The genus Gibbsiella, which was isolated from oak trees displayin

The genus Gibbsiella, which was isolated from oak trees displaying extensive stem bleeding, was recently reported by Brady et al. (1). The genus Gibbsiella consists of only one species named Gibbsiella quercinecans (NCPPB 4470T).

The genus Gibbsiella, which is a Gram negative, rod-shaped, non-spore forming and non-motile bacterium, has been closely related to genera Serratia, buy BIBW2992 Kluyvera, Klebsiella, and Raoultella (> 97%) by 16S rRNA sequence analysis. However, the genus Gibbsiella forms a distinct lineage within the family Enterobacteriaceae, this having been confirmed by both gyrB and rpoB gene sequencing. Streptococcus mutans is known as a primary pathogen of dental caries in humans (2). One of its virulence properties is the

ability to produce exopolysaccharides from sucrose (3, 4). The oral cavities of many animals are colonized by a large number of bacteria, including DAPT exopolysaccharide-synthesizing strains such as S. mutans. In this study, the microflora of the bear (Ursus thibetanus) oral cavity was investigated, focusing on exopolysaccharide-synthesizing strains. The exopolysaccharide-synthesizing strains selected for this study were Gram negative isolates from the oral cavities of bears. The strains formed large, raised, sticky colonies with irregular margins on mitis salivarius agar (Difco Laboratories, Detroit, MI, USA). During this research, a non-pigmented, non-motile, non-spore-forming Gibbsiella like strain, designated NUM 1720T, was isolated from the oral cavity of bears. The strain

was grown at 37°C under aerobic Plasmin conditions on brain-heart infusion agar (Difco Laboratories). The isolates were subjected to further taxonomic study. DNA was extracted from the bacterial cultures by using the Promega Genome kit (Promega, Madison WI, USA) according to the manufacturer’s instructions. To determine the phylogenetic affinity of the isolate, the almost-complete 16S rRNA gene was sequenced and subjected to a comparative analysis. The 16S rRNA gene was amplified using a PCR with primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′; E. coli positions 8–27) and 1525r (5′-AAAGGAGGTGATCCAGCC-3′; E. coli positions 1543–1525) according to the method described by Shinoda et al. (5). The PCR products were directly sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Stockholm, Sweden) and automatic DNA sequencer (3130 genetic analyzer; Applied Biosystems). The closest known relatives of the novel isolates were identified by performing database searches. Identification of the closest phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server ( (6). The topologies of the trees were evaluated by performing a bootstrap analysis of the sequence data, using CLUSTAL W software (7). Sequence similarity values were calculated manually.

In the murine-Langerin-DTR models, developed originally to target

In the murine-Langerin-DTR models, developed originally to target only LCs, it was realized subsequently that both CD207/Langerin+ DDCs and LCs were ablated by diphtheria toxin treatment. Because the two DC

subsets reconstituted Caspase inhibitor with different kinetics, interpretation of the effect on T cell responses was complex [63-65]. Finally, depletion of CD205+ DCs in CD205-DTR mice dramatically reduced CD4+ and CD8+ T cell responses to bacterial and viral infections [48]. However, given that the steady-state frequency and distribution of Tregs, Th1 and Th17 cells was grossly altered by diphtheria toxin treatment, it was difficult to attribute the effect solely to CD205+ DCs, without considering the effect of the altered immune environment [48]. CD11c-cre and Langerin-cre mice have also been used to generate targeted knock-outs of multiple immune signalling molecules, including recombination signal binding protein for immunoglobulin kappa J (RBPJ) [66], signal transducer and activator of transcription 3 (STAT3) [67], tumour necrosis factor, alpha-induced protein 3 (TNFAIP3) (A20) [68] and myeloid differentiation primary response gene 88 (Myd88) [69]. These applications suffer from the same subset specificity issues as the DTR models, due to model-dependent artefacts

and the complex expression patterns of Langerin and the CD11c transgene [70, 71]. Administration of horse cytochrome c is an alternate strategy used to ablate cross-presenting DCs via specific induction of the apoptosis pathway in

cells possessing cross-presentation machinery [72]. Experiments using this treatment have suggested that cross-presentation is NVP-LDE225 purchase limited to a subset of splenic CD8+ cDCs, although the GPX6 model was complicated by the partial depletion of CD11b+(CD4+) cDCs, which are usually considered to be incapable of cross-presentation [73]. In addition to inducible ablation, transcription factor knock-out mice have been used to define in-vivo DC subset function, as they show complete or partial deficiencies in well-defined DC subsets (reviewed in [1, 74]). For example, the comparison of interferon regulatory factor 4 (IRF4–/–) mice (lacking CD11b+ DCs) with Id2–/– or IRF8–/– mice (both lacking CD8+ DCs) has supported the paradigm that CD11b+ DCs promote Th2 cytokine production, while CD8+ cDCs promote Th1 cytokine production [75, 76]. Similarly, basic leucine zipper transcription factor, ATF-like 3 (BATF3–/–) mice have been used to demonstrate that cross-presentation is confined to the CD8+ cDC and CD103+ mDC subsets, which are selectively deficient in these mice [77]. Interestingly, while both CD205-DTR [48] and BATF3-deficient mice [77] lack CD8+ cDCs, only in the CD205-DTR model were splenic CD4+ T cell responses affected. An additional complexity in transcription-factor knock-out mice is that the targeted transcription factors are expressed, albeit at lower levels, in the remaining DC subsets [74, 78].

In the monocytic cell line THP-1, where upregulation of EpoR expr

In the monocytic cell line THP-1, where upregulation of EpoR expression occurred very early (Fig. 1), reduction of IL-8 mRNA was accordingly detected already 1 h after costimulation with ARA290. To establish infection, E. coli firmly adheres and eventually invades the epithelial cells in the urinary bladder (Wu et al., 1996; Martinez et al., 2000). Intracellular MAPK Inhibitor Library price bacteria are able to multiply and persist in the bladder epithelium, likely constituting the reservoir for recurrent infection (Mysorekar & Hultgren, 2006). We therefore investigated whether ARA290 influenced these two crucial steps of bacterial infection. In 5637 bladder epithelial cells, the

total number of E. coli did not differ after any treatment. In contrast, invasion was reduced when selleck screening library cells were costimulated with inactivated bacteria and 100 nM ARA290 (P<0.05; Fig. 4). A similar effect was obtained in the bladder epithelial cell line T24 by costimulation with 10 nM ARA290 (data not shown). To understand the mechanism underlying reduced bacterial invasion, we investigated the pathways known to be activated during E. coli invasion into bladder epithelial cells. Type 1 fimbriae expressed by virtually all UPEC bind to different cell surface markers on uroepithelial cells, including β1 integrins (Martinez et al., 2000; Eto et al., 2007). Activated β1 integrin signals to FAK, which becomes phosphorylated and further activates phosphoisonitol-3-kinase.

Eventually, bacterial binding induces rearrangement of the cellular actin cytoskeleton and uptake into the cell (Martinez & Hultgren, 2002). We assessed the influence of ARA290 on the activation of this pathway by determining the content of phosphorylated FAK (pFAK) at 5, 15 and 25 min after infection with E. coli CFT073. As expected, infection with CFT073 induced

increased levels of pFAK (Fig. 5). Interestingly, activation of FAK was diminished in cells costimulated with ARA290, indicated by lower levels of pFAK compared with cells exposed to bacterial stimuli only. The total FAK Cepharanthine levels were not affected by this treatment as determined by reprobing the blot with anti-FAK antibody. It thus remains to be determined whether reduced FAK activation was due to the specific inhibition of FAK phosphorylation, or whether upstream signals, i.e. β1 integrin signaling was impaired. However, we did not observe changes in β1 integrin mRNA expression, nor could we detect changes on the protein level, either in the total or in the membrane protein fraction (data not shown). With emerging resistance against conventional antimicrobial therapy, new treatment strategies are needed. In this study, we investigate whether the nonerythropoitetic Epo analogue ARA290 might be a candidate for such an approach. Using an in vitro model of E. coli UTI, we reveal two mechanisms by which ARA290 modulates E. coli infection.

9 ± 0 5 mm) at the 15-cm site and 0 8 to 2 0 mm (1 2 ± 0 4 mm) fo

9 ± 0.5 mm) at the 15-cm site and 0.8 to 2.0 mm (1.2 ± 0.4 mm) for the vein at the 10-cm site and 1.0 to 3.0 mm (1.9 ± 0.5 mm) at the 15-cm site. Under clinical

conditions, the two case flaps survived well without major complications. The clinical follow-up period Copanlisib concentration was from 12 to 14 months (mean: 13 months). The advantage in using this recipient pedicle lies not only in its superficial aspect but also in the protection offered by the surrounding muscle. Thus the defect could be reconstructed efficiently without stress upon the surgeon; if the ALTP flap of the ipsilateral side was used, the defect could be reconstructed efficiently within the same surgical field. © 2009 Wiley-Liss, Inc. Microsurgery 2010. “
“Replantation of amputated body parts is a highly specialized, cost-intensive procedure and can offer significantly increased quality of life in

selected cases.[1] Continued technical find more innovation and experience have been reflected in a number of successful personal operative series being reported in the literature.[2] In the absence of custom made devices for storage of the amputated part, prehospital preparation is often determined by the referring practitioner, prior to contact with the referring department. To optimize chances of successful replantation, appropriate preparation and transfer to the replantation center are critical. However, literature regarding perceptions about correct preoperative storage and transfer by referring practitioners is limited. Our intital study reported significant deviations from the advanced trauma life support (ATLS) guidelines in this regard, excluding suitable patients from replantation.[3, 4] In consideration of the increased penetrance of ATLS and equivalent courses in the medical community and the recent nationwide reconfigurations in health service delivery, we performed a 5-year follow-up survey (reaudit) to determine any changes in referring practitioner perceptions of this procedure. The survey was conducted on centers

referring to the Welsh Centre for Burns and Plastic Surgery (n = 16) between November 2012 and February 2013. To facilitate comparisons, the same semi-structured telephonic questionnaire and best practice guidelines (ATLS) as our earlier study[3] were adopted 17-DMAG (Alvespimycin) HCl (Table 1). A total of 68 healthcare practitioners were invited, of whom 51 responded (78% respondent rate), from 90% of referring units. The respondents included the following grades: consultant (14%), specialist registrar (12%), and core trainee/senior house officer (50%); foundation year/house officer (4%); nurse practitioner (10%); and acute care GP (10%). Of the respondents, only 25% described the entire procedure correctly. Of the remainder, only 4% remarked they would seek advice on storage of the amputated part before preparing for transfer. Labeling of the amputation with any identification details was mentioned by only 10% of respondents.

In our earlier study we demonstrated co-regulation of


In our earlier study we demonstrated co-regulation of

inflammatory with anti-inflammatory CD4+ T cells in CL disease [10]. In order to understand more clearly the possible role of the specific Vβ CD4+ T cell subpopulations in CL disease, correlation analyses were performed between the frequency of proinflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-10) cytokine-producing cells for each of the specific Vβ CD4+ T cell subpopulations following stimulation with SLA. Among the three Vβ families that demonstrated higher frequencies of TNF-α-, IFN-γ- and IL-10-producing cells, two of them, selleck chemical Vβ 5·2 and 24, demonstrated strong positive correlations between the frequency of cells producing IL-10 and TNF-α or IFN-γ (Vβ 5·2) (Fig. 7). In addition, the Vβ 8 subpopulation (P = 0·02, data not shown) demonstrated

a positive correlation. Our earlier data demonstrated a direct correlation between the frequency of both activated T cells and IFN-γ-producing lymphocytes and the size of ulcerated cutaneous lesions in CL disease [15]. Thus, it was of great interest to verify if any of the specific CD4+ Vβ subpopulations also correlated with lesion size as a method of identifying possible T cell subpopulations involved with the local immune response and possible tissue damage. Interestingly, correlation analyses revealed a positive correlation between higher frequencies of Vβ 5·2 CD4+ T cells and larger lesion areas (Fig. 8). Thus, three Vβ subpopulations (Vβ 5·2, 11 and 24) were identified as having a significant and consistent bias towards involvement with the anti-Leishmania response as measured by a variety of indicators, such as overall frequency, portion of cells committed to an ‘experienced’ phenotype and cytokine production.

One of these, Vβ 5·2, also showed a positive correlation with lesion size. Given that there is intense trafficking of lymphocytes from the local draining lymph nodes through the blood and to lesions, we have seen that the blood often reflects what is happening at lesion sites in CL and mucosal disease when considering the overall immunoregulatory profile [10,12,13,34]. However, specific T cell GBA3 subpopulations could be expected to accumulate in lesions if they express receptors specific for a prevalent antigen. This preferential accumulation would be identified by a higher percentage of cells expressing a given TCR Vβ segment in the inflammatory infiltrate compared to the percentage of these same TCR Vβ-expressing cells in the blood. Given the positive correlation of CD4+ Vβ 5·2-expressing T cells with lesion size and their greater frequency of activation and cytokine production as measured by all criteria examined in this study, we analysed the percentage of these cells among CD4+ T cells in the inflammatory infiltrate of lesions from a group of CL patients.

The resulting supernatant was resuspended in 10 μL of Solution A

The resulting supernatant was resuspended in 10 μL of Solution A. The protein concentration of the nuclear and mitochondria/cytoplasm fractions was determined using the Biorad Protein Assay. These procedures were done as previously described 20. Quantitation of the Western blots was performed using Adobe Photoshop CS3 as described (

Briefly, the Adobe Photoshop lasso tool was used to outline each protein band and a background region on the membrane. The mean gray value and the pixel value were multiplied to determine the absolute intensity of the band. When no band was visible, the outlined region was made equal in pixel number to that of the background region. The background to be subtracted from a given band was determined by multiplying the mean gray value of the outlined background region by the pixel

measurement for the corresponding band. The authors thank Victor E. Marquez for his generous gift of HK434 and Yuefang Sun for taking care of the mouse colonies. find more This study was supported by a grant from the National Institute of Health (to A. W.) and the Research Supplement for underrepresented minorities from the National Cancer Institute (to J. T.). Conflict of interest: The authors oxyclozanide declare no financial or commercial conflict of interest. “
“Monocytes are blood leukocytes that can differentiate into several phagocytic cell types, including DCs, which are instrumental to the inflammatory response and host defence against

microbes. A study published in this issue of the European Journal of Immunology by Balboa et al. [Eur. J. Immunol. 2013. 43: 335-347] suggests that a shift of the CD16− monocyte population toward a CD16+ subpopulation may represent an immune evasion strategy that ultimately favors persistence of Mycobacterium tuberculosis. Together with other recent reports, the article by Balboa et al. sheds new light on the function of CD16+ monocytes in health and disease; in this commentary, we discuss the implications stemming from these findings. Immunity to pathogens and inflammatory reactions relies on the coordinated action of several immune cell populations including lymphoid cells and monocyte-derived phagocytes, such as macrophages and DCs. Monocytes are generated in the marrow and circulate in the blood where they can patrol the whole body for signs of infection or inflammation, and migrate to injured tissues upon attraction by several chemokines and microbial ligands. Monocytes exhibit high plasticity and can differentiate into a variety of cell subsets depending on their microenvironment in infected or inflamed tissues [1, 2].

Furthermore, LCMV infection in vivo or LCMV-infected DCs in vitro

Furthermore, LCMV infection in vivo or LCMV-infected DCs in vitro rendered, via TLR2, CD4+CD25+ Tregs capable of diminishing T1D. We identify novel mechanisms by which TLR2 promotes immunoregulation and controls autoimmune diabetes in naïve or infected hosts. This work should help understand T1D etiology and develop novel immune-based therapeutic

interventions. Type 1 diabetes (T1D) is a genetic disease resulting in the destruction of insulin-producing β cells by autoreactive T cells in the pancreatic islets of Langerhans RAD001 1. The importance of additional environmental factors such as infections in the development of this disease has long been reported, but to date whether and how these might trigger or prevent T1D is not understood 2. It has been proposed that the inflammatory events induced upon anti-infectious immunity enable enhanced presentation of β-cell antigens to autoreactive T cells. Pro-inflammatory Cell Cycle inhibitor cytokines cause the up-regulation of class I MHC molecules on β cells, and may thereby “unmask” them for recognition by CD8+ T cells 3. In addition, concomitant damage to β cells and activation of APCs by the infection may promote the presentation of β-cell antigens to CD8+ T cells. This has notably been demonstrated in NOD mice using Coxsackievirus B4 4, or in RIP-LCMV mice, which transgenically

express lymphocytic choriomeningitis virus (LCMV) antigens on their β cells and develop autoimmune diabetes following LCMV infection 5–7. Inflammatory signals not only promote DC and T-cell activation but might also directly cause β-cell destruction 8–10, therefore strongly contributing to T1D development. On the other hand, studies in humans and mice suggest that infections and inflammation might play a protective role in T1D; notably, disease can be prevented in

NOD mice by infection with a number of viruses 2. Antiviral immunity may increase resistance to diabetogenic infections or “distract” the immune system from their detrimental effect 11. In addition, see more as we reported recently 12, viral infections may shape the immune system such that diabetogenic T cells are impaired or kept under control by immunoregulatory mechanisms. We found that viral infection triggered the expansion of invigorated CD4+CD25+ Tregs that produced TGF-β and protected from autoimmune diabetes by synergizing with programmed death-ligand 1 (PD-L1). These findings indicated a beneficial role of virally induced inflammation in T1D. A number of studies in mice have underscored the capacity of pro-inflammatory agents to prevent rather than induce T1D when intervening in the absence of β-cell damage and autoantigen 13. TLRs are usually referred to as “danger-sensing” molecules that play a central part in triggering inflammation and immunity in response to infection 14.

These cells are able to present antigens to lymphocytes, and play

These cells are able to present antigens to lymphocytes, and play a role in the up-regulation of homing molecules such as DC [4,5]. In contrast to immune response induction, tolerance is the unresponsiveness of the immune system via suppression of T and B cell activation by regulatory Selumetinib molecular weight T cells, deletion or anergy. However, there are many open questions about the function

of the LN, including the migration of cells from the draining area, the role of the LN in the induction of immune responses, the control of parasites or tolerance. It is possible to use knock-out mice, e.g. lymphotoxin α or retinoic acid-related orphan receptor (Ror)-γt knock-out mice to study the function of LN. These mice have reduced or no LN, but they all have further disorders, particularly in the spleen [6,7]. To circumvent the problems of immune

system dysfunction caused by these gene knock-outs, a second method of studying LN function is to remove only the LN of interest. This LN dissection technique permits identification of the role of a specific LN without affecting further organs or areas. Therefore, in this review learn more different research areas are illustrated where LN dissection was performed to identify the function of LN or the consequences of a missing LN. LN dissection is an experimental surgical technique which has been used for many years not only to from analyse the role of LN in the immune system and lymph fluid transport, but also in different diseases in animal models. LN were removed from many different draining sites such as the skin-draining site (for example the axilliary LN [8], the brachial LN [9], the popliteal LN [10–12] or the inguinal LN [13,14]), the head–neck region (cervical LN [15–19]) or the peritoneal area (the mesenteric LN [20–23] and the coeliac LN [24]). For dissection of the mesenteric

LN (mLN), for example, the abdomen was opened and the gut was taken out so that the mLN were visible (Fig. 2a). The mLN were excised carefully in order not to injure the superior mesenteric artery lying behind, whereas the connection of the lymph vessels and small blood vessels to the LN was disturbed. Afterwards, the gut was replaced in the abdomen and the abdomen was closed. LN are integrated as central organs in the lymph vessel system. The afferent lymphatics coming from the draining area, which could be the gut system or the skin, transport fluid, proteins, lipids and different cell populations of the immune system to the LN sinus. The efferent lymphatics leave the LN at the medullar site to greater LN or veins of the blood system. After LN dissection, the lymph vessel system is destroyed and the afferent and efferent system vessels are reconnected.

It is well established that the innate immune system changes with

It is well established that the innate immune system changes with aging or immune senescence.62–65 In elderly patients, NK cells, macrophages, dendritic cells, and neutrophils show impaired function as well as decreased toll-like receptor (TLR)-mediated cytokine responses. Aging has been shown to impair responses Doxorubicin to viral infections including HIV, HSV, CMV, and Influenza; one mechanism is thought

to be the functional impairment of plasmacytoid dendritic cells, the major producer of type I interferons, which are essential for combating viral infections.66 Several studies have demonstrated that innate immune factors are compromised in the FRT of post-menopausal women. A general decline in several immunomodulatory factors has been reported that appear to be age related as well as attributed to the loss of endocrine responsiveness.67 As multiple immune factors of the FRT are estrogen responsive, the loss of estrogen with aging results in loss of TLR function, secretory antimicrobial components, commensal lactobacilli, and acidity of vaginal microenvironment.68 Vaginal epithelium thins significantly in the non-estrogenic post-menopausal state. There is also lack of production

of cervical mucus, which itself is a protective barrier against pathogens.69 Gender-specific STA-9090 mouse decline of immune responses in the elderly have been described (reviewed by Refs 62,70). Post-menopausal women show higher chronic levels of proinflammatory cytokines IL-6, MCP1, and TNFα as well as a reduced ability to respond to pathogens or stimuli (Reviewed by

Refs 62,70). Mselle et al.71 have shown that inactive endometrium has lower numbers of NK cells compared to endometrium of cycling Thalidomide women. A few studies have addressed the loss of specific antimicrobials in the FRT of post-menopausal women. Production of defensins has been shown to change under the influence of sex hormones.72 Han et al.,73 demonstrated that estradiol can enhance the production of HBD2 whereas progesterone can decrease it. Fahey et al.74 reported a loss of antibacterial activity against both Gram-positive and Gram-negative bacteria in the uterine secretions of post-menopausal women and correlated this with a loss of SLPI secretion, a molecule well known for bactericidal and viricidal activity.74,75 Shimoya et al.76 confirmed lower SLPI levels in cervical vaginal secretions from post-menopausal women and further showed that hormone replacement therapy in elderly women increased SLPI levels. In our studies (M. Ghosh, J. V. Fahey, S. Cu-Uvin, C. R. Wira, unpublished observations), we observed a reduction in anti-HIV activity in CVL from post-menopausal compared to pre-menopausal women. Using Luminex analyses we found that post-menopausal CVL contained higher levels of proinflammatory IL1α and lower levels of Elafin (Ghosh, unpublished observation) when compared to pre-menopausal controls.

Measurement of cell viability by 7-AAD staining 24 h after thawin

Measurement of cell viability by 7-AAD staining 24 h after thawing demonstrated that Teffs had a viability of 90%, whereas 70% of Tregs were viable (data not shown). We tested whether selleck the expression of any of the markers affected by GAD65 stimulation was related to clinical outcome of treatment. We found no significant correlation between expression of Treg markers used in this study and changes in stimulated C-peptide measured as ΔAUC or AUC 4 years after treatment. C-peptide secretion was not significantly different in patients where an FSChiSSChi population was induced by

GAD65 stimulation compared to those who did not respond in this way (data not shown). To test whether the function of Tregs in T1D children included in the Phase II trial was affected by GAD-alum or placebo administration, suppression assays

using sorted and expanded Tregs (CD4+CD25hiCD127lo) and Teffs (CD4+CD25–CD127+) were performed. Gates used to sort Tregs and Teffs are illustrated in Fig. 3a. Expanded Tregs from patients treated with GAD-alum suppressed proliferation of autologous Teffs to the same extent as Tregs from placebo patients Selleckchem MG 132 (Fig. 3b). Tregs from both groups of patients displayed dose-dependent suppression of proliferation. As reported previously [25, 27, 28], we further found that suppression in autologous cultures of Tregs and Teffs was reduced in all patients (n = 7, placebo and GAD-alum

combined) compared to a healthy control (seven repeated measurements, Fig. 3c, P < 0·0001). To determine whether this attenuated suppression was intrinsic to Tregs or Teffs, we tested the suppression of Tregs from T1D patients (either GAD-alum- or placebo-treated, with similar results; Fig. 3b,d), and from a healthy control in autologous and cross-over culture suppression assays. As shown in Fig. 3c, T1D Tregs exerted the same level of suppression on Teffs coming from either T1D or healthy subjects. Interleukin-2 receptor In the reverse experiment, healthy Tregs were able to suppress Teffs from healthy or T1D subjects to a similar degree. Taken together, these results suggest that attenuated suppression from Tregs of T1D patients is due to reduced Treg efficacy rather than to increased Teff resistance to suppression. To determine whether there was a difference in reduced Treg-mediated suppression due to treatment, we tested if the suppression exerted in cross-over cultures of T1D Tregs versus healthy Teffs and healthy Tregs versus T1D Teffs was different between treatment arms. There was no difference in suppression exerted by Tregs from GAD-alum-treated patients compared to placebo Tregs in cross-over cultures with healthy Teffs, nor in the suppression exerted by healthy Tregs cultured with Teffs from GAD-alum-treated patients and Teffs from placebo subjects (Fig. 3d).