For CD3ζ immunoprecipitation, insoluble material was pelleted, an

For CD3ζ immunoprecipitation, insoluble material was pelleted, and the supernatant was incubated with 5 μg anti-CD3ζ (clone 6B10.2; Santa Cruz Biotechnology, SantaCruz, CA) and 50 μl 50% protein G–Sepharose (Pharmacia, Uppsala, Sweden). Cell lysates or immunoprecipitates were solubilized in reducing Laemmli sample buffer (BioRad), resolved by 10% SDS–PAGE and blotted onto nitrocellulose membranes (BioRad, Benicia, CA). Blots were probed with anti-phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY) or anti-CD3ζ (6B10.2; Santa Cruz Biotechnology) and horseradish peroxidase-conjugated secondary antibody

followed by detection with Super Signal Chemiluminescent Reagent (Pierce, Rockford, IL). For measurement of phosphorylation of p56Lck, stimulated cells were lysed and transferred to the nitrocellulose membrane before probing them with specific antibodies (PY 416; Cell Signaling) and blots were developed

Wnt cancer Ibrutinib as described above. Quantification was performed using multi gauge V3.0 software. For intracellular analysis of phosphorylation events in stimulated CTL,31 cells were fixed with 4% paraformaldehyde (PFA) for 10 min at 37° after stimulation and permeabilized with 90% ice-cold methanol. For staining of total protein, resting cells were permeabilized with ice-cold methanol. Permeabilized cells were washed extensively with PBS and stained with anti-CD8 antibody and one of the following: anti-p56Lck-phycoerythrin conjugate (BD phosflow, SanJose, CA), anti-LAT (Cell Signaling), anti-pLAT (PY 191; Cell Signaling), or anti-ppERK AF647 conjugate (p44/42 MAPK; Cell Signaling). For detecting unconjugated antibodies, anti-rabbit IgG-allophycocyanin secondary antibody (1 : 1000; Molecular Probes, Carlsbad, CA) was used. Measurement of intracellular free Ca2+ was carried out using the calcium-sensitive dye Fluo-3 acetoxymethyl ester (Fluo-3

AM). Resting high or low avidity CD8+ T cells were loaded with 5 μm Fluo3 AM (Invitrogen Life Technologies, Carlsbad, CA) in sterile and degassed FACS buffer (1 × PBS with 5% FCS) at 37° for 1 hr before. Dolutegravir order After washing, cells were incubated in the same medium at 37° for the indicated times. Samples were acquired on a FACSCalibur cytometer (Becton Dickinson, San Jose, CA). Basal Fluo3 fluorescence levels were measured for 60 seconds following which EL4 cells or EL4 cells loaded with Ova257–264 peptide (10−6, 10−9 and 10−12 M) were added. Calcium measurements were acquired for 60 seconds, followed by addition of CaCl2 (1 mm) to measure extracellular uptake. Data were analysed with flo jo software (Treestar, Ashland, OR). Our previous studies employing splenocytes from a TCR-transgenic mouse have shown that, at the population level, CTL of high or low avidity could be generated by stimulation with APC bearing low versus high amounts of antigen.

A strategy for a successful hookworm vaccine may use a cocktail o

A strategy for a successful hookworm vaccine may use a cocktail of potential vaccine candidates, including APR-1, targeting both the larval and blood-feeding stages (63). In the early 1990s, gastroenterologist John Croese and medical parasitologist Paul Prociv identified a series of cases of eosinophilic gastroenteritis in Caucasian residents of North Queensland selleck screening library (69). Initially, the disease was of unknown

aetiology but as more cases were diagnosed, solitary adult hookworms were identified from a few patients and were subsequently identified as the canine hookworm, A. caninum, a parasite that was previously thought not to reach maturity in the human gut (69) (Figure 2). As awareness spread amongst clinicians, cases were diagnosed in other areas where A. caninum was prevalent, including southern Queensland (70) and the south of the USA (71). While solitary adult A. caninum were identified CH5424802 purchase in only a handful of patients, infection was suspected

in many more, so we developed assays to detect circulating IgG and IgE antibodies to adult A. caninum excretory/secretory proteins and confirmed that many of the suspected cases of eosinophilic gastroenteritis where there was no parasitologic evidence of infection (i.e. no detection of adult worms or faecal eggs) were likely caused by occult infection with A. caninum (70,72). It is also noteworthy that in at least one patient, an adult A. caninum was observed in the absence of any overt pathology PtdIns(3,4)P2 or symptoms

(70). These findings pose an intriguing scenario whereby human enteric infection with the zoonotic A. caninum might be far more common than appreciated, and many of these infections might go unnoticed because of mild to no detectable pathology/symptoms. The Hygiene Hypothesis states that as populations become more hygienic and therefore virtually eliminate childhood parasitic infections (which have been constant partners through human evolution), there has been a concurrent increase in immune dysregulatory syndromes, such as autoimmunity, allergy and inflammatory bowel diseases. Diseases such as these are substantially less common in parts of the world with high helminth endemicity, and within endemic areas, the prevalence of allergic atopy is significantly lower in individuals with chronic worm infections (73–78). In epidemiologic studies, there is a good case for hookworm infection suppressing immune dysregulation.

The activation of NK cells observed in response to LASV- and MOPV

The activation of NK cells observed in response to LASV- and MOPV-infected MΦs is particularly interesting in that it is almost as robust as for positive controls, regarding the expression of NKp30 for instance, but nevertheless presents a different phenotype. We show here that LASV and MOPV do not infect NK cells. This result was expected and consistent

with previous studies showing that α-dystroglycan, the LASV and MOPV entry receptor, is expressed preferentially PLX4032 research buy on DCs and poorly on lymphocytes and that lymphocytic choriomeningitis virus, the prototypic Arenavirus that is closely related to LASV and MOPV, can infect only a few types of lymphocyte. Moreover, after direct contact with the viruses, NK cells were not activated and displayed no change in their effector functions. A slight downregulation of NKp30 expression and an increase in the expression of CXCR3 on the cell surface was even

observed in the presence of LASV or MOPV. Interestingly, TLR7 stimulation induced NKp30 downregulation as well. These results suggested that NK cells can detect both viruses, possibly through TLR7 stimulation requiring further investigation. NK cells display a rapid decrease in surface CXCR3 when cocultured PXD101 mouse with LASV- or MOPV-infected MΦs. However, the significance of this downregulation is unclear. It is unlikely to be accounted for by the modulation of CXCR3 mRNA synthesis, as analysis of the mRNAs revealed no change during LASV or MOPV infection (data not shown). Tideglusib CXCR3 is the receptor for the inflammatory chemokines CXCL9, 10, and 11. These chemokines, initially described as attracting activated T lymphocytes, are secreted in large amounts during the infection of MΦs with LASV and MOPV in vitro (Pannetier et al., manuscript in preparation). Moreover, the transcripts for CXCL10 and CXCL11 are found in PBMCs and lymph nodes from infected Cynomolgus monkeys [18]

and we show here that CXCL11 is detected in LASV- and MOPV-infected NK/MΦ cocultures. CXC chemokines, such as CXCL11, have been reported to induce the rapid desensitization and internalization of their receptor, CXCR3 [20]. Thus, the downregulation of surface CXCR3 expression could partly be accounted for by receptor internalization. This hypothesis is consistent with our observations, showing that CXCR3 surface expression is also downregulated when cell contact is prevented, implying that soluble factors are involved. Moreover, it is also consistent with our results with neutralizing Ab directed against CXC chemokines that abolish or reduce the downregulation of CXCR3 at the surface on NK cells in the presence of LASV- or MOPV-infected MΦs respectively.

In contrast, ATCC33650, known to lack perosamine (Perry & Bundle,

In contrast, ATCC33650, known to lack perosamine (Perry & Bundle, 1990), did not exhibit this phenotype. A recent

study (Sheng et al., 2008) showed that the deletion BMN 673 concentration of per in E. coli O157:H7 resulted in a mutant lacking the O antigen with a concomitant nonmotile, autoaggregative phenotype. The liquid cultures of this mutant also showed more rapid sedimentation than that of the parent strain. When we compared the turbidity of spent culture media obtained from strains YS-11 (wild type), 455 (wzt-deleted mutant), 455-LM (complemented strain), and ATCC33650 (per negative) cultures, both strains 455 and ATCC33650 cells showed rapid sedimentation in the medium (data not shown). Because strains YS-11 and 455-LM induced greater abscess formation in mice than did strains 455 and ATCC33650, it is likely that the biofilm-like structures as described above for these strains might be important for the pathogenicity of E. hermannii. However, it is important to note that the data presented were derived from the study of one clinical isolate; therefore, the results might not be representative of the overall pathogenic potential of this organism. As for future

studies, we will examine other strains of E. hermannii for the presence of the per cluster. More thorough investigations are also needed to determine the role of this gene cluster in biofilm formation by this organism, although the data obtained from this study strongly suggest that the wzt is involved in the exopolysaccharide production. We are grateful to Mr Hideaki Hori (the Institute of Dental Research, Osaka Dental University) for his excellent assistance with the electron microscopy. A part of this research was performed at the Institute of Dental Research, Osaka Dental University. This study was supported in part by the Osaka Dental University Research Fund (A05-09) and Osaka Dental University Joint Research Funds (B08-01). T.Y. and Y.S.-S. contributed equally to this study. “

oligodendrocyte glycoprotein (MOG), a minor protein of the central nervous system myelin, is recognized as a potential target in multiple sclerosis and neuromyelitis optica. The extracellular domain of MOG is commonly used in a wide range of mouse strains and other animals to induce cAMP experimental autoimmune encephalomyelitis (EAE), an autoimmune animal model of multiple sclerosis, because it is a target for antibody-mediated attack. Previous studies, using selected peptides, have indicated that MOG35–55 peptide is an encephalitogenic epitope in C57BL/6 (H-2b) mice. A more systematic analysis of both T-cell and B-cell responses following immunization of C57BL/6 mice with either recombinant extracellular mouse MOG protein (1–116) or with overlapping peptides spanning the whole sequence of MOG, before assessment of responses to 15 mer and 23 mer peptides was undertaken.

The mortality risk was equal in the two groups by day 106 of foll

The mortality risk was equal in the two groups by day 106 of follow-up, and improved in the transplanted group thereafter. McDonald and Russ have reported similar findings using ANZDATA.14 An analysis of the period 1991–2000 found an 80% lower long-term risk of mortality between those transplanted and those remaining on the waiting list. Cameron et al. have performed a meta-analysis examining

the effect of transplantation on overall quality of life.15 Successful kidney transplantation was associated with improved Selleckchem Daporinad general wellbeing and less distress, when compared with continued haemodialysis or peritoneal dialysis. There are several individual studies that have examined quality of life issues in more detail. Evans et al. reported that 79.1% of transplant recipients describe near normal physical function, compared with only 50% of dialysis patients.16 Mental function scores were also higher in transplant recipients. Studies by both Gorlen et al.17 and Laupacis et al.18 found that the quality of life improvements associated with transplantation were sustained long term. However, transplantation continued to affect quality of life relative to normal.18 This was attributed to the side effects of immunosuppression,

comorbid conditions and the stress associated with the possibility of losing graft function. A detailed analysis of the relative costs of Bumetanide dialysis and Ferroptosis inhibitor drugs transplantation has been performed by Kidney Health Australia.19 Estimates of the cost of home or satellite-based dialysis (haemodialysis and peritoneal) for an individual are approximately $45 000–$60 000 per year. Hospital-based haemodialysis is estimated to cost approximately $83 000 per year. Although the initial cost of transplanting an individual is estimated to be relatively high ($62 000 for the first year) the cost falls significantly thereafter (approximately $11 000 per year for year 2 and onwards). The estimated costs associated

with an individual live donor transplant are similar to those for an individual deceased donor transplant.19 A Canadian report estimated that transplanting an individual would result in savings of CAN$104 000 over a 20-year period.20 Only a brief account of the overall safety data will be summarized here. A much more detailed analysis of the literature regarding donor safety will follow in subsequent sections of these Living Kidney Donor guidelines. By and large, live kidney donation is considered to be safe for the majority of healthy donors. This contention, however, is based predominantly on large retrospective studies, which demonstrate that unilateral nephrectomy in healthy subjects is generally associated with a very low level of long-term risk.21–27 A meta-analysis published by Garg et al. has examined the development of proteinuria in donors.

We found Tem cells highly expressing OX40, even if at lower level

We found Tem cells highly expressing OX40, even if at lower level than Treg cells (Fig. 3C); thus intratumoral OX86 injection could directly target Tem cells at this site. Conversely, Tem cells obtained after immunization of naïve BALB/c mice with two consecutive injections of BM-derived dendritic cells (BMDCs) activated with LPS, as previously described 17, expressed low or absent OX40, even after in vitro activation (Supporting Information Fig. 3). In BMDC-injected animals, Tem cells were shown to constitutively express CD40L at sufficient levels to induce DC activation 17. The CD40/CD40L interaction is crucial for DC activation,

survival and proliferation 26. Many data suggest that this axis is involved in inducing protective anti-tumor immune response 18, 19, 27, 28 and that activation of this pathway may represent a strategy for tumor treatment. To investigate whether OX86-induced tumor rejection was dependent on the CD40/CD40L axis, WT and CD40−/− mice were inoculated subcutaneously

with CT26 cells and treated intratumorally with OX86. As shown in Fig. 4A, OX86 treatment was ineffective in CD40-deficient mice, while inducing tumor rejection or impaired tumor growth in CD40-sufficient mice. Such failure to induce anti-tumor response in the absence of CD40 could be either due to defective licensing of DC reactivation and migration from the tumor or due to impaired T-cell priming at the dLNs by DCs licensed, but not competent, for optimal T-cell costimulation. 3-mercaptopyruvate sulfurtransferase To discriminate between these

two possibilities, an in vivo DC migration assay was performed. Tumors progestogen antagonist growing in WT and CD40−/− mice were treated with OX86 or rat IgG co-injected with green fluorescent microbeads. Only DCs that have up-taken the beads at the tumor site can be detected as fluorescent in dLNs. Although OX86 rescued DC migration from tumor to dLNs in WT mice, the same treatment was ineffective in CD40−/− mice (Fig. 4B), a finding implying that in absence of the CD40/CD40L axis tumor-associated DCs cannot be reactivated. We also confirmed that, in CD40−/− mice, the OX40 expression by tumor-infiltrating Tem cells was not defective, indicating that the CD40/CD40L system did not affect the T-cell activation status at the tumor site or Tem-cell responsiveness to OX86 treatment (Supporting Information Fig. 4). We hypothesized that, in the immunosuppressive tumor microenvironment, Tem cells were inhibited in their ability to license DCs via CD40L, and that OX40 triggering might provide the right signal for Tem cells to supply an effective CD40/CD40L mediated co-stimulation. Although the intratumoral injection of OX86 did not change the percentage of Tem cells (Fig. 4C), it significantly upregulated CD40L expression on Tem cells (Fig. 4D). Such CD40L up-modulation was specific for Tem cells, as no other T-cell subsets and especially CD44lowCD62Llow recently activated T (Tact) cells responded accordingly (Fig. 4E).

Depletion of dendritic cells from CD3-activated PBMC or from unst

Depletion of dendritic cells from CD3-activated PBMC or from unstimulated PBMC reduced cancer cell destruction by approximately 50%. It has been reported that signals from activated CD4+ T cells enable dendritic cells to instruct bystander dendritic cells to prime naïve CD4+ T cells [50, 51]. However, CD3-activated T cells could not initiate this dendritic circuit without monocytes; furthermore, monocytes were required in unstimulated PBMC cultures that were added to CD3-activated PBMC. Depletion of monocytes from CAPRI cells immediately before their coculture with cancer cells did not significantly reduce lysis. However,

depletion of dendritic cells decreased cancer cell destruction by 50% (Fig. 5A, B). This suggests that dendritic cells may provide a continuous flow of cytokines Histone Acetyltransferase inhibitor and/or of tumour-immunogenic information by building an information bridge between cancer cells and effector T cells to maintain cancer cell destruction by T effector

cells. Supplementary professional antigen presentation by activated dendritic cells may prevent rudimentary TCR signalling by cancer cells leading to multiple immunosuppressive effects, such as default secretion of IL-10 by Th1 cells [52]. Taken together, optimal priming for cancer destruction required cell-mediated bidirectional cooperation among a cellular quartet consisting of CD14+ monocytes, CD14−CD1a+CD83+ dendritic Methocarbamol cells, CD4+ T cells and CD8+ T cells, whereas

a cellular trio comprising dendritic cells, helper T see more cells and cytotoxic T cells achieved optimal cancer cell lysis without monocytes. Carcinomas often escape from recognition by downregulating their own HLA expression [32, 33]. Increased HLA expression of cancer cells correlates with increased survival of patients [53–56]. Could CAPRI cells, which lyse HLA-restricted tumour cells, influence the HLA expression of cancer cells? Examination of CFSE-stained carcinoma cells showed that cocultured CAPRI cells did indeed increase the expression of HLA class I and class II molecules in autologous cancer cells (Fig. 3), and they most likely do so in many cancer types lysed by CAPRI cells (listed in Table 3, lysis not shown). Of particular note was the successful CAPRI cell-mediated lysis of carcinoma cells of Bowen’s disease. These intraepidermally growing carcinoma in situ cells are commonly recalcitrant to therapy because they are enveloped by fibroblasts. Less than 1% of Bowen’s cancer cells bound keratinocyte antibodies in cytospins (not shown). This cancer is an excellent example of the proposed inhibitory role of tumour stroma, as this stroma can prevent direct lysis by T cells [57].

The increase in larvae from day 3 to 7 days post-challenge was pr

The increase in larvae from day 3 to 7 days post-challenge was probably due to the gradual migration of L3 from the stomach to the different sections of the small intestine (24,25). Individuals never completely cleared the infection, and nematodes were still present, although with very low numbers, in the first section at 120 days post-challenge. Graphidium strigosum: Abundance was consistently higher in the fundus compared to the antrum, and no temporal changes were Vismodegib ic50 observed between sampling points (or the interaction between sampling point and

organ section), when differences among individuals and the nonindependent sampling of the two parts of the stomach from the same individual were considered (Figure 1b, Table 2). All infected individuals maintained a constant number of nematodes up to 120 days post-infection. The drop in parasite number find more in the antrum at day 40 and 60 post-challenge was caused by a sampling procedure and should not be considered biologically relevant. These

results were consistent with our long-term observations on the intensity of infection of these nematodes in free-living rabbits of different age, specifically, rabbits can reduce or clear T. retortaeformis but not G. strigosum. Trichostrongylus retortaeformis: A strong IFN-γ expression in the first section of the small intestine of infected rabbits was observed during the first 30 days post-challenge; thereafter, no dominant pattern was observed (Figure 2a). Analysis based on the normalized Ct values (that differs from the 2−ΔΔCt transformation in Figure 2) found that changes in IFN-γ and IL-4 significantly Progesterone differed between treatments (infected and controls) and time post-infection (DPI): IFN-γ decreased while IL-4 increased in transcription with the infection course, IL-10 exhibited constant expression over time although was significantly higher in infected compared to controls (Table 3). Graphidium strigosum: A robust IL-4 expression was observed in the top section of

the stomach of infected rabbits; however, the between-individual variability was high as highlighted by the large standard error bars (Figure 2b). Based on the Ct values, the expression of the three cytokines was higher in the infected compared to the controls but no significant changes were recorded during the course of the infection (Table 3). The two infections clearly showed different cytokine profiles, which imply differences in the effectors and timing of their activation as well as their dynamical consequences. The somatic antibody response of infected rabbits to L3 and adult stage was similar both for IgA and IgG against the two nematodes supporting the hypothesis that the two parasite stages cross-react at the antibody level. As such, we only present the results for the adult stage (Figures 3 and 4) and summarize in the supplement the findings for the L3 stage (Figures S1 and S2).

4, Supplementary Fig  S2) TF release by cells stimulated with Ig

4, Supplementary Fig. S2). TF release by cells stimulated with IgG fractions from SN-APS, LPS or IgG fractions from APS was increased significantly compared to untreated endothelial cells, as well as cells stimulated with human control IgG. TF release was PD-0332991 nmr inhibited significantly by preadsorption of SN-APS IgG with CL or LBPA (Fig. 4). To our knowledge, this is the first study showing aPL detected by TLC immunostaining associated with clinical features of APS in patients repeatedly negative for the laboratory criteria of APS, i.e. aCL, aβ2-GPI and LA. Moreover, the results suggest that the biological activity of these antibodies is able to trigger a signal transduction pathway(s) in endothelial cells with consequent proinflammatory

and procoagulant effects. Current laboratory criteria for the classification GS-1101 cell line of APS include aCL and aβ2-GPI measured by standardized ELISA and LA, detected by clotting assays [1,21]. However, the term SN-APS has been suggested recently for patients with a clinical profile suggestive of APS who are persistently negative for the routinely used assays [2,22,23]. We studied here a cohort of patients affected mainly by autoimmune systemic diseases presenting a clinical picture suggestive of APS, i.e. vascular thrombosis and/or pregnancy morbidity associated with several non-criteria APS features, persistently negative for the routinely used aPL. Interestingly, a statistically significant

correlation was observed between thrombosis and pregnancy morbidity in these patients. In the absence of positive blood tests, more clinical features would make a diagnosis of SN-APS more convincing. We identified the presence of aPL in about 60% of such patients using a method (TLC immunostaining) in which the antigen was run on aluminium-backed silica gel plates; in this way it may mimic phospholipid exposure after protein binding [8,12]. Interestingly, a strong correlation GBA3 was observed between these aPL specificities demonstrated by TLC immunostaining. The prevalence of aPL detected by TLC test in SLE patients without APS was similar to that showed by ELISA (61% and 78%, respectively). Although these aPL antibodies are probably

of low affinity and are not associated with any clinical manifestation, long-term prospective studies could clarify their clinical relevance. With regard to the so-called SN-APS patients, the discrepancies between ELISA and immunostaining on TLC plates in detecting antibodies against CL, LBPA and PE may be due to the different antigenic presentation of phospholipids on chromatograms compared to the surface of microtitre wells. In addition, six (16·7%) of the SN-APS patients showed serum IgG antibodies against annexin II detected by ELISA. Anti-annexin II have been associated recently with thrombosis in patients with APS, even though they can be detected in serum of patients with rheumatoid arthritis and other autoimmune systemic disorders [14,24].

Our studies revealed that responses to linear epitopes of MOG fol

Our studies revealed that responses to linear epitopes of MOG following immunization with recombinant MOG were absent in peptide-immunized animals. Moreover, the use of peptides to the full sequence revealed novel epitopes for antibody responses. These findings are relevant to study aspects that control disease progression

and to test tolerogenic therapeutic regimens in H-2b mice. In addition, they reveal information of the relevance of B-cell populations that will be key to understanding the mechanisms by which these B-cell populations could contribute to disease. Despite the reports that MOG transcripts are expressed in lymphoid organs, both MOG-deficient and WT mice show similar T-cell and B-cell responses against the extracellular RG7204 price domain of MOG, including the immunodominant MOG35–55 T-cell epitope. Also, no differences in the fine specificity of the T-cell responses

to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG−/− mice. As Doxorubicin cell line we have reported previously,[9] this lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE. In CNS myelin MOG comprises 2·5% of the total myelin proteins[4] compared with proteolipid protein, which represents about 50% of the total myelin protein. Despite the relatively low levels of protein, MOG is a major target of the immune responses that lead to chronic demyelinating disease in mice, rats and marmosets.[4, 5] The pathogenic properties of MOG, particularly induction of demyelination, are commonly associated with antibody responses to Amoxicillin the extracellular immunoglobulin-like domain making MOG a readily accessible target of the immune attack on compact myelinated axons.[17, 18] Many EAE studies make use of recombinant MOG proteins corresponding to residues 1–125 of hMOG or 1–116 of mMOG to understand

the role of antibodies to conformational epitopes in disease.[2, 4, 8, 19] As well as being a target for pathogenic antibodies, the immunoglobulin-like domain contains the promiscuous peptide residue MOG35–55, which is encephalitogenic in several mouse strains including C57BL/6 (H-2b), Biozzi ABH (H-2dq1), NOD(H-2g7) and PL/J (H-2u) mice, as well as in outbred monkeys.[3, 10, 20, 21] This promiscuous peptide also contains an epitope for induction of disease in Lewis rats[6] and MOG35–55 is also pathogenic in HLA-DR2 transgenic mice, providing a strong rationale for its potential pathogenic effect in humans.[22] However, in MS patients the T-cell responses and epitope specificity of the human B-cell response to MOG is not only heterogeneous, but may also be restricted to a subset of patients.