Sciatic nerve was transected, and end-to-end neurorrhaphy was performed on 32 male Sprague-Dawley rats, which were randomly divided into four groups (n = 8 per group): nerve coaptation without treatment (group I); nerve coaptation covered with HA film sheath (group II); nerve coaptation with intramuscular VEGF gene in plasmid injection (group III); and nerve coaptation combined with HA film IDH assay sheath and intramuscular VEGF gene in plasmid injection (group IV). Contralateral sciatic nerves were used as control. VEGF

expression was verified from gluteal muscle biopsies surrounding the sciatic nerve by reverse transcriptase-PCR. Electrophysiological, histopathological, and electron microscopic evaluations were performed after 4 weeks. Mean peak amplitude of groups I–IV and nonoperated sciatic nerve were 4.5 ± 0.6 mV, 6.4 ± 0.4 mV, 6.7 ± 0.5 mV, 8.5 ± 0.4 mV, and 9.8 ± 0.5 mV, respectively. Mean myelinated axonal counts of groups I–IV and nonoperated sciatic nerve were 105 ± 24, 165 ± 19, 181 ± 22, 271 ± 23, and 344 ± 17, respectively. Treatment with HA film sheath coverage combined with intramuscular VEGF gene in plasmid injection yielded statistically significant

higher peak amplitudes and myelinated axonal counts selleck chemical (P < 0.001). In addition, significantly less scar formation with HA administration (groups II and IV; P < 0.001) was found. Thus, it was found that VEGF might crucially regulate nerve regeneration processes and that HA can reduce the scar

formation. This study showed that the combination of HA film sheath and VEGF gene may synergistically promote peripheral nerve regeneration. © 2013 Wiley Periodicals, Inc. Microsurgery 34:209–216, 2014. “
“Venous flow-through flaps are well-described options for Glycogen branching enzyme small defects where donor site morbidity is undesirable or in areas where useful local veins are in close proximity to the defect, particularly in the extremities. However, higher rates of flap loss have limited their utility. The saphenous venous flap in particular has been widely sought as a useful flap, and while arterialization of this flap improved survival rates, congestion has remained a limiting feature. We describe report a modification in the design of saphenous venous flaps, whereby an arterialized flap is provided with a separate source of venous drainage, and demonstrate survival of substantially larger venous flaps than previously reported. In five consecutive patients, we describe three main modifications to the saphenous venous flap as previously described: (a) Using arterialized flaps only; (b) Reversing the flap to allow unimpeded flow during arterialization; and (c) Anastomosing additional vein(s) that are not connected to the central vein—especially at the periphery of the flap for true venous drainage.

Interestingly, serum vitamin A was dependent on serum Vitamin B12. Immunohistochemistry showed that megalin and cubilin were accumulated at the apical surface of the proximal tubules in B12-Def., and restored in 24 hrs and 7days-CNB12. However megalin expression was not changed at protein and RNA level. Therefore, it is suggested that vitamin B12 deficiency

suppresses the endocytosis via megalin. As a result of confocal imaging, RBP reuptake-vesicles were decreased size and numbers in B12-Def. and restored in 7days-CNB12. RBP expression at protein level was dependent on serum Vitamin B12 level, whereas RBP mRNA was not changed. Conclusion: The selleck products present data shows that vitamin B12 status is linked to endocytosis via megalin, and reabsorption of vitamin A in the kidney. EIAM-ONG SOMCHIT1, SINPHITUKKUL KITTISAK2, MANOTHAM KRISSANAPONG3, EIAM-ONG SOMCHAI4 1Chulalongkorn

University; 2Chulalongkorn University; 3Lerdsin Hospital; 4Chulalongkorn University Introduction: Previous in vitro study showed that aldosterone rapidly stimulates PKC alpha that could activate alpha1 isoform of Na, K-ATPase and then enhances its activity. There are no in vivo data demonstrating the rapid effects of aldosterone on renal protein expressions of PKC alpha and alpha1-Na, K-ATPase simultaneously. The present study further investigates the expression of these proteins. Methods: Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 mg/kg BW). After 30 minutes, abundances Lumacaftor and localizations of PKC alpha and alpha1-Na, K-ATPase proteins were determined by western blot analysis and immunohistochemistry, respectively. Results: Aldosterone administration significantly increased Calpain plasma aldosterone levels from 1,251.95 ± 13.83 to be 6,521.78 ± 209.92 pmol/L. By western blot analysis, aldosterone enhanced renal protein abundances of PKC alpha (tissue homogenate) and alpha1-Na, K-ATPase (plasma membrane) approximately 50% and 30%, respectively (P < 0.05). From immunohistochemistry examination in sham group, the protein

expression of PKC alpha was prominent in the medulla. Aldosterone stimulated the expression both in cortex and medulla with the translocation from basolateral to luminal side of proximal convoluted tubule. For alpha1-Na, K-ATPase protein expression, the sham rats showed a strong immunostaining in the distal convoluted tubule, collecting duct, and thick ascending limb. Aldosterone elevated the expression in the proximal convoluted tubule and medullary collecting duct. Conclusion: This in vivo study is the first to demonstrate simultaneously that aldosterone rapidly elevates PKC alpha and alpha1-Na, K-ATPase protein abundances in rat kidney. Both immunoreactivities were stimulated in cortex and medulla. The greater affected areas were noted for PKC alpha expression, whereas the alterations of alpha1-Na, K-ATPase were observed only in the proximal tubule and medullary collecting duct.

Furthermore, the striking prognostic value of the analysis of immune infiltrates in tumors has firmly established the capacity of adaptive immunity to control tumors [2, 4]. There are at least two major hurdles to

overcome in efforts to generate vaccines to cancer: the generation of sufficiently strong and long-lasting selleck inhibitor tumor-specific T-cell responses that do not destroy healthy self-tissues, and the recruitment of sufficient numbers of effector T cells into tumor sites and metastases. In order to address the first issue, one approach is to take advantage of the ability of CD4+ T helper cells to potently synergize with CD8+ T cells, promoting their activation and memory [5]. Although much of the effort in identifying T-cell epitopes for immunization in cancer has focused on self- or modified Ruxolitinib ic50 self-antigens

[6], given the issue of self-tolerance which is further compounded by the ability of tumors to generate tolerance to themselves, it is difficult to generate sufficient T-cell help via the (modified) self-antigen route. A strategy that has long been considered to overcome this obstacle is the addition of foreign (e.g. xeno) antigens into cancer vaccines to boost immunity [7, 8], and more recent studies have provided direct evidence that the beneficial effects of this procedure are through the provision of T-cell

help [9-11]. A substantial advantage of employing foreign helper determinants physically linked to Rho determinants recognized by CD8+ T cells, rather than tumor-associated helper determinants, is that the tumor cannot use either downregulation of their own helper epitopes, or induction of tolerance against these foreign epitopes, as a means of escape. Interestingly, it has been theorized that MHC class II-restricted T cells are likely to be more self-tolerant than MHC class I-restricted T cells or B cells [12]. It would seem an insurmountable task for our immune system to become tolerant of all of the various self-antigens throughout our body. The task would be made much simpler if extensive tolerance were only needed for T cells recognizing antigens presented on the limited number of cells that express MHC class II; expression of MHC class II is restricted to several hematopoietic lineages and endothelial cells while the vast majority of cells in the body, the various parenchymal tissue cells, generally lack expression. This concept is consistent with observations of a state approaching ignorance to some self or neoself antigens by CD8+ T cells and B cells [13-15], while CD4+ T cells remain robustly tolerant [9, 13].

Results:  It was

observed that urinary proteins from FSGS patients more significantly induced the expression of α-SMA and vimentin and reduced cytokeratin-18 expression than those from MCD patients in HK-2 cells. Both ERK1/2 and p38 were activated by urinary proteins from MCD or FSGS patients. Pretreatment of the cells with SB203580 or PD98059 abolished the effect of urinary proteins from FSGS patients on the expression of α-SMA, vimentin and cytokeratin-18, while only SB203580 elicited this effect Ponatinib purchase when cells were treated with urinary proteins from MCD patients. Conclusion:  The urinary proteins from MCD and FSGS patients induced significant changes of EMT-related proteins through activation of distinct mitogen-activated protein kinase-related signalling pathways. Quality of proteinuria may play an important role in determining the severity and progression of tubular injury associated with different kidney

diseases. “
“Acute renal injury (AKI) is a relatively common clinical condition, reported to be associated with high rates of in-hospital mortality. Although here is an extensive literature on the LDE225 mw nature and consequence of AKI in the developed World, much less is known in the developing World and more specifically in sub-Saharan Africa, which is addressed directly in this study. We describe the prevalence, clinical characteristics and impact of AKI in patients admitted to a single centre in Ethiopia with no dedicated renal services. Renal function tests are not preformed routinely in many Ethiopian hospitals. This occurred in 32% of all patients in this study, falling to 23% on surgical wards. As a consequence no cases of AKI were identified in the context of surgical admissions. AKI was only identified in a cohort of patients on medical wards, with a prevalence of roughly 20% of medical patients in which renal function was measured. The patients with AKI were younger Exoribonuclease than those at risk of AKI in studies from the developed

World but were older than those who did not develop AKI in this study. In the majority of cases AKI could be considered to be pre-renal in its origin. In contrast to studies in the developed World, AKI did not adversely impact on either duration of hospital stay or on patient mortality. Residual renal impairment was, however, common at the point of discharge. The data suggest subtle differences in the nature and impact of AKI between those published and mainly derived from the developed world and patients in sub-Saharan Africa. “
“Plasma cell dyscrasias (PCD) are a spectrum of diseases characterized by clonal proliferation of plasma cells secreting a monoclonal immunoglobulin.

Apoptotic cells were identified as the cells, which were Annexin V positive. The method is based on the selective binding of Annexin V to the phosphatidylserine displayed at the external membrane of cell surface in apoptotic cells. Propidium iodine was not used as an identifying agent because we evaluated the early stage of apoptosis. The staining procedure was performed according to the description provided by the company. Intracellular, cytoplasmic Bcl-2 protein was identified in 1 × 106 cells, which were fixed and permeabilized using Cytofix/Cytoperm

kit (BD Biosciences, Pharmingen) and PD0325901 then incubated with PE-conjugated hamster anti-Bcl-2 antibody (BD Biosciences, Pharmingen) followed by either PE hamster IgG isotype control antibody (BD Biosciences, Pharmingen) for 30 min in the dark at 4°C. The cells were acquired on a BD FACS Calibur Flow Cytometer, and the data were analysed using

lysis II Software (BD, Le Pont de Claix, France). MLN cells were cultured at a concentration of 1 × 106 cells per well in 96-well flat-bottom plates (Costar) and incubated with 10 μg/mL of somatic complete antigen, (AgS) or 5 μg/mL antigenic fractions (F9, F13, F17). After 72 h of culture cells were collected, washed with PBS and used in further analyses. FLIP protein was identified in 8 × 106 cells, which were lysed in 1% Triton X-100 (Serva, Germany) for 1 h on ice. Then lysed cells were centrifuged for 10 min at 18 000 g and supernatants were diluted in the same volume

of Laemmli’s sample buffer. The proteins from each sample were separated on 12% SDS–polyacrylamide click here gel and were transferred onto a nitrocellulose membrane (0.2 μm; Whatmann Inc., Dassel, Germany) for 1.5 h (17 V, using the Trans-Blot SD Semi Dry Transfer Cell; Bio-Rad, Hercules, CA, USA). The membrane was than blocked using Immune system 5% nonfat dry milk in PBS for 1 h at RT, treated with antisynthetic peptide rabbit polyclonal IgG. The antibody specificity, corresponding to amino acid 447-646 of human FLIP with mouse cross-reactivity, which recognized the long form of FLIP, molecular size 55 kDa (Upstate, Millipore, NY, USA), kept overnight at 4°C, and then incubated with peroxidase-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories Inc., Baltimore, MD, USA) for 60 min. Immunoblot was developed by the DAB (Sigma-Aldrich). Samples without the primary antibody were used as negative control. For NF-κB expression, cells from cultures were lysed with Nuclear Extraction Kit (Upstate), and both fractions, cytoplasmic and nuclear, were used in the Universal Colorimetric Transcription Factor Assay (Upstate). Levels of p50 and p65 proteins in fractions were determined according to manufacture instructions. Absorbance was measured in spectrophotometer at λ = 450 nm. SDS-PAGE electrophoresis was performed according to the method of Laemmli.

5%) had hypertension, 07 (13.5%) had diabetes, mellitus, 04 (7.7%) had renal disease 03 (5.8%) had liver disease and 15 (28.8%) had arthralgia 07 (13.5%), 14 had gastrointestinal problems (46.1%), 07 (13.5%) had headache/migraine, 02 (3.8%) had suffered hemiparesis. Mean blood pressure was 133.99 ± 40.89/82.76 ± 27.79 mmHG in males and 132.10 ± 16.20/ 83.46 ± 7.85 mmHG in females. Based on American Heart Association classification for hypertension, 19 patients had normal blood pressure, 8 were in

prehypertensive stage, 16 patients were in hypertension stage 1, 6 were in hypertension stage 2 and 2 had crisis hypertension. Mean serum creatinine for males was 0.94 ± 0.14 and 0.91 ± 0.84 for females. Mean of BIA derived TBW was 33.7 ± 6.6 and that derived using selleck compound equation was 34.8 ± 6.18. There was no statistically significant difference between the two (F 0.001, t 1.317 and p 0.189). Mean creatinine clearance was 97.39 ± 28.98

in males and107.60 ± 34.03 in females, GFR was74.1 ± 25.98 ml/min/1.73 m2 in males and 65.17 ± 21.14 ml/min1.73 m2 in females. Based on GFR we classified subjects into chronic kidney stages (CKD) 1–5. Out of 52 subjects 8 were in CKD stage 1, 23 were in CKD stage 2, 18 were in CKD Everolimus mw stage 3, 1 each in CKD stage 4 and CKD stage 5 respectively. Conclusion: Since there was no significant difference in total body water calculated by BIA and Hume’s equation, therefore, BIA can be safely used for estimating water compartments in healthy and in diseased subjects and as a tool for screening general population for presence of chronic kidney disease. OKADA RIEKO1,2,3,4, YASUDA YOSHINARI2, TSUSHITA KAZUYO3,

WAKAI KENJI1, HAMAJIMA NOBUYUKI4, MATSUO SEIICHI2 1Preventive Medicine, Pregnenolone Nagoya University; 2Nephrology /CKD Initiatives, Nagoya University; 3Comprehensive Health Science Center, Aichi Health Promotion Foundation; 4Young Leaders’ Program in Health Care Administration, Nagoya University Introduction: Renal hyperfiltration (early-stage kidney damage) and hypofiltration (late-stage kidney damage) are common in populations at high risk of chronic kidney disease. This study investigated the associations of renal hyperfiltration and hypofiltration with the number of metabolic syndrome (MetS) components. Methods: The study subjects included 205,382 people aged 40–74 years who underwent Specific Health Checkups in Aichi Prefecture, Japan. The prevalence of renal hyperfiltration [estimated glomerular filtration rate (eGFR) above the age-/sex-specific 95th percentile] and hypofiltration (eGFR below the 5th percentile) was compared according to the number of MetS components. Results: We found that the prevalence of both hyperfiltration and hypofiltration increased with increasing number of MetS components (odds ratios for hyperfiltration: 1.20, 1.40, 1.42, 1.41, and 1.77; odds ratios for hypofiltration: 1.07, 1.25, 1.57, 1.89, and 2.21 for one, two, three, four, and five components, respectively, compared with no MetS components).

Very interesting data published by Man et al. [22] suggest that IRF4 contributes to effector CD8+ T-cell differentiation by regulating metabolic pathways, in particular glycolysis. T cells need high energy supply for their strong proliferative burst after activation. To meet this demand, they switch their metabolism from oxidative phosphorylation to aerobic glycolysis [72]. This process seems to be greatly impaired in the absence of IRF4,

because activated Irf4–/– CD8+ T cells demonstrated lower uptake of glucose and produced less l-lactate as compared Akt inhibitor to WT CD8+ T cells. Moreover, oxygen consumption and extracellular acidification rate were lower in Irf4–/– as compared to WT CD8+ T cells. Consistently, the authors found direct binding of IRF4 to regulatory regions of genes encoding transcription factors that regulate cellular metabolism, including hypoxia-inducible factor α (HIF1α) and forkhead box O 1 (FOXO1), as well as of several genes encoding regulators of glycolysis such as the glucose transporters GLUT1 and GLUT3 [22]. However, it is still possible that the disturbed metabolic switch in Irf4–/–CD8+ T cells is secondary to their impaired expansion and effector differentiation, which is regulated by IRF4 by other means. Therefore, these attractive data need further evaluation, including

identification of the mechanisms through which IRF4 integrates strength of TCR ligation and metabolic pathways. Besides its requirement for effector CTL differentiation, IRF4 also participates in the formation of the memory Small molecule library CD8+ T-cell pool. In L. monocytogenes infected Irf4–/– mice, the numbers of antigen-specific memory CD8+ T cells and the production of the cytokines IFN-γ and TNF-α were significantly lower than those observed in L. monocytogenes infected WT mice [23]. Similarly in response to influenza Arachidonate 15-lipoxygenase infection, mice with conditional deletion of IRF4 in CD8+ T cells generated significantly lower numbers of antigen-specific memory cells [25]. Taken together, IRF4 is a fundamental regulator of effector and memory CTL formation by acting upstream of other transcription factors, including BLIMP-1 and T-BET (Fig. 2),

which regulate these processes, and by connecting the strength of TCR ligation to aerobic glycolysis. Similarly to Th9 cells, Tc9 cells produce the cytokines IL-9 and IL-10 upon in vitro induction, whereas expression of the Th2-cytokines IL-5 and IL-13 is strongly reduced as compared to Tc2 cells. In comparison to CTLs, Tc9 cells express diminished amounts of the transcription factors EOMES and T-BET and, accordingly, they display low cytotoxic activity in vitro [63, 68]. In adoptive T-cell transfer experiments, Tc9 cells showed IL-9-dependent antitumor activity [68]. In an allergic airway disease model, Tc9 cells alone were not pathogenic by themselves, but promoted airway inflammation when combined with Th2 cells [63].

Finally, glycans from schistosomes are known to have a major role in the stimulation of innate immune responses [35]. We previously reported that the cytokine-inducing activity of 0–3 h RP is heat labile (declining at

temperatures above 50°C), and glycan dependent [8], with a key role for the mannose receptor [9]. Here we show that the production of all 3 cytokines assayed (IL-8, TNFα and IL-10) in WB cultures was reduced after 0–3 h RP was treated with sodium meta-periodate to disrupt the glycosylated moieties. This shows that glycans influence both pro-inflammatory and regulatory cytokine induction in S. mansoni-infected humans. see more However, as molecules released by the mature schistosome egg are also glycosylated [7], and as there is sharing of glycan moieties between different life cycle stages [36], it is possible that innate immune cells that respond to 0–3 h RP (e.g. through C-type lectins such as the macrophage mannose receptor) [9] are also responsive to antigens released by other parasite stages (e.g. the egg)[37], which maintain or down regulate cell responsiveness after initial parasite infection. Therefore, production of cytokines in response to cercarial glycosylated E/S material may be reinforced in response to egg deposition,

which may in turn feedback to affect the response to subsequent exposure to cercariae. It is also possible that the Th2-polarized adaptive response dominant after chronic infection in turn influence the Saracatinib ability of innate immune cells to produce IL-10 to cercarial E/S products. It is therefore likely that there will be ongoing communication, or crosstalk, between the innate and adaptive immune systems to regulate reactivity to both

Liothyronine Sodium cercariae and eggs released by adult worm pairs. In conclusion, this study is the first to examine immune responses to cercarial E/S antigens, specifically the early production of cytokines indicative of the innate or early adaptive immune response, in human subjects. Our data show that cercarial E/S material induces the production of IL-10 in S. mansoni-infected individuals and suggests that cercarial E/S antigens are initial stimulants of a ‘regulated’ immune phenotype, which is prevalent after repeated and chronic infection with schistosomiasis. We gratefully thank the population of Diokhor Tack and the village chief, Daoure Mbaye, for their hospitality and participation in this study. This study would not have been possible without the field workers in Richard Toll, Abdoulaye Yague, Mankeur Diop, Moussa Wade and Ngary Sy, who assisted in the blood sample collection and microscopic analysis. We would also like to thank the medical and technical staff of the Health Centre in Richard Toll for their support. The authors would also like to thank Ann Bamford for help in preparation of antigen material. This study was supported by The European Union (EU INCO-CT-2006-032405 to APM, SM, and K P).

There were no operative complications, no flap-related complications, and at two years follow-up, the patient subjectively described bilateral soft and supple breasts, which were symmetrical in a bra, and with which she has reported high satisfaction. An account of the “split DIEP flap” is provided, highlighting the planning, technique, GW-572016 concentration and vascular rationale. The technique comprises partition of a previously transferred DIEP flap breast reconstruction into two parts based on preoperative computed tomographic angiography, performed to guide surgical planning in avoiding pedicle

damage and identifying the portion of the flap to island. The split DIEP flap for staged bilateral autologous breast reconstruction offers two soft-tissue flaps for the price of one donor site, offering new possibilities in breast reconstruction and the broader field of tissue transplantation. © 2013 Wiley Periodicals, Inc. Microsurgery, 2013.

“
“Reconstruction of distal thumb injuries still remains a challenge for hand surgeons. Surgical treatment includes the use of local, regional, and free flaps. The purpose of this report is to present the results of the use of a sensitive reverse flow first dorsal metacarpal artery (FDMA) flap. The skin flap was designed on the radial side of the proximal phalanx of the index finger based on the ulnar and radial branch of the FDMA and a sensory branch of the superficial radial nerve. This neurovascular flap was used Stem Cell Compound Library in five patients to cover distal soft-tissue thumb

defects. All flaps achieved primary healing except for one patient in whom superficial partial necrosis of the flap occurred, and the defect healed by second intention. All patients maintained the thumb original length and were able to return to their previous daily activities. The reverse flow FDMA flap is a reliable option to cover immediate and delayed defects of distal thumb, offering acceptable functional and cosmetic outcomes in respect to sensibility, durability, and skin-match. © 2013 Wiley Periodicals, Inc. Microsurgery 34:283–286, 2014. “
“To investigate the relationship between ischemic time and rejection against allotransplants, vascularized cutaneous flaps from the groin IKBKE of Brown Norway rats were transplanted to Lewis rats. The ischemic time was set at 1 hour and 6 hours for comparison. Cycrosporine A was used as the immunosuppressant. The results showed more severe rejection in the 6 hours ischemic time group in vivo, and in vitro examination using mixed lymphocyte reaction assay also demonstrated a greater antidonor response in 6 hours-ischemic group than that in 1 hour-group. Immunohistochemical study demonstrated more MHC class II antigen expression in 6 hours-ischemic group than in 1 hour-group. These results suggest that longer ischemic time induces more severe rejection against allo-transplanted tissue compared with the shorter one through an upregulation of MHC class II antigen.

Splenocytes were removed from both CatG-deficient mice and C57BL6 control mice, and cell surface and total expression of MHC II (I-Ab) was analysed by flow cytometry. Levels of surface (Fig. 6d) or total (not shown) I-Ab in B cells, DCs, and resting or activated Belnacasan macrophages did not differ between CatG-deficient and control mice. Analysis of peritoneal macrophages also revealed no differences in

I-Ab expression between CatG−/− and C57BL6 control mice (data not shown). We concluded that, by several criteria, CatG lacks the ability to modulate steady-state MHC II levels in vivo and in live, cultured APCs. Our findings provide information on the mechanisms by which MHC II molecules resist endosomal proteolysis, a key biochemical requirement for their function in presentation of peptides captured in endocytic compartments. In their native conformation, purified, detergent-solubilized MHC II molecules failed to be degraded by most lysosomal proteases tested (cathepsins D, L, S, H, and B). The resistance of MHC II molecules to these

proteases thus is an inherent property selleck products of the folded MHC II ectodomains. In contrast, purified MHC II molecules were susceptible to proteolytic attack by CatG at a single cleavage site, which is broadly, but not universally, conserved amongst MHC II molecules. However, using several independent approaches, we were unable to detect any involvement of CatG in the turnover of MHC II molecules embedded in membranes of live APCs. These results

show, on the one hand, that proteolytic resistance of MHC II molecules is not absolute, allowing some scope for regulated turnover; on the other hand, they suggest that the CatG cleavage site is inaccessible in the membrane-embedded native MHC II protein, in vivo. The resistance of MHC II molecules to many endosomal proteases is structurally plausible: the immunoglobulin superfamily domain fold, which is adopted by the membrane-proximal domains, is well known to be highly protease-resistant, and the peptide-loaded antigen-binding groove is highly compact. Initiation of HLA-DR proteolysis by CatG in vitro involved site-specific cleavage between leucine (L) and glutamine (Q) within fx1 and fx2 of the lower loop of the β domain, isothipendyl which may be one of very few sites with sufficient flexibility to allow proteolytic attack. These findings are reminiscent of previous studies, in which CatG was demonstrated to initiate cleavage within the flexible hinge regions of immunoglobulins.39 The membrane-proximal location of the cleavage site, away from the antigen binding groove, is consistent with our observation that CatG cleaves peptide-loaded MHC II molecules, and that peptide binding is retained by CatG-cleaved DR molecules. The fact that peptide-loaded molecules are substrates for CatG supports the notion that CatG is capable of initiating proteolysis of MHC II molecules in their native conformation.