterial genes and Fenbendazole derived components are essential to specific immunomodulatory properties (Borchers et al., 2009; Fink, 2010; Kleerebezem et al., 2010; Lebeer et al., 2010; Meijerink et al., 2010). Large numbers of candidate strains are often tested as probiotics for immunomodulating properties in a variety of in vitro models

to select those strains with the best characteristics. In these in vitro studies, effects of heat-killed bacteria may not be directly extrapolated to effects of viable bacteria. Nevertheless, recent literature shows similar effects comparing live bacteria with heat-killed bacteria or even with components from the respective bacteria (Ghadimi et al., 2008; Li et al., 2009; van Hoffen et al., 2010). Very limited information is available with respect to the in vivo molecular responses to probiotic bacteria in human mucosal tissues; however, a recent study of van Baarlen et al. (2009) showed a considerable overlap between in vivo human responses to live and heat-killed L. plantarum, provided that these bacteria were harvested from the same phase of growth. Systematic studies to link in vitro data to in vivo effects have rarely been performed so far and results are also not found to be consist (Foligne et al., 2007). Based on the limitations of the in vitro model, extrapolations to in vivo effects must therefore be considered with caution.

Thus anti-CD33 antibodies eliminate malignant myeloid cells selectively while sparing normal stem cells [70]. The first humanized CD33 molecule approved by the Food and Drug Administration (FDA) was conjugated with calicheamycin (gemtuzumab). Trials exploring single-agent use of gemtuzumab have achieved

remission only in the in the range of 15%, but gemtuzumab used together with other agents to treat selleck chemical relapsed or refractory leukaemia are promising [71–77]. The most significant toxicity reported is liver injury, occurring most commonly when gemtuzumab is used in combination with thioguanine or in the setting of allogeneic stem cell transplantation [78]. Antibody treatment has been reviewed recently [79]. AML cells are weak stimulators of T cells and often possess mechanisms that prevent induction of T cell response and induce resistance to cytotoxicity (see above). Simple vaccination

with irradiated blasts with BCG or other cytokines resulted in prolongation of remission but with no improvement in survival [1]. To increase the susceptibility of AML to immune attack, investigators have sought to improve antigenicity of the leukaemia by transfection of genes for co-stimulatory molecules such as 4-1BB ligand [80], combinations of CD80 and IL-2 [81] or by differentiating the blasts into leukaemic DC. In a study of 22 AML patients, DC were generated successfully in five and used to treat patients in remission. However, only selleckchem two of these patients were long-term survivors [82]. Alternatively, DC have been generated from AML patients in remission and made more antigenic by Exoribonuclease fusion with AML blasts [83], exposure to AML lysates or peptide antigens or transfection

with RNA [84]. A clinical trial with a monocyte-derived DC loaded with mRNA for Wilms tumour-1 (WT1) antigen is under way [85]. Although immune responses to AML can be enhanced in vitro with these approaches, clinical data are scanty and clinical responses in small diverse patient series is still very preliminary (reviewed in [86]). A recent review listed more than 14 candidate leukaemia-associated antigens expressed by AML, some of which have formed the basis for developing antigen-specific vaccines using DNA or peptides [87]. Most widely researched and developed as peptide vaccines in clinical trials are the HLA-A2 peptide epitopes of WT1 (WT1126), proteinase 3 (PR1) and hyaluronan-mediated motility receptor (RHAMM)/CD168 (receptor for hyaluronic acid mediated motility), and an HLA A24-specific epitope of WT1 [88]. Vaccines have been combined with the BCG-based adjuvant, montanide, keyhole limpet haemocyanin (KLH) or incomplete Freund’s adjuvant, with or without concurrently administered GM–CSF [89]. All these peptides induce immune responses with increases in tetramer-positive T cells producing gamma-interferon after peptide stimulation.