8,9 The T reg cells function to dampen immune responses through a variety of approaches, including contact-mediated inhibition, secretion of perforin and granzyme A/B, sequestration of key growth factors such as IL-2, and secretion of suppressive cytokines including TGF-β, IL-10 and IL-35.7 Interleukin-10 in particular plays an important role in immune homeostasis, both in mice10 and humans,11 suggesting that it has several non-redundant find more roles in regulating inflammatory responses. Many cell types in addition to Foxp3+ cells12 can produce IL-10, most notably several lineages of CD4+ T cells,13 including Th1,14–16 Th214,17 and Th1718–20

cells, as well as various types of Treg cells.21 In a feed-forward mechanism, IL-10 can drive its own expression through the induction of an IL-10-producing Treg-cell population termed Tr1 cells.22,23 Conversely, IL-10 can also be induced independently of IL-10 signalling in both Foxp3+ and Foxp3− Treg-cell populations.24 Given its potent anti-inflammatory effects, various strategies are being explored to target IL-10 for therapeutic intervention.25 The intimate interplay between the critical factors in development

of Treg and Th17 cells, along with the dual reliance on TGF-β signalling for MI-503 datasheet their differentiation,26 has led to conceptualization of a Treg–Th17 axis. From a therapeutics perspective, the identification of drugs that promote pro-inflammatory or anti-inflammatory responses by influencing differentiation along this axis has gained momentum as examples of T-cell plasticity continue to be characterized,27 in particular within the Treg-cell and Th17-cell populations.28 Moreover, several reports have characterized ‘hybrid’ T-cell populations where Foxp3 is expressed in various effector T-cell populations,29 and IL-10

can be produced by Th1, Th2 and Th17 cells.12 These results PD184352 (CI-1040) suggest that it may be possible to treat disease by shifting the balance along the Treg–Th17 axis in situ during ongoing immune responses. For example, one mechanism to dampen inflammation would be to induce IL-10 expression within Th17 cells participating in pathological inflammation. To that end, targeting non-cytokine signalling pathways may be a viable option. For example, ATP,30 sphinogosine-1-phosphate31 and vitamin D32 can modulate Th17 development, whereas antigen-presenting cell (APC)-derived indolamine 2,3-dioxygenase33 and retinoic acid34 can promote Treg-cell populations, highlighting the importance of non-cytokine signalling pathways to this paradigm. Estrogen is a well-documented modulator of immune function in humans and mice, capable of increasing the expression of Foxp335 and IL-10.

Deterioration

of the renal allograft function after the biopsies was seen in 31 patients (62%), of which 11 lost their graft. We suggest that histopathological changes of transplant glomerulopathy might be accompanied by inflammation of the microvasculature, such as transplant glomerulitis and peritubular capillaritis, thickening of the peritubular capillary basement membrane, and circulating anti-HLA antibodies. C4d deposition in the PTC is not always present in biopsy specimens of TG. We speculated that C4d deposition in the GC, rather than that in the PTC might be a more characteristic manifestation of TG. Many of the patients with TG had a history of AR. Anti-HLA antibody Class II, particularly when the antibody was DSA Class II, appeared to be associated with the development of TG. The prognosis of grafts exhibiting TG was not too good even under the currently used immunosuppressive protocol. Transplant glomerulopathy (TG) is Akt inhibitor a morphologic pattern of chronic kidney allograft injury and is

generally associated with poor renal allograft survival.[1] TG is characterized by double contours of the glomerular basement PLX-4720 order membranes (GBM), often accompanied by increased mesangial matrix.[2] TG is included as a criterion of chronic active antibody-mediated rejection (c-AMR) in the Banff ‘09 classification.[3] In this report, we discuss the clinical and pathological analyses of patients developing TG after renal transplantation. During the period from January

2006 to October 2012, TG was diagnosed in 86 renal allograft biopsy specimens (BS) obtained from 50 renal transplant recipients who were followed up at our institute. The data of these 86 BS and 50 patients were retrospectively reviewed from the clinical records in this study. The immunosuppressive protocol mainly consisted of triple-drug therapy, including methylprednisolone (MP), cyclosporine (CYA) or tacrolimus (TAC) and mizoribine (MZ), azathioprine (AZ) or mycophenolate mofetil (MMF) (Table 1). In some cases, basiliximab and rituximab had been given in addition (Table 1). Renal allograft biopsy was performed as part of the diagnostic workup for graft dysfunction 4��8C and proteinuria, or as protocol biopsy. The biopsy specimens were examined by light, electron and immunofluorescence microscopy. The biopsies were diagnosed and scored according to the Banff ‘09 classification.[3] TG was diagnosed by light microscopy based on the finding of double contours of the GBM.[2] Patients with hepatitis C virus-associated glomerular disease and thrombotic microangiopathy were excluded from this study. We used the ptcbm score, which showed thickening of the peritubular capillary basement membrane and was evaluated by light microscopy (LM) in place of diagnosing of peritubular capillary basement membrane multilayering (PTCBMML) by electron microscopy (EM).

Production of immunoglobulins was lower in ST subjects as a result of reduced survival and not lower proliferation SB203580 nmr of B cells. Increased apoptosis of B cells in the MB0 group can result in fewer cells developing into antibody-secreting cells upon stimulation, hypogammaglobulinaemia and poor humoral response to antigens. For CVID MB1 patients a different mechanism should be responsible, because their B cells behave like control B cells in their sensitivity

to apoptosis. This holds true for the two evaluated CVID MB2 patients. Their B cell apoptosis rescue was similar to CVID MB1 patients and controls (data not shown). In a recent paper, Borte et al. [35] suggested that IL-21 restores immunoglobulin production in patients with CVID. Using purified B cells, they found that IL-21 reduced apoptosis from naive and memory B cells from 14 CVID patients. However, no CVID group distinction was made; stimulation with anti-CD40 and IL-21 also included IL-4, and they considered only the CD27– naive and CD27+ IgD– memory B cell populations (excluding CD27+IgD+). The proportion of MB1/MB2 to MB0 patients in their studied cohort

might have influenced the final result and explain the apparently distinct conclusions. We cannot exclude the possibility that the peripheral blood B cells with increased apoptosis found in CVID MB0 could be the result of incomplete activation by follicular CD4+ T cells. In keeping www.selleckchem.com/Akt.html with this, Hagn et al. [36] have demonstrated that human B cells co-cultured with incompletely activated CD4 T cells that secrete IL-21, but do not express CD40L, differentiate into granzyme B (GzmB)-secreting and potentially cytotoxic Endonuclease cells, able to induce slowly developing apoptosis of several cell lines. Activation of human B cells by IL-21 and BCR engagement in the absence of CD40 ligation results in their differentiation into GzmB-secreting

cytotoxic cells rather than into plasma cells. In summary, our findings reinforce the fact that (in humans) the net effect of different stimuli on B cells depends upon both the B cell subpopulation studied and the activation status of the B cell and underscore the relevance of these features in CVID physiopathology. We suggest that higher levels of apoptosis of CVID MB0 CD27+ B cells during an immune response can result in lower levels of immunoglobulin production, irrespective of their proliferation. The results highlight the heterogeneity among CVID patients, where distinct molecular mechanisms underlie common clinical symptoms, and highlight the need to classify and study CVID patients separately when evaluating B cell responses. A.C., J.P., N.L. and J.M.F. designed and performed the experiments and analysed the data. N.M. and J.P. contributed to patient selection. All authors contributed to writing the manuscript.

On day −1, mice were injected i.p with 0.5×106 BM-derived DC, were pulsed with either 10 μg/mL of TCR peptide B5 (group one) or the control B1 peptide (group two). A third group

of mice were injected with PBS only. On day 0, mice were challenged with MPBAc1-9/CFA/PTx and EAE was monitored. Injection of DC pulsed with peptide B5 was associated with significant protection from EAE compared with mice injected with B1-pulsed DC or PBS only (Fig. 5). The disease scores of mice treated with B5-pulsed Cilomilast manufacturer DC were significantly lower (p<0.0001) than mice treated with B1-pulsed DC. Collectively, these data demonstrate that DC loaded with TCR peptide B5 activate CD4+ Treg, resulting in protection against MBP-induced EAE disease. It has been widely demonstrated that CD4+ T cells with regulatory function can be harnessed to protect against inflammatory diseases. However, pathways leading to the priming or activation of antigen-specific CD4+ Treg have yet to be fully defined. Here the mechanism for the natural priming of antigen-specific CD4+FOXP3− Treg to a defined self-antigen derived from the conserved framework 3 region of the TCR is presented. This mechanism of CD4+ Treg priming is dependent on APC engulfing apoptotic Vβ8.2+CD4+ T cells, and processing and presenting a conserved TCR-derived antigenic determinant to the CD4+ Treg population. Notably, DC activation is required for

optimal priming of the Treg and CD8α+ DC seem to be most efficient in this priming. It was indicated by earlier studies that selleck chemicals llc the CD4+ and CD8+ Treg that suppressed the anti-MBP response in humans and mice were recognizing antigenic determinants associated with the disease-mediating CD4+ T-cell population 30–34. However, due to the lack of knowledge concerning the exact antigenic determinants recognized on the disease mediating cells, the unknown role of APC, and the paucity of defined CD4+ and CD8+

Treg clones, the mechanism of natural Treg priming had not been delineated. Studies presented here show that the naturally occurring TCR-peptide-reactive CD4+ Treg were stimulated upon co-culture with large numbers BCKDHA of irradiated spleen cells form naïve H-2u mice (Fig. 1). Stimulation of Vβ8.2 TCR peptide-reactive CD4+ Treg, but not irrelevant CD4+ T cells, indicated that APC (especially DC) within the splenocyte population present an MHC class II-associated TCR peptide. We have recently delineated the mechanism by which DC acquire TCR antigenic determinants from Vβ8.2+ T cells and present another TCR-derived antigenic determinant in the context of the non-classical MHC class I molecule Qa-1 to novel subset of CD8αα+TCRαβ+ Treg 24. As Vβ8.2TCR peptide-reactive CD4+ and CD8αα+TCRαβ Treg work in unison to down-regulate the Vβ8.2+ T-cell response 3, 15, 30, it is not surprising that DC are able to process and present different TCR-derived peptides in the context of class II and class Ib MHC molecules.

enterica Enteritidis African invasive isolate D24954 and laboratory strain PT4. The differential kinetics between cell-free killing and phagocytosis of invasive nontyphoidal Salmonella allows these bacteria to escape

the blood and establish intracellular infection before they are killed by the membrane attack complex. “
“Th1 cell-mediated adaptive immune response is very important but may not be sufficient to control Mycobacterium tuberculosis (M. tuberculosis) infection. The roles Rucaparib price of the various T cell subsets and cytokines in the inflammatory processes are not clearly elucidated. We investigated whether Th1, Th22 and Th17 cells mediated cellular immunity at the local site of M. tuberculosis infection in patients with tuberculous pleurisy (TBP).

The results showed that the cytokines IFN-γ and IL-22 but not IL-17 were elevated in tubercular pleural fluid. Following stimulation with immune-dominant peptides of early secreted antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) or Bacille Calmette–Guerin, pleural fluid mononuclear cells expressed high levels of cytokines IFN-γ, IL-22 and IL-17 as revealed by mRNA and protein measurements. In addition, we showed that cytokines IFN-γ, IL-22 and IL-17 were produced in M. tuberculosis-specific immune response by distinct subsets of CD4+ T cells with the phenotype of CD45RA−CD62L−CCR7+CD27+. Our results demonstrated for the first time that ESAT-6- and CFP-10-specific Th1, Th22 and Th17 STI571 cost cells existed in the patients with TBP and might

play an essential role against M. tuberculosis infection. The findings of this study raised the possibility of unravelling the critical targets for therapeutic intervention in chronic inflammatory diseases such as TBP. Tuberculosis (TB) is considered Selleckchem Bortezomib to be a global emergency. Approximately nine million people worldwide develop TB and 1.6 million people die of TB each year [1]. The vaccine administered to infants for TB is Bacille Calmette–Guerin (BCG), which has only limited efficacies. BCG protects against disseminated forms of TB in children, but it fails to protect against highly prevalent pulmonary tuberculosis (PTB) infection in adults [2, 3]. Importantly, it has been reported that approximately 90% of Mycobacterium tuberculosis-infected people develop a latent infection with no apparent clinical consequences. TB develops in approximately 10% of M. tuberculosis-infected individuals [3]. Therefore, there is an urgent need to understand the mechanisms of immune defence to help to control the epidemic. The Th1 cell-mediated adaptive immune response to M. tuberculosis infection is very important but not enough to control the disease [4–7]. However, the roles of the various T cell subsets and cytokines in the inflammatory processes are not clearly elucidated. In addition to IFN-γ, both IL-22 and IL-17 may contribute to the local immune response against M. tuberculosis infection.

Using immunohistochemical staining, we found no difference in cell infiltration between healthy skin and skin from challenge sites of non-responders after 48 h, indicating that the lack of response was not due to active down-regulation. In agreement with this, we did not find any significant up- or

down-regulation of gene expression in the challenge sites of non-responders after 48 h. Furthermore, mRNA expression in elicitation responses of patients with psoriasis and healthy controls could not be distinguished, either in the positive or negative reactions, indicating that the clinical selleck difference between the groups is not due to a difference in down-regulation at the elicitation site. Regulatory T cells have been found to be dysfunctional in suppressing auto-antigen specific effector T cells in various autoimmune diseases [22–25], but their regulation of environmental antigens were not investigated in these studies. It is theoretically possible that some sort of down-regulatory event took place before 48 h at which the biopsy was taken; however, a cellular response would be expected to be present at this time-point. The significance of a T helper type 17 (Th17) profile in

autoimmune diseases is well established through multiple areas of research, as reviewed by Steinmann [26], and patients with autoimmune diseases have Ku-0059436 been demonstrated to have higher than normal levels of circulating Th17 cells and cytokines such as interleukin (IL)-17, IL-6, IL-21, IL-22 and IL-23 [27]. We hypothesize that the highly Th17-directed cytokine milieu in patients with autoimmune diseases interferes with the mounting of a contact allergic response. This could be due to interference in the differentiation of naive T cells to become effector or memory T cells necessary for the contact allergic reaction or to regulation of antigen-presenting cells. Interestingly, in a murine study, Brandt et al. demonstrated that short-term incubation of in vitro-generated dendritic cells Casein kinase 1 (DC) with IL-21 significantly reduced their potential to induce an antigen-specific CD8+ T cell proliferation [28,29].

Antigen-presenting cells, particularly Langerhans cells, play a pivotal role in the sensitization phase of contact allergy, as they are responsible for the processing, transport and presentation of allergens to naive T lymphocytes in the skin, draining lymph nodes. Cumberbatch et al. found that the function of epidermal Langerhans cells, specifically Langerhans cells mobilization and migration, is profoundly impaired in the uninvolved skin of psoriasis patients compared with the skin of healthy volunteers [30]. The authors hypothesized that this could be due to disease progression characterized by systemic changes that affect Langerhans cell function. The systemic changes could be due to a Th17-skewed milieu found not only in psoriasis patients, but also in patients with other autoimmune diseases.

3F). In order to determine if miR-21 directly targeted PDCD4 expression, we performed a luciferase assay. Specifically, overexpression of miR-21 in Jurkat T cells transfected EPZ-6438 in vitro with a luciferase vector harboring the 3′UTR of PDCD4 resulted in reduced transcriptional

activity, suggesting that miR-21 targets directly PDCD4 expression in Jurkat cells (Fig. 3G). In addition, miR-21 overexpression resulted in inhibition of PDCD4 protein expression (Fig. 3H). These findings suggest that miR-21 regulation controls PDCD4 expression in Jurkat cells. Finally, we assessed the expression of pSTAT5 and PDCD4 in OVA-stimulated LNCs isolated from OVA-primed PD-1−/− and WT mice. Western blot analysis showed upregulation of pSTAT5 protein expression in OVA-stimulated MEK inhibitor LNCs from PD-1−/− mice as compared with WT controls, whereas the protein levels of PDCD4 were downregulated in the respective LNCs (Fig. 3I). These results indicate that the PD-1-STAT5-miR-21-PDCD4 regulatory pathway

is functional in pathogenic Ag-specific T cells. To verify the involvement of miR-21 in the regulation of the immune response in PD-1−/− mice, we isolated OVA-primed LNs from PD-1−/− mice and transfected them with anti-miR-21 inhibitor (as-miR-21) prior to in vitro stimulation with OVA. As shown in Fig. 4A, as-miR-21-transfected PD-1−/− lymphocytes showed decreased proliferation in response to OVA compared with nontransfected cells (stimulation index=22.1 for nontransfected cells versus 8.6 for miR-21-transefected cells at 13.3 μg/mL OVA). Inhibition of miR-21 activity in OVA-stimulated LNCs resulted in threefold and twofold decreased IFN-γ and IL-17 production respectively, compared with nontransfected OVA-stimulated LN cells

(Fig. 4B and C). Finally, adoptive transfer of OVA-specific cells, that were transfected to overexpress miR-21, into syngeneic recipients resulted in significantly higher severity of arthritis as compared Phospholipase D1 with mice that received control-transfected effector cells (Fig. 4D). In conclusion, we demonstrate that breakdown of tolerance and development of autoimmunity in the absence of the PD-1 pathway is regulated by the expression of miR-21 on Ag-specific T cells and the effect of this microRNA on PDCD4 expression. The PD-1 pathway has an important role in the regulation of peripheral tolerance since its deficiency leads to the development of autoimmunity. Here, we demonstrate for the first time that the development of T-cell-mediated autoimmunity in PD-1−/− mice is regulated by aberrant expression of miR-21 in Ag-specific T cells. Deficiency of PD-1 pathway resulted in markedly increased and sustained severity of induced arthritis, indicating increased intensity of the immune response.

PCR products were separated by agarose gel electrophoresis and transferred onto Zeta-Probe nylon membranes (Bio-Rad). Oligonucleotide probes were end-labeled with (γ-32P)ATP (MP Biomedicals) using OptiKinase as described by the manufacturer (USB) and purified by NucAway Spin Columns (Ambion) before hybridization at 42°C in 3× SSC/0.1%SDS/10× Denhardt’s solution/50 μg/mL salmon sperm DNA (Roche) hybridization ICG-001 supplier buffer. The following probes were used: TND, located in the VDJ junctions of the VV29 transgene 30, endogenous Cμ probe, located in exon 1 of the C57BL/6 Cμ gene (5′GCAAAAACAAAGATCTGC),

and the Transgene Cμ probe, located in exon 1 of the BALB/c Cμ gene (5′GCAAAAACAGAGATCTGC). All the blots were washed once in 3× SSC/5 mM EDTA/0.1% SDS/5× Denhardt’s solution/50 μg/mL salmon sperm DNA (Roche) and once in 1× SSC/0.1% SDS/5 mM EDTA for 15 min each at 42°C. For Cμ probes, the blots were further washed twice in 0.1× SSC/0.1% SDS/5 mM EDTA for 30 min each at 42°C. Cγ transcripts containing transgene VDJ segments or endogenous VDJ segments were PCR amplified from serially diluted cDNA (Fig. 2A) with primers L3RI and CγRI. The PCR annealing temperature was 55°C

for 30 s and an extension temperature at 72°C for 1 min for 40 cycles. The PCR products were transferred onto Zeta-probe nylon membranes (Bio-Rad) and hybridized with a transgene-specific probe (TND) to identify transgenic VV29-Cγ transcripts. BMS-777607 order Amplifications of β-actin with the β-actin primers listed above were used as loading controls. The β-actin PCR was performed with cDNAs that were diluted at 1:6400, 1:12 800, and 1:25 600. Quantitation was performed by measuring band intensities from Southern blots for transgene-specific Cγ transcripts (VV29-Cγ), or band intensities from ethidium bromide-stained agarose gels for β-actin, followed by dilution factor correction. The mean values from three independent experiments were normalized by dividing the values for the VV29-Cγ to the values obtained

SB-3CT for β-actin. Cγ transcripts from in vitro-stimulated B-cell cultures using L3RI and the CγRI primers were amplified using Platinum Taq DNA Polymerase (Invitrogen). The PCR products were cloned into pGEM vectors (Promega) and plasmids containing the PCR inserts were isolated as described previously 32. Forty plasmids were spotted onto a Zeta-probe nylon membrane for dot blot hybridization with the TND probe using the method described above. All clones (both TND-positive and TND-negative) were sequenced at the Tufts University Core Facility (Tufts University School of Medicine). The sequence analyses confirmed the association of transgene VDJ sequences with endogenous Cγ sequences for TND-positive clones and provided a frequency of 27.

Assays were performed in triplicate for each sample. The optical densities (ODs) of the blanks were less than 0.1. The levels of serum IgM and IgG were determined by ELISA. Microtitre plates (MaxiSorp, Nunc) were coated with 50 μL of antihuman IgG or antihuman IgM, at 2 or 5 μg/mL, respectively. Serum Igs (IgM and IgG) levels were determined using alkaline phosphatase-coupled goat antihuman IgM or anti-human IgG (Sigma-Aldrich). The absorbance was measured at 405 nm in

an ELISA reader (Organon Teknia). Absorbance values were quantified into milligrams per millilitter using the standard dilution curves of the corresponding purified human Igs (Sigma-Aldrich). Glycosphingolipid extraction from L1210 tumor

LDE225 clinical trial cells was performed as reported previously [49]. The acidic glycosphingolipid fraction was desiccated and then dissolved in chloroform/methanol (2:1; v/v) for developing on high-performance thin-layer chromatography (HPTLC) on precoated thin-layer plates (Merck, Darmstadt, Germany) in the solvent system consisting of chloroform/methanol/0.25% selleck chemicals llc KCL and 2.5 M NH3 (5:4:1; v/v). Gangliosides were visualized with orcinol stain [50]. Immunostaining with 14F7 mAb on HPTLC plates was performed as previously reported [50]. The plates were incubated with biotinylated goat antimouse IgG (Jackson Immunoresearch Laboratories) and strepdavidin-alkaline phosphatase (Jackson Immunoresearch Laboratories). Color was PRKD3 developed with an alkaline-phosphatase (AP)-conjugated substrate kit (Biorad, CA, US). Serum IgM and IgG fractions were isolated using a protein G mini column (Pro-Chem Inc., MA, USA) following the manufacturer’s instructions. Purity and reactivity against gangliosides of the eluted (IgG) and unbound (IgM) fractions were tested by ELISA as described above. The column fractions were screened both for binding and cytotoxic activity against L1210 tumor cells (see below). To assess the binding of anti-NeuGcGM3

Abs present in human sera, the cells were blocked in PBS containing 1% FCS for 20 min on ice. Human serum samples, diluted 1/5, were incubated with 105 cells for 30 min on ice. After washing with cold PBS, cells were incubated with PE-conjugated goat antihuman Igs (IgM + IgG), FITC-conjugated goat antihuman IgG or FITC-conjugated goat antihuman IgM (Jackson ImmunoResearch Laboratories), for 30 min on ice. The percentage of positive stained cells was determined in a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The WinMDI 2.9 program was used to analyze a total of 104 cells acquired on every assay. To be considered positive, a serum sample percentage of binding had to be ≥15% and at least two times the percentage obtained by incubating the cells only with the secondary antibody.

It has been reported that German cockroach extract is capable of activating protease-activated receptor

(PAR)-2 and provoking IL-8 secretion from bronchial epithelial cells [7], indicating that cockroach allergen may affect the expression of PARs and hypersecretion of cytokines. Indeed, we recently demonstrated that recombinant Per a (rPer a) seven can upregulate the expression of PARs and provoke Th2 cytokine, IL-4 and IL-13, production in P815 cells [8]. As Per a 1s are major allergens in American cockroach and their functions in provoking allergic reactions remain obscure and mast cells play a key role in allergic reactions, we generated rPer a 1.0101 and rPer a 1.0104 and investigated their influence on the expression of PARs and cytokine production in P815 cells in the current study. Patients and samples.  A total of 21 allergic rhinitis patients with positive skin prick to allergen extracts selleck compound and four healthy controls (HC) were recruited in the study. mTOR inhibitor Among the allergic patients, 15 of them were allergic to American cockroach and six of them to ragweed. The informed consent from each volunteer

according to the declaration of Helsinki and agreement with the ethical committee of the First Affiliated Hospital of Nanjing Medical University was obtained. Serum (2 ml) from peripheral venous blood was collected from each patient and HC for Western blot analysis. Expression of Per a 1.0101 and 1.0104 proteins in E. coli.  The procedures were mainly adopted from the one described previously for Per a 7 [8]. Briefly, pMD-Per a 1.0101 and pMD-Per a 1.0104 plasmids were digested and then ligated into unique Nde I and Hind III sites Cytidine deaminase in a pET-28a expression vector, respectively. The resulting plasmids were transformed into E. coli BL21 (DE3) for the expression of proteins. The final expression condition, under which the proteins were expressed mostly in soluble form, was at 25 °C for 12 h in the presence of 0.6 mm of IPTG. rPer a 1.0101 and rPer a 1.0104 proteins

were purified using BugBuster Ni-NTA His bind purification kit according to manufacturer’s protocol as described previously [8]. Endotoxin contamination was examined with the LAL assay according to the manufacturer’s instructions. The endotoxin levels detected with limulus amebocyte lysate chromogenic endpoint assay for endotoxin (Hycult Biotech, Uden BV, The Netherlands) were very low, being <0.01 EU/mg in rPer a 1.0101 and rPer a 1.0104 proteins. Evaluation of solubility of American cockroach allergens.  In order to express American cockroach allergens in a soluble form in E. coli, a statistical model for prediction of solubility of protein expression in E. coli was used [9]. A composite parameter canonical variable (CV), which is dependent on the contribution of each of the individual amino acid, was calculated as follows: CV = 15.43 (N + G + P + S)/n−29.