Injections were started on the third day after arthritis induction and were performed three times a week. In a second set of experiments, D8, the anti-eotaxin-2 antibody showing best protective

results, was tested in a dose–response model. Adjuvant arthritis was induced according to the above-described protocol. Animals (six rats per each condition) were treated with D8 intraperitoneally at a dose of 20 µg, 100 µg or 1000 µg, starting on day 3, three times weekly (D8 prevention group). A separate set of animals (six per condition) were treated with identical doses after arthritis onset (D8 treatment group). In order to compare the anti-inflammatory effect of D8 with that of a traditional anti-inflammatory agent of known efficacy, one group was treated with intraperitoneal methotrexate MG-132 in vitro (MTX), 0·25 mg/kg, once weekly, starting on day 3 after arthritis induction (MTX prevention group). An additional group was treated with MTX, 0·25 mg/kg once weekly, in combination with D8, 100 µg intraperitoneally given three times a week, starting on day 3 (combined D8–MTX prevention group). A control group was treated with PBS throughout the experiment. Body weight in grams was measured every other day as an indicator of systemic inflammation. For evaluation of paw swelling, ankle and wrist diameter in mm (to one place after the decimal point) were recorded

selleck chemical three times a week. Each paw was scored on a scale of 0–4 for the degree of swelling, erythema

and deformity (maximum score 16 per animal) as follows: 0 = normal, 1 = slight erythema and/or swelling of the ankle or wrist, 2 = moderate erythema and/or swelling of ankle or wrist, 3 = severe erythema and/or swelling of ankle or wrist and 4 = complete erythema and swelling of toes or fingers and ankle or wrist and inability to bend the ankle or wrist. Finger and toe swelling was recorded according to their partial contribution: ankles, each toe scored 0·2; wrist, each finger scored 0·25; the sum of all joints was calculated. Whole animal mobility was scored between 0 and 4 according to the following definitions: 0 = normal, Y-27632 2HCl 1 = slightly impaired, 2 = major impairment, 3 = does not step on paw and 4 = no movement. Data were analysed using spss software version 16·01. Student’s t-test was performed to identify significant differences between experimental groups. Three or more group means were compared by one-way analysis of variance, with an assumed significance level of P < 0·05. In these experiments, treatment was given before the appearance of clinical arthritis (prevention group). Effect of treatment with anti-eotaxin-2 antibodies on arthritis score.  Treatment with anti-eotaxin-2 monoclonal antibodies caused a significant reduction in arthritic score severity, compared to rats treated with PBS. This protective effect was evident in all three antibodies tested (G7, G8 and D8).

1 mM nonessential amino acids, and 1 mM sodium pyruvate (Life Technologies, Grand Island, NY)]. All cultures were maintained at 37 °C in a humidity-controlled incubator with 5% CO2 and were grown to confluence over 5–6 days before addition of pathogenic bacteria C. rodentium. The cells were washed and placed in antibiotic-free medium for 1 h. Confluent stock monolayers were subcultured by trypsinization. In this study, we utilized mouse intestinal epithelial cell line CMT93 to better elucidate cell

signaling responses to enteric pathogens in vitro. Nine experiments were conducted independently with similar results. To determine the time-dependent intracellular changes of NF-κB and Smad 7 in response to pathogen exposure, CMT93 cells were exposed with Cr (2.5 × 107 CFU per well) for 1 h Gefitinib in PKC412 mw antibiotic-free DMEM at 37 °C. Subsequently, the media and cell lysates were collected at 0, 15, 30, 60, 90, and 120 min and 14 and 24 h postpathogen exposure. Cells were washed and lysed [(1% Triton-X-100 supplemented with 0.1 μM phenylmethylsulphonyl fluoride, 0.1 μM sodium orthovanadate, and Halt protease inhibitor (10 μL mL−1,

Pierce cat# 78410, Thermo Scientific, Rockford, IL)]. The lysates were kept at −80 °C for future Western blot analysis. The culture supernatants were stored at −20 °C for future measurement of TNF-α cytokine production. Total RNA was isolated from frozen colonic tissue (distal part of the colon) and treated CMT93 cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer’s protocol. First-strand cDNA was synthesized using 2 μg of extracted total RNA (Ready-to-Go kit; Amersham Pharmacia Biotech, Piscataway, NJ). IL-10 and TGF-β colonic expression was determined by real-time PCR using QuantiTect SYBR green real-time PCR kit (Qiagen, Valencia, CA) on the Opticon II DNA thermocylcer (MJ Research, Waltham, MA). A PCR master mix was prepared according to the manufacturer’s aminophylline protocol with a reaction volume of 50 μL, using the following real-time cycler conditions: 95 °C for 15 min, 94 °C

for 15 s, 55 °C for 30 s, 72 °C for 30 s for 38 cycles. GAPDH was used as internal controls. LightCycler relative quantification software was used to normalize data to the same GAPDH mRNA level. Samples were run in duplicate. Mouse IL-10 and TGF-β commercially available PCR primers were purchased from Biosource International, Inc. (Camarillo, CA) for detection, while GAPDH commercially available upstream and downstream PCR primers were utilized for detection (R&D Systems, Minneapolis, MN). Mouse colonic tissue and treated CMT93 cells were homogenized with lysis buffer prepared as previously mentioned. The suspensions were centrifuged at 4 °C, and the supernatant was collected, and protein content was determined using DC protein assay (Bio-Rad Laboratories, Hercules, CA).

However, the mechanism of cyst formation in the AQP11(-/-) mouse is still unknown. Methods: To enable the analyses of AQP11 at the protein level in vivo, AQP11 BAC transgenic mice (TgAQP11) that express 3 × HA-tagged AQP11 protein were generated. In addition, to investigate the mechanism of cyst formation in the AQP11(-/-) mouse, we analyzed the AQP11(-/-) mouse, by focusing on the polycystic kidney disease-related gene products such as polycystins. Results: Immunofluorescence of the kidney from

TgAQP11 mice revealed that AQP11 localizes to the endoplasmic reticulum (ER) of proximal tubule cells. Since ER is essential for quality control and trafficking of newly synthesized GDC-0449 datasheet proteins, we hypothesized that the absence of AQP11 in ER could result in impaired quality control and aberrant trafficking

of polycystin-1 (PC-1) and polycystin-2 (PC-2). An increased protein expression level of PC-1 and a decreased protein expression level of PC-2 in AQP11(-/-) mouse kidneys were found, compared with wild-type mice. Moreover, PC-1 had a higher molecular weight in AQP11(-/-) mouse kidneys, caused by impaired selleck chemicals N-glycosylation processing of PC-1. In addition, density gradient centrifugation of kidney homogenate and in vivo protein biotinylation revealed impaired membrane trafficking of PC-1 in AQP11(-/-) mice. Finally, it was demonstrated that the Pkd1(+/-) background results in increased severity of cyst formation in

AQP11(-/-) mouse kidneys, indicating that PC-1 is involved in the mechanism of cyst formation in AQP11(-/-) mice. Conclusion: Our data demonstrated that impaired glycosylation processing and aberrant membrane trafficking of PC-1 in AQP11(-/-) mouse could be a key mechanism of cyst formation in AQP11(-/-) mice. ZHAO YE1,2,3,4, ZHAO HONG1,2, ZHANG YUN1,3, ZHANG JIANLIN2, TSATRALIS TANIA1, WANG CHANGQI1, WANG YA1, WANG YIPING1, WANG YUANMIN4, LEE VINCENT1, ALEXANDER STEPHEN I.4, ZHENG GUOPING1, HARRIS DAVID C.1 1Centre for Transplant and Renal Research Westmead however Millennium Institute, the University of Sydney, Sydney, NSW, Australia; 2Dept. of Biochemistry and Molecular Biology, Shanxi Medical University, P. R. China; 3Experimental Center of Science and Research of First Teaching Hospital, Shanxi Medical University, P. R. China; 4Centre for Kidney Research, Children’s Hospital at Westmead, Sydney, NSW, Australia Introduction: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblast formation in kidney fibrosis. Previously we have shown that MMP-9 induced EndoMT in glomerular endothelial cells. This study investigated whether Notch signaling plays a role MMP-9-induced EndoMT of peritubular endothelial cells in kidney fibrosis. Methods: Mouse renal peritubular endothelial cells (MRPEC) were isolated by magnetic microbead separation using anti-CD146 Ab.

jejuni were isolated from patients with enteritis (food poisoning); of those, 14 strains belonged to ST21 (CC21), ST22 (CC22), ST42 (CC42), ST400 (CC353), ST407, ST545 (CC22), ST922, ST4052

(CC353), ST4060 (CC460), ST4063 (CC283) and ST4108 (CC607) [12]. Seven strains of C. jejuni (serotype Penner HS:19) were from patients with GBS and belonged to ST22 (CC22), ST2140 (CC574), ST4049 (CC464), ST4051 (CC22), and ST4053 (CC353) [12]. C. jejuni also included strain ATCC33560. Five strains of C. coli were isolated from patients with enteritis and belonged to ST860 (CC828), ST1068 (CC828), ST1593 (CC828), and ST4059 (CC828) [12]. Four strains of C. fetus and two strains of C. lari, which were isolated from the feces of patients with food poisoning, were kindly provided by Dr Akemi Kai (Tokyo Selumetinib mouse Metropolitan Institute of Public Health, Tokyo, Japan). V. cholerae O1 strain EO8 [13], V. cholerae O139 strain T16 [14], and H. pylori strain C7M [15] were also assessed. All bacterial strains were stored at −80°C in 3% skim milk (Difco; Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 5% glucose (Difco). The other bacterial strains used were from our laboratory stock. For Campylobacter growth, blood-agar plates (trypticase soy agar supplemented with 5% sheep blood; Becton Dickinson, Tokyo, Japan) were inoculated and incubated for 1–2 days at 37°C in a microaerophilic atmosphere (6–12% O2

and 5–8% CO2; the remaining gases being mostly N2 from air). BHI broth CHIR-99021 (Difco) supplemented with 10% Dimethyl sulfoxide FBS (Gibco, Carlsbad, CA, USA) was used as

a liquid medium (at 37°C in a microaerophilic atmosphere). Bacterial strains other than Campylobacter were also grown in BHI broth supplemented with 10% FBS (at 37°C). Prior to motion analysis, test bacteria were grown in BHI containing 10% FBS at 37°C for approximately 3 hrs (to a log phase). Bacterial motility was then examined under an inverted, phase-contrast microscope with a Micro Warm plate (Kitazato, Tokyo, Japan) that regulated the temperature of the specimens. The motility speed (μm/s) was measured using a motion analysis system with the program C-Imaging C-MEN (Complix); the limit of resolution of swimming speeds was 100 μm/s. Bacterial swimming in a liquid layer of BHI broth containing 10% FBS (106 to 107 colony forming units/mL) between a glass slide and a glass cover (pre-coated with FBS) was continuously recorded 15 times in 0.05 s analysis segments (a total of 0.75 s) and the swimming speed (μm/s) of each bacterial cell in a specimen obtained, essentially as described previously [15]. Pre-coating the glass surface with FBS is important because it prevents attachment of test bacteria to the glass surfaces. Measurements were performed in at least five different fields of each specimen, swimming speeds for approximately 300 bacterial cells being measured for each specimen (within a few mins), and the percentage of motile bacteria determined.

We proposed a parsimonious hypothesis for the dynamics of the rabbit–nematode system where the seasonal dynamics of T. retortaeformis were driven primarily by the host acquired immune response affecting helminth development and fecundity (10,14,15), while G. strigosum was not constrained by immunity, so that parasite abundance increased exponentially

https://www.selleckchem.com/products/Rapamycin.html with host age (11). Previous studies supported the hypothesis of an immune-regulated T. retortaeformis infection and noted that third-stage larvae may enter arrested development under adverse immunological conditions (16). The tendency to arrest the development in the mucosa and the evidence of intestinal pathology were more recently confirmed in laboratory experiments (17,18). Laboratory infections of rabbits with G. strigosum showed a clear increase in serum

IgG but this was not sufficient to clear the infection, and high intensities were still observed 3 months after the initial challenge (19). VX-809 order No clinical symptoms but chronic asthenic gastritis were also reported in rabbits exposed to different infection doses (20). Overall, these studies indicate that rabbits develop different immune responses against T. retortaeformis and G. strigosum, which can explain the different patterns of infection observed in free-living rabbit populations. The identification of the processes affecting host–parasite interactions can be challenging in natural animal systems if more than one mechanism is taking place and, even more, when there are confounding variables that

cannot be ruled out (10,21). Motivated by our epidemiological work and to gain a better understanding of the immuno-parasitological mechanisms influencing the interaction between the host and its parasites, we undertook a comprehensive study to quantify changes in the rabbit’s immunological components and associated helminth intensities, during a primary infection of T. retortaeformis and G. strigosum. Laboratory infections were performed, wherein rabbits were challenged with third-stage larvae (L3) and the dynamics of the systemic and local immune response quantified for 120 days post-challenge. Our prediction was that the immune response to the two helminths differed fundamentally in the intensity but not the triclocarban type of components activated, so that T. retortaeformis would elicit a stronger response than G. strigosum, and this would lead to the clearance of the first but not the second nematode. The ultimate goal of this study was twofold: first, to identify the most common immunological processes and essential components affecting the epidemiology of these gastrointestinal infections and second, to highlight the immunological differences between these helminths and discuss how they can explain the epidemiology of infection in free-living rabbit populations. Trichostrongylus retortaeformis and G.

Transmitted subclinical glomerulonephritis is noted in approximately 15% of Japanese donors.[10] IgA nephropathy accounts for over 90% of transmitted glomerulonephritis. The follow-up protocol biopsy shows early disappearance of IgA deposition within the first 3 months after transplantation in many recipients. On the contrary, early recurrence of IgA nephropathy develops within

1 to 2 months’ post-transplant in a small number of recipients with IgA nephropathy. In the overlapping period between transmission and early recurrence, it would be impossible to correctly detect recurrence of IgA nephropathy. Recurrence of IgA nephropathy is usually confirmed at the protocol biopsy performed 3 months post transplant or later, and deteriorated graft function is absent at the protocol biopsy. The majority of recurrent IgA nephropathy cases involve only histological recurrence without TSA HDAC chemical structure Sorafenib proteinuria and microscopic haematuria. Protocol biopsy makes it possible to study the detailed progression of recurrent glomerulonephritis from a very early change to typical glomerular

disease. We learned about many interesting recurrent cases of both primary glomerulonephritis and secondary glomerulopathies, which were presented at the annual conference of the Japanese Clinicopathological Conference on Renal Allograft Pathology. Some of the important case reports were published in Clinical Transplantation as the proceedings of the Japanese Clinicopathological Conference on Renal Allograft Pathology. Almost all the reports SSR128129E of recurrence of rare renal disease presented details of both histological changes based on protocol biopsies and clinical course. These reports included recurrence of light chain deposition disease,[25] fibronectin nephropathy,[26] atypical HUS caused by complement regulatory factor H disorder,[27] HSPN,[28] IgA nephropathy,[29, 30] C-ANCA-associated glomerulonephritis,[31] mixed

cryogloburinemic glomerulonephritis,[32] FSGS[33, 34] and others. We strongly encourage learning from these papers for a better understanding of the detailed changes in recurrent glomerular diseases. Understanding the pathogenesis of recurrent glomerulonephritis is critical to optimizing prevention as well as treating individual cases of recurrent glomerulonephritis. The study of recurrent glomerulonephritis will contribute not only to improving long-term graft survival but also to clarifying the pathogenesis of each case of glomerulonephritis. Protocol biopsy is one the most effective methods for achieving the ultimate goal of elucidating the pathogenesis of recurrent glomerulonephritis. “
“Date written: April 2009 Final submission: April 2009 Blood glucose control should be optimized aiming for a general HbA1c target ≤7%. (Grade A*).

[7] demonstrated that DNA vaccines, initially designed to

prevent infection, also have a pronounced therapeutic action. DNA hsp65 switches the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria in heavily infected mice [8]. Ha et al. demonstrated that immunotherapy using either a plasmid DNA encoding mycobacterial 85A antigen or interleukin-12 (IL-12) DNA vaccine combined with conventional chemotherapy was highly effective for the prevention learn more of Mycobacterium tuberculosis (M. tb) reactivation and reinfection in mice [9]. Immunotherapy with plasmid DNA is also a valuable adjunct to antibacterial chemotherapy to shorten the duration of treatment and improve the treatment of latent TB infection [10]. Like Ag85A DNA vaccine, single Ag85B DNA vaccine is effective in treating TB in mice; however, Hsp70, ESAT6 or MPT64 DNA vaccine has much smaller or no effect on mice TB [7, 11]. Recently,

a combined DNA vaccine encoding Ag85B, MPT64 and MPT83 along with chemotherapy showed strong potential for TB immunotherapy [12]. A combination of the DNA vaccines expressing mycobacterial hsp65 and IL-12 delivered by the hemagglutinating PD0325901 manufacturer virus of Japan (HVJ)-envelope and liposome (HSP65 + IL-12/HVJ) exerts therapeutic efficacy (survival and immune responses) in TB-infected monkeys [13]. Our previous study showed that the immunotherapy with Ag85A DNA vaccine in combination with rifampin (RFP) results in effective treatment of MDR-TB infected mice [14]. In this study, MDR-M. tb strain sensitive to pyrazinamide (PZA) was used as the positive control to further confirm the immunotherapeutic effects almost of Ag85A DNA vaccine on MDR-TB-infected mice. The application of such immunotherapy in combination with first line anti-TB drugs might result in cure of MDR-TB. Mice.  A total of 110 pathogen-free female BALB/c mice 6–8 weeks of age were purchased

from the Academy of Military Medicine and Science, China, maintained under barrier conditions in an animal room at the 309th Hospital of Chinese PLA, Beijing, China, and fed on a sterile commercial mouse diet (Beijing KeAoXieLi Company Limited, Beijing, China). MDR-TB strain.  The MDR-TB strain M. tuberculosis HB361 used for mice infection was isolated from a TB patient in the Tuberculosis Department of Thorax Disease Hospital of Hebei province, China. The drug resistance was determined again by conventional species identification and conventional drug susceptibility test using the absolute concentration method on Lowenstein-Jensen medium in line with Chinese Laboratory Science Procedure of Diagnostic Bacteriology in tuberculosis [14, 15]. Strain HB361 was resistant to RFP and isoniazid, but sensitive to PZA. Immunogenicity of DNA vaccines.  A total of 40 female BALB/c mice were immunized intramuscularly with saline, plasmid vector pVAX1, M. vaccae vaccine (Longcom Biological Pharmacy, Anhui, China), and Ag85A DNA for three times at 2-week intervals. M.

05). The CTA-guided duplex ultrasonography could direct the perforator-complex selection according to the size of the venous-perforator, and may reduce the intraoperative problems and the incidence

of fat necrosis. © 2013 Wiley Periodicals, Inc. Microsurgery 34:169–176, 2014. “
“This selleck chemical study was performed to review our 16-year experience in acute finger ischemia. A review of the literature was also performed. A retrospective chart review of 17 patients, 14 men and 3 women, was conducted. Etiologies were ulnar aneurysm in 11 cases, atrial fibrillation in five cases and thoracic outlet syndrome in one case. Upto the palmar superficial arch, embolus due to atrial fibrillation Selleck Deforolimus or thoracic outlet syndrome could be loosened by a Fogarty catheter. In cases of aneurysm of the ulnar artery, we performed each time an aneurysm resection followed by direct anastomose

alone, while three patients had additional grafts: artery graft (epigastric artery) or reversed vein grafts (superficial forearm vein). Microsurgical dissection of the digital collateral arteries enabled us to perform a thrombectomy. The transversal arteriotomies were closed after the collateral arteries were washed. The immediate perfusion of digit after the reconstruction of the aneurysm was each time excellent. The disoccluded vessels, investigated by Allen testing and Doppler ultrasound, were all patents. Two patients suffered from a small ulcer of the small fingertip that disappeared after

2 weeks. One patient had a 30° ischemic flexion contracture in the metacarpophalangeal joint and 25° flexion contracture in the proximal interphalangeal joint of the third digit. With regards to long-term Tolmetin outcomes, no secondary amputations were necessary and there was no recurrence after a mean follow-up of 10.7 years. Diagnostic of acute digital ischemia is often neglected. An early recognition and an aggressive microsurgical treatment are necessary to ensure low morbidity. © 2009 Wiley-Liss, Inc., Microsurgery, 2010. “
“Osteonecrosis of the femoral head is a disease in which bone death occurs and usually progresses to articular incongruity and subsequent osteoarthritis. To delay the process of the disease and the conversion to total hip arthroplasty, many surgical techniques have been described. Core decompression, nonvascularized autologous bone grafts, porous tantalum implant procedure, and various osteotomies have been used for the management of early precollapse stage osteonecrosis of the femoral head. However, none of these procedures is neither entirely effective nor can obtain predictable results. With the progress of microsurgery, the implantation of a free vascularized fibula graft to the necrotic femoral head has provided the most consistently successful results.

We also thank the contributions of the animal caretakers. This study was supported

by a Grant-in-Aid from BSE Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan. “
“Hepatitis C virus infection affects more than 170 million people worldwide. More than 80% of the patients are not able to eliminate the virus and progress to a chronic infection that usually culminates in complications such as cirrhosis FK506 chemical structure and/or hepatocellular carcinoma. Although the adaptive immune response has been widely shown to be essential for viral clearance, the role of natural killer (NK) cells is not clearly understood. In this study, the effect of HCV core protein is examined on NK cell function, i.e., cytotoxicity and cytokine secretion. The expression of core protein in the YTS NK cell line led to an increase in the percentage of apoptotic cells CP-690550 purchase soon after transduction. The surviving cells exhibited decreased cytotoxicity associated with decreases in perforin and granzyme B expression. Furthermore, the HCV core protein–transduced YTS NK cells had reduced IFNγ production as well as an altered surface receptor expression pattern. These features may correspond to a state of functional anergy similar to that seen in T cells transduced

with HCV core protein. Together, these data suggest that HCV core protein may alter NK cell function. Hepatitis C virus infection affects more than 170 million people worldwide. More than 80% of the patients are not able to eliminate the virus and progress to a chronic infection that usually culminates in some other complications such as Nintedanib (BIBF 1120) cirrhosis and/or hepatocellular carcinoma [1]. It has previously been reported that the host cellular immune response is essential in the outcome of the disease [2]. However, few studies have addressed the role of innate immune cells in responses

to HCV infection. Natural killer (NK) cells are lymphocytes of the innate immune system that provide protection against infections and tumours [3]. They express a variety of activating and inhibitory cell surface receptors that control their activation. When activated, NK cells are able to initiate a response that involves both cellular cytotoxicity and secretion of cytokines such as IFNγ and TNF, which may have direct antiviral effects and also serve to recruit other cell types involved in host defences [4]. In HCV infection, there is no consensus about the frequency of NK cells in patients [5–7], but most authors have observed that NK cells from chronically infected individuals show a weakened cytotoxic activity [6, 8] and decreased expression of perforin [8], as well as altered IFNγ secretion [9, 10]. This functional inactivation of NK cells could be one of the multiple mechanisms that the virus uses to interfere with and evade host antiviral immune response, and thus persist in the individual. Recent works have focused on the importance of NK cells in the course of the disease [11, 12].

In order to determine their tolerogenic activity,

as characterized by anergy induction and change in the cytokine secretion profile, Tg4 mice were treated with a minimum of ten i.n. doses of Ac1–9[4K], [4A] or [4Y] and the extent of tolerance induction was examined in vitro. The proliferative response of CD4+ T cells from untreated and peptide-treated BMS-777607 Tg4 mice to Ac1–9[4K], [4A] and [4Y] in vitro is shown in Fig. 3A. Naïve CD4+ T cells responded optimally to the cognate Ac1–9[4K] peptide at a concentration of 100 μg/mL, while Ac1–9[4A] and [4Y] acted as superagonists, requiring 100- and 10 000-fold lower concentrations than MBP Ac1–9[4K] to optimally stimulate naïve Tg4 CD4+ T cells, respectively. Administration of either of the three peptides i. n. resulted in a reduced proliferative response of the treated compared with the untreated Tg4 CD4+ T cells.

Paclitaxel supplier CD4+ T cells from mice treated i.n. with Ac1–9[4K], [4A] or [4Y] required 10-, 100- and 1000-fold higher concentration of Ac1–9[4K], respectively, to proliferate (Fig. 3A). The maximum proliferation of CD4+ T cells from treated mice remained below half the value observed from untreated Tg4 mice over a wide range of peptide concentration and affinity. Furthermore, Fig. 3A shows that neither could the hierarchy be altered nor could the relative degree of unresponsiveness be overcome by stimulating with higher affinity analogues. Changes in the cytokine secretion profiles of CD4+ T cells from untreated compared with peptide-treated Tg4 mice in response to in vitro peptide stimulation are shown in Fig. 3B. Supernatants from the above cultures were collected and analyzed for levels of IL-2, IFN-γ and IL-10 by sandwich ELISA. CD4+ T cells from untreated mice responded to in vitro stimulation with Ac1–9[4K], [4A] and [4Y] by increasing IL-2 secretion (top row, Fig. 3B), correlating directly with the proliferative response shown in Fig. 3A. these This was also the case for IFN-γ secretion (middle row, Fig. 3B). No IL-10 was detected in cultures of untreated CD4+ T cells upon Ac1–9[4K], [4A] or [4Y] stimulation in vitro (bottom row, Fig. 3B). The cytokine secretion profile

of CD4+ T cells from mice treated with i.n. Ac1–9[4K] was similar to that of untreated mice, albeit with lower IL-2 production. CD4+ T cells from mice treated with i.n. Ac1–9[4A] and [4Y] responded by much reduced IL-2 production in response to Ac1–9[4K], [4A] or [4Y] stimulation compared with those from untreated and Ac1–9[4K]-treated mice. IFN-γ was produced by CD4+ T cells from mice treated with i.n. Ac1–9[4K] and [4A] but not [4Y]. CD4+ T cells from both the i.n. Ac1–9[4A]- and [4Y]-treated mice produced large amounts of IL-10 in response to stimulation with Ac1–9[4K], [4A] or [4Y]. These results suggest that an active Th1 response is the dominant or default effector pathway in the Tg4 mouse model in response to MBP Ac1–9 peptides.