13 Intriguingly, we found that treatment of BL cells Small molecule library with proteasome inhibitors partially restores their capacity to present the EBNA1 epitope, thereby suggesting that proteasomes from BL cells, although less active against prototype substrate peptides, which only partially indicate the in vivo proteasomal activities, degrade the HPV epitope during the processing of EBNA1. It

remains to be elucidated whether other EBNA1-derived CTL epitopes may be more efficiently generated and presented after partial inhibition of proteasomes or whether this effect is restricted to the HPV epitope. In conclusion, our study, together with previous reports, strongly supports the idea selleck chemicals llc that EBNA1-specific CTLs might be exploited therapeutically to target EBV-positive malignancies in combination with chemotherapy and protocols designed to restore antigen-presenting capacity in the tumour. In this context, it has been recently demonstrated that tubacin, a molecule that inhibits histone deacetylase 6, demonstrates a fairly selective capacity

to induce apoptosis in BL cells, but not in LCLs.37 Furthermore, the combination of tubacin with a proteasome inhibitor induced efficient killing of BL cells,37 which are known to be resistant to proteasome inhibitor-induced apoptosis.21,38 These findings, together with those reported in this study, suggest that the use of proteasome inhibitors, alone or in combination with other drugs such as tubacin, may represent a strategy Molecular motor for the treatment of EBNA1-carrying

tumours, because proteasome inhibitors, in addition to their effect as pro-apoptotic drugs, may also increase the immunogenicity of EBNA1, thereby resulting in the efficient elimination of EBNA1-positive malignancies. This work was supported by grants from the University of Ferrara and Fondazione Cassa di Risparmio di Ferrara. We are grateful to A. Forster for editorial assistance and to Dr A. Balboni for HLA typing. The authors have no financial conflicts of interest. Table S1. MHC class I expression in lymphoblastoid cell line and in Burkitt’s lymphoma cells. “
“EAE, an animal model for multiple sclerosis, is a Th17- and Th1-cell-mediated auto-immune disease, but the mechanisms leading to priming of encephalitogenicTcells in autoimmune neuroinflammation are poorly understood. To investigate the role of dendritic cells (DCs) in the initiation of autoimmuneTh17- andTh1-cell responses andEAE, we used mice transgenic for a simian diphtheria toxin receptor (DTR) expressed under the control of the murineCD11c promoter (CD11c-DTRmice onC57BL/6 background).EAEwas induced by immunization with myelin oligodendrocyte glycoprotein (MOG) protein in CFA.

, 2004; Kuula et al., 2009). The findings presented in this paper support the therapeutic

usefulness of the nonantibiotic properties of doxycycline in the treatment of chronic inflammatory diseases such as rheumatoid arthritis and periodontal disease, where suppression of interstitial collagenase and 92-kDa gelatinase (gelatinase B) may be beneficial to reduce pathologically excessive degradation of the ECM. It is noteworthy, as shown in this and previous studies (Hanemaaijer et al., 1997), that the inhibition/reduction of MMP-8 and -9 expression and activities by doxycycline and CMTs is not complete, thus allowing these MMPs to carry out the protective actions (McMillan et al., 2004; Sorsa & Golub, 2005; Kuula et al., 2009). Both doxycyclines and chemically modified tetracyclines, when used in conjunction with other chemotherapy agents, ICG-001 may not only lead to more successful periodontal treatments but may reduce the risks for other significant medical conditions including diabetes, heart attack, stroke and other CVDs (Golub et al., 2009; Payne et al., 2009). This study was supported by grant no. A43273 from the New York State Office of Science, Technology and Academic Research

(NYSTAR), through NYSTAR’s Center of Advanced Technology, Stony Brook University. The authors would like to acknowledge Dr Mary Truhlar, Chair of Department of General Dentistry, Stony Brook University, for her support and encouragement of this project. “
“The complement system is regulated

by inhibitors such as factor PD0325901 I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms Ibrutinib price in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complementfactor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. In conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS. Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure 1.

7 cells. The cellular uptake of ODN1668 was highly dependent on the concentration of ODN1668 after a 4-h-incubation of ODN. The addition of ODN1720 or DNase I-treated ODN1720 hardly altered the cellular uptake of ODN1668 (Fig. 5A). Thus, the cellular uptake of ODN1668 was not affected by DNase I-treated

ODN1720, so it would not be involved in the mechanism of increased TNF-α production by DNase I-treated DNA. Next, we focused on the stability of ODN1668 against DNases, because the presence Panobinostat concentration of DNA or DNA fragments could increase the stability of ODN1668, which would result in increased cytokine production. To evaluate the effect of DNase-treated DNA on the stability of ODN1668 against DNases, ODN1668 was incubated with DNase I or DNase II in the presence of DNase-treated ODN1720. Unexpectedly, the degradation of ODN1668 by DNase I was markedly accelerated by the addition of DNase I-treated ODN1720 (Fig. 5B). Similar experiments were performed at lower DNase I concentrations of 0.1 and 0.5 U/mL, which Kinase Inhibitor Library purchase could better reflect the situation of cultured macrophages. Under the DNase I concentration of 0.5 U/mL,

the degradation of ODN1668 by DNase I was also accelerated by the addition of DNase I-treated ODN1720, whereas no significant degradation of ODN1668 was observed at a concentration of 0.1 U/mL DNase I for the experimental period of 4.5 h (Supporting Information Fig. 3). Therefore, it was postulated that the increased CpG motif-induced TNF-α production by DNase

I-treated DNA was not mediated by the increase in the stability of CpG DNA against DNase I. On the other hand, the degradation of ODN1668 by DNase II was retarded by the addition of DNase I-treated ODN1720 (Fig. 5C) or DNase II-treated ODN1720 (Fig. 5D). Taking into consideration that the DNase II-treated ODN1720 did not increase the ODN1668-induced TNF-α production (Fig. 3B), it seems that the ODN stabilization to DNase II did not contribute to the increase in TNF-α production by ODN1668. Therefore, the effects of DNase I-treated ODN1720 on the degradation of ODN1668 by DNase II would not be important for the ODN1668-induced TNF-α production. To evaluate whether DNase I-treated DNA increases the CpG DNA-induced inflammatory 3-oxoacyl-(acyl-carrier-protein) reductase response in vivo, ODN1668 was subcutaneously injected with intact or DNase I-treated ODN1720 into the footpad of the right hind leg of mice. The injection of ODN1668 alone did not induce significant footpad swelling (Fig. 6A), and the co-injection of ODN1720 had little effect on it. However, co-injection of DNase I-treated ODN1720 significantly increased the footpad swelling. Moreover, the infiltration of mononuclear cells and neutrophils into the footpad was evaluated using the paraffin sections of the footpad of mice receiving a subcutaneous injection of ODN1668 (Fig. 6B).

4 Importantly however, the origin of myofibroblasts in DN remains largely unknown. It is generally believed that myofibroblasts are derived from resident fibroblasts, epithelial cells (through epithelial-mesenchymal transition, EMT), mesangial cells or bone marrow. The role of EMT in the development and progression selleck chemicals of renal fibrosis has been thoroughly and extensively investigated. EMT is a process whereby polarized epithelial cells lose their epithelial markers such as E-cadherin, acquire de novo mesenchymal or myofibroblast markers such as SPF1 and -α-SMA, and convert to motile mesenchymal cells. EMT is an important process during histogenesis and organogenesis,

including gastrulation and the formation of neural crest, heart, the musculoskeletal system, craniofacial structures and the peripheral nervous system. EMT can also play a critical role in tumour progression,5 and EMT is now regarded as the first step in cancer metastasis.6 Powerful imaging techniques that allow tracing of individual cell

migration from primary tumours has provided direct evidence that EMT occurs in mouse and human carcinomas.7–9 In addition, there is increasing evidence demonstrating the existence of EMT in the development X-396 and progression of organ fibrosis during tissue injury. EMT is found to be associated with fibrosis occurring in kidney, lung and liver.10–12 Recently, endothelial-mesenchymal transition, or endothelial-myofibroblast transition (EndoMT) has emerged as another mechanism involved in both developmental and pathological processes.13 Kisanuki et al. used the Tie2-Cre × ROSA26R genetic approach in mice clearly showed that endocardial cushion mesenchyme at E12.5 is derived entirely from endothelial progenitors.14 Arciniegas et al.15 demonstrated that transforming growth factor (TGF)-β1 can induce aortic endothelial cells (EC) to differentiate 6-phosphogluconolactonase into α-SMA positive cells in vitro suggesting a novel role for TGF-β1 in atherogenesis. Moreover, embryonic EC have been observed to transdifferentiate

into mesenchymal cells expressing α-SMA in vitro and in vivo,16 and vascular endothelium-derived cells contain α-SMA in restenosis,17 inflammation and hypertension.18 Taken together, these findings suggest the potential importance of EndoMT in cardiovascular development and fibrosis. In fact, EndoMT also plays an important role in pulmonary fibrosis,19 idiopathic portal hypertension20 and corneal fibrosis in a rat corneal scrape injury model.21 Zeisberg et al. identified EndoMT as a mechanism for the accumulation of carcinoma-associated fibroblasts and suggested that anti-angiogenic treatment of tumours may have a direct effect in decreasing activated fibroblasts that likely facilitate cancer progression.22 Zeisberg et al.

Undoubtedly, the laboratory mouse has proven to be an invaluable model for biological research and most of what we know today about mammalian biology is derived from research carried out with Mus musculus. Nonetheless, to reject other animal models is to ignore the

need to address evolutionary divergence among mammals by studying biology across an array of genotypes. Moreover, the opportunity to exploit unique biological models or intriguing insights can be squandered. Clarity about the nature of immunologic tolerance was developed because Owen and Medawar capitalized on the unique properties of the placental vasculature of twin calves. Rowson’s frustrations with uterine infections in embryo transfer recipients gave impetus to his fruitful RG7420 solubility dmso studies that established progesterone as a key hormone regulating uterine immunity. The papers in this special issue of the American Journal of Reproductive Immunology highlight additional examples whereby farm animals are being used to develop

concepts pertinent to a wide range of mammalian species. Domestic farm animals are not the only mammalian species that can make useful research models, of course, but they offer advantages of availability, ease of handling, cost, and a well-described biology and husbandry. When Medawar was struck with the idea of using the calf in his research, he IWR-1 mw turned to colleagues at the Animal Breeding and Genetics Research Organization in Edinburgh. Today, unfortunately, the infrastructure for conducting farm animal research is eroding.21,22 For example,

the number of scientist years working in animal production or protection in the United States declined 22% from 1985 to 2006 and doctorates awarded in the animal sciences in the United States declined by 30% from 1985 to 2004. An increase in Resveratrol investments in basic research using farm animals will have a positive impact not only on agricultural productivity but on understanding mammalian biology and enhancing human health. During the initial preparation for this paper, I was fortunate enough to attend the celebrations surrounding the 100th Anniversary of the Dept. of Genetics at the University of Florida. In the course of the event, I heard details of the contributions of Ray Owen to the idea of immunologic tolerance that I was unaware of previously. Medawar had acknowledged his debt to Owen in his Nobel Lecture but, until I heard the details in Madison, I knew little about Owen or his work. I acknowledge Millard Susman, James Crow and Ray Owen for sharing images and information about this important time in reproductive immunology. “
“This study investigated whether angiotensin II type 1 receptor agonistic autoantibodies (AT1-AAs) mediate the increased release of soluble endoglin (sEng) in women with preeclampsia. Serum samples were obtained from women with normal pregnancies or with preeclampsia.

All-cause death and cardiovascular (CV) events were recorded as the main outcome. Among the UCG records, left atrial diameter (LAD), left ventricular ejection fraction (LVEF), were determinants of log-transformed (ln) BNP; UFR, age and sex were also significant. There was a positive

correlation between BNP and LAD (r = 0.285, P < 0.001). Receiver operating characteristic (ROC) analysis Dorsomorphin in vivo revealed that BNP had 90% and 80% sensitivity to predict the presence of LA enlargement of 77.9 pg/mL and 133.2 pg/mL, respectively. Higher BNP and lower LVEF were associated with higher risk for developing all-cause death and CVD. In the adjusted model, patients with BNP higher than 471 pg/mL had hazard ratio of 2.18 (95% confidence interval (CI) 1.20–3.96, P = 0.01), compared to those with BNP <109 pg/mL. B-type natriuretic peptide was determined by LAD, LVEF, UFR, age and sex. BNP and LAD had positive correlation and BNP could become a useful tool for estimating the presence of LA enlargement. RG7420 research buy BNP and

LVEF was a strong risk factor for predicting all-cause death and CV events among patients undergoing haemodialysis. “
“Recurrence of native kidney disease following kidney transplantation affects between 10% and 20% of patients, and accounts for up to 8% of graft failures. In a considerable number of recipients with transplant glomerulopathy, it is impossible to distinguish between recurrent and de novo Farnesyltransferase types. An accurate estimate of the incidence of recurrence is difficult due to limitations in the diagnosis of recurrent glomerulonephritis. De novo glomerular lesions may be misclassified if histological confirmation of the patient’s native kidney disease is lacking. Asymptomatic histological recurrence in renal allografts may be missed if protocol biopsies are not available. Studies based on protocol biopsy are pivotal to accurately estimate the incidence of recurrence. Many factors are known to influence recurrence of kidney disease after

transplantation, including the type and severity of the original disease, age at onset, interval from onset to end-stage renal disease, and clinical course of the previous transplantation. Early recognition of recurrence is possible in several glomerular diseases. Factors such as the existence of circulating permeability factors, circulating urokinase receptor and anti-phospholipase A2 receptor antibody, as well as disorders of complement regulatory proteins like factor I mutation and factor H mutation factors are expected to be useful predictors of recurrence. Peculiar clinical course of atypical haemolytic uremic syndrome after kidney transplantation is an informative sign of recurrent glomerular disease. These factors play pivotal roles in the development of recurrence of certain types of glomerulopathies.

vulnificus from the bacteriological viewpoint. After confirming the efficacy of HBO therapy in a mouse footpad infection model, we showed that cells of V. vulnificus, but not those of E. coli, lose their colony-forming ability in HBO, whereas both species grow equally well in ambient air. Furthermore, we obtained evidence

that HBO-induced killing of V. vulnificus cells can be accounted MK-2206 purchase for by their low tolerance to DNA damage induced by ROS, as well as their inability to inactivate ROS. Vibrio vulnificus strains L-1, 371 and 374 (8), and E. coli K-12 strain MG1655 were obtained from our own laboratory stocks. The yeast extract broth (pH 7.2) used contained per liter: 5 g of yeast extract (Difco, Franklin Lakes, NJ, USA), 10 g of polypeptone (Wako, Osaka, Japan), and 5 g of NaCl. Cultures in this medium were grown with shaking, and cells from log-phase cultures were used throughout the study. When needed, the broth medium was solidified with agar (Wako) added at 15 g/L to make yeast extract agar. All bacterial cultures were incubated aerobically at 37°C. An HBO chamber of 15.2 L capacity (Barotec Hanyuuda, Tokyo, Japan) was used throughout this study. HBO experiments

were carried out essentially as previously described (9, 10). After flushing the chamber containing test materials with O2 at a flow rate of 10 L/min for 5 mins, the pressure in the chamber was raised at a rate of 0.2 atm/min by adjusting the outlet valve. After the pressure had reached the desired value, O2 flow was maintained at 1.0 L/min. Small molecule library solubility dmso After each treatment, decompression was performed slowly, at a rate of 0.1 atm/min, to avoid complications, namely, bubble formation in the blood

of the animals or in the culture media. Animal experiments were carried out in the chamber at room temperature, whereas in vitro cultural studies were done by placing the chamber in a room kept at 37°C. Experiments using N2 gas were carried out in essentially the same way. Log-phase cells of V. vulnificus grown in Isotretinoin yeast extract broth were washed twice in PBS, centrifuged, and resuspended in PBS containing approximately 106 cells/mL. The right hind footpads of 6-week old female mice of Kud:ddY strain (specific pathogen free, Kyudo, Saga, Japan) were inoculated with 0.1 mL aliquots of the suspension. The footpad swelling index, used to indicate the degree of inflammation (11), was defined as the difference in size between the inoculated and the control (left hind) footpads of each animal, and the size of the footpad was approximated by the product of thickness × width, both measured in mm with Peacock dial thickness gauge calipers (Ozaki, Tokyo, Japan). Finally, the mice were killed by cervical dislocation and the infected feet cut off at the level of the knee and homogenized with PBS (1.0 mL per foot) in a Multi-Beads Shocker MB601 (Yasui Kikai, Osaka, Japan).

In addition, LAG-3 is a negative regulator of T cell receptor (TCR)-mediated signal transduction in effector

T cells and functions in the same manner as cytotoxic T lymphocyte antigen-4 (CTLA-4) [9–12]. Finally, LAG-3 controls activated regulatory T cells (Tregs), while it is not expressed by unstimulated natural Tregs[13]. However, LAG-3 is expressed by interleukin Talazoparib chemical structure (IL)-10-secreting early growth response (Egr)-2+LAG-3+CD4+ Tregs associated with Peyer’s patches [14]. We have shown previously that depleting anti-LAG-3 antibodies prevented the development of alloreactive effector T cells in a heart allotransplant model in rodents and represents an effective treatment for allograft rejection [15]. In this study, we have characterized a cytotoxic anti-LAG-3 chimeric antibody (chimeric A9H12) and evaluated its potential for selective therapeutic depletion in a non-human primate model of delayed-type hypersensitivity (DTH), a low-invasive Enzalutamide cell line and non-terminal model based on the induction of local T helper type 1 (Th-1)-mediated cellular immune responses [16]. Our investigation demonstrated

that LAG-3+ T lymphocytes could be depleted in vivo in primates and that this resulted in a long-lasting inhibition of immune responses in this preclinical model. C57/B6 mice were immunized three times with Chinese hamster ovary (CHO) cells transfected with human LAG-3 cDNA, followed by an intravenous (i.v.) booster injection of a recombinant hLAG-3Ig protein purified from the supernatant of transfected CHO cells. Three MTMR9 days after the boost, splenocytes were fused with the X63.AG8653 fusion partner [17] to obtain hybridoma cells, using traditional techniques. The A9H12 hybridoma was selected for its antibody-dependent cell cytotoxicity (ADCC) activity towards LAG-3 expressing cells and subcloned to yield a stable cell line. A bicistronic vector coding for the variable heavy (VH) and variable light (VL) domains of A9H12 fused to human CL kappa and CH1-hinge-CH2-CH3 immunlglobulin (Ig)G1 regions was

generated and used to transfect CHO-S cells (Invitrogen, Illkirch, France). After antibiotic selection and limiting dilutions, a stable subclone was selected to produce the chimeric A9H12 in ProCHO5 medium (Lonza, Vervier, Belgium). The product in the supernatant cell was purified by adsorption on a HiTrap recombinant Protein A FF column (GE Healthcare, Velizy, France), eluted by acid pH (Glycin HCl, 0·1 M, pH 2·8) and dialysed against phosphate-buffered saline (PBS; Invitrogen). LAG-3+ CHO cells or human peripheral blood mononuclear cells (PBMCs) stimulated with 1 µg/ml of Staphylococcal enterotoxin B (SEB; Sigma Aldrich, L’Isle D’Abeau Chesnes, France) for 48 h were used as targets. Chimeric A9H12 binding was revealed with a fluorescein isothiocyanate (FITC)-conjugated goat F(ab′)2 anti-human IgG (Southern Biotech, Birmingham, AL, USA).

Renal hyperfiltration was associated with prehypertension and prediabetes, while hypofiltration was associated with dyslipidemia, abdominal obesity, overt hypertension, and overt diabetes. Conclusion: The number of MetS components is a good risk indicator of early- and late-stage kidney

damage. Therefore, kidney function should be monitored in subjects with MetS components. MetS components should be treated as early as possible to prevent the development of kidney damage and cardiovascular diseases in people with hyperfiltration, regardless of their body weight. YATABE JUNICHI1, Talazoparib solubility dmso MATSUNAGA SHIGERU3, OGAWA ATSUSHI4, YATABE MIDORI2, TAKANO KOZUE2, ASAHI KOICHI1, TERAWAKI HIROYUKI1, NAKAYAMA MASAAKI1, WATANABE TSUYOSHI1 1Department of Chronic Kidney Disease Initiatives, Fukushima Medical University; 2Department of Pharmacology, Fukushima Medical University School of Medicine; 3Department of Biological Production, Akita Prefectural University; 4Aizufujikako Co., LTD Introduction: Advanced-stage renal disease patients have potassium restriction on their diet. In a survey on 38 hemodialysis patients, a majority (52.6%) of patients answered they are not eating

as much vegetable as they like and many (73.7%) answered that they would like to try low-potassium vegetables. Therefore, Aizufujikako, Co. Ltd. has developed low-potassium vegetables and fruits to meet this selleck screening library need. Methods: Low-potassium lettuce is grown hydroponically in clean rooms of what used to be semiconductor factories using the cultivation method patented by Akita Prefectural University. The lettuce seeds are planted one by one in plastic pots for germination then the seedlings were transferred to water culture system. After 14–21 days, control solution in the growth chamber

was substituted with a “no potassium” solution, and the seedlings were cultivated for another 10–21 days with controlled 4-Aminobutyrate aminotransferase light cycles. Testing for potassium content, microbes and metals were performed for quality control. One hundred and eighty healthy volunteers tasted the low-potassium lettuce and answered the questionnaire. Results: The newly developed low-potassium lettuce contained 44.7 ± 20.0 mg potassium per 100 g, close to 90% less potassium compared to regular lettuce (approximately 400 mg potassium per 100 g). There was no significant difference in dietary fiber and vitamin contents between the low-potassium lettuce and regular lettuce. However, low-potassium lettuce contained significantly greater amount of sodium compared to regular lettuce. In the taste testing by healthy volunteers, 73.6% answered that the low-potassium lettuce tasted good, 63.9% wished to purchase the lettuce for themselves to eat, and 84.9% would suggest to buy the low-potassium lettuce if people close to them were on potassium restriction.

2b), as detected by SDS-PAGE. Strikingly, there was only minimal loss of binding of the AMCA-HA peptide to HLA-DR1 upon digestion with CatG, and this slight loss was unaffected by the CatG inhibitor (Fig. 2c). Thus, peptide-loaded HLA-DR molecules are susceptible to CatG proteolysis, and cleavage of the β chain does not disrupt the integrity of the antigen-binding groove occupied by the peptide. To determine

the exact CatG cleavage site within the HLA-DR β chain, we performed N-terminal sequencing as well as peptide mapping AZD1208 order of the digestion products of purified soluble HLA-DR1 (sDR1). For these experiments we used sDR1 expressed in either insect cells or E. coli. Neither of these have a transmembrane domain and E. coli purified sDR1 is not glycosylated, which led to the fragments being smaller on gels (10 and 15 kDa). sDR1 expressed in insect cells (not shown) was used for identification of the N-terminal sequence of both fragments by Edman degradation (underlined italic sequence, Fig. 3a). The first residue of the larger fragment corresponds to the glycine (G) in position 1 of the mature protein. The first residue of the smaller fragment was selleck identified as glutamine (Q) at position 110. In order to define the boundaries

of both fragments, we also digested sDR1 expressed in E. coli (Fig. 3a), which is not glycosylated and was therefore used for MALDI-TOF analysis. The two bands were excised from a gel and digested with trypsin, Staphylococcus aureus V8 protease, or Arg-C protease. All peptides of these digests identified by mass spectrometry are indicated in black text in Fig. 3a. The peptide SFTVQRRVEPKVTVYPSKTQPL (underlined in Fig. 3a) was identified from a V8 digest and the peptide RVEPKVTVYPSKTQPL was identified from an Arg-C digest of the larger fragment, indicating that CatG did Loperamide not cleave after the arginine (R), but did cleave after leucine 109 (L109). Based on the masses of the two fragments and on the fact that their sequences were contiguous, these fragments appear to represent the complete β chain, which therefore has only a single CatG cleavage site. The cleavage site, between HLA-DRβ L109 and glutamine 110 (Q110,

L/Q), is located on a loop between fx1 and fx2 of the membrane-proximal, immunoglobulin-like domain, as indicated on the crystal structure of HLA-DR (Fig. 3b). To explore whether HLA-DR β chain polymorphism might influence CatG susceptibility, we first compared the amino acid sequences of several HLA-DR β chains [DRB1*0101 (DR1), DRB1*1501 (DR2b), DRB1*0301 (DR3), DRB1*0401, and DRB1*0404] and found conservation of the L/Q cleavage site (Fig. 4a). We then subjected various recombinant soluble HLA-DR allelic variants to digestion with CatG and used HLA-DR-specific rabbit serum (CHAMP) to measure residual levels of DRβ and detect the 18-kDa DRβ fragment (Fig. 4b). As predicted from sequence alignment, CatG degraded the β chain of all HLA-DR molecules tested.