T-cell Mediated Disease Research Articles

Modern approaches to multiple sclerosis

This article will summarise aspects of ‘modern approaches to multiple sclerosis (MS)’, focusing on areas where there has been significant change in practice within the last decade, and in particular the new therapeutic agents available for relapsing MS which have posed new dilemmas for neurologists prescribing disease modifying therapies (DMT) for the condition. The basics: aetiology, pathology, and classification MS is a common cause of neurological disability in young and middle age adults, second only to trauma in the UK. Approximately 80,000 individuals in the UK have MS. MS is considered to be a predominantly autoimmune, T-cell mediated disease occurring in genetically predisposed individuals after one or more environmental ‘triggers’. The identity of the trigger(s) remains unknown, but currently favoured candidates include childhood Epstein-Barr virus infection, and low vitamin D levels. The result is the onset, usually in adulthood, of lesions affecting the white matter of the central nervous system (CNS), as in the brain, spinal cord and optic nerves. The pathology is characterised by demyelination and axonal loss in the CNS white matter (Fig 1). 1 MS is classified into subtypes according to a simple, but useful scheme: it is purely clinical, based upon the observations of the patient’s pattern of disease (Fig 2). Most patients present with a relapsing-remitting disease (RRMS), with relapses (‘attacks’) followed by recovery (partial or complete), with marked variability in both severity of relapses, and interval between them. A large proportion of patients with RRMS will eventually develop ‘secondary progressive’ disease (SPMS), characterised by a reduction or cessation of acute relapses but instead a gradual worsening of disability (Fig 2). The length of time spent in the relapsing phase is markedly variable, but once secondary progression is established the rate of progression is fairly predictable, and essentially irreversible. 2 There are no DMT for progressive forms of MS.

Maturation-Resistant Dendritic Cells Ameliorate Experimental Autoimmune Uveoretinitis

BackgroundEndogenous uveitis is a chronic inflammatory eye disease of human, which frequently leads to blindness. Experimental autoimmune uveoretinitis (EAU) is an animal disease model of human endogenous uveitis and can be induced in susceptible animals by immunization with retinal antigens. EAU resembles the key immunological characteristics of human disease in that both are CD4+ T-cell mediated diseases. Dendritic cells (DCs) are specialized antigen-presenting cells that are uniquely capable of activating naïve T cells. Regulation of immune responses through modulation of DCs has thus been tried extensively. Recently our group reported that donor strain-derived immature DC pretreatment successfully controlled the adverse immune response during allogeneic transplantation.MethodsEAU was induced by immunization with human interphotoreceptor retinoid-binding protein (IRBP) peptide1-20. Dendritic cells were differentiated from bone marrow in the presence of recombinant GM-CSF.ResultsIn this study, we used paraformaldehyde-fixed bone marrow-derived DCs to maintain them in an immature state. Pretreatment with fixed immature DCs, but not fixed mature DCs, ameliorated the disease progression of EAU by inhibiting uveitogenic CD4+ T cell activation and differentiation.ConclusionApplication of iBMDC prepared according to the protocol of this study would provide an important treatment modality for the autoimmune diseases and transplantation rejection.

Potent orally bioavailable purine nucleoside phosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lymphocytes—A novel treatment approach for autoimmune diseases, organ transplantation and hematologic malignancies

The profound suppression of T-cell immunity seen in purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficient patients supports potential application of inhibitors of PNP in the therapy of T-cell mediated diseases. BCX-4208 is a novel potent transition state analog inhibitor of human PNP with an IC 50 of 0.5 nM. PNP inhibition leads to elevation of dGuo which is converted to dGTP mainly in lymphocytes causing imbalance in deoxynucleotide (dNTP) pools and cell apoptosis. In in vitro studies, neither BCX-4208 nor dGuo alone inhibits proliferation of lymphocytes. BCX-4208 in the presence of 10 µM deoxyguanosine (dGuo) inhibits lymphocyte proliferation induced by MLR, IL-2 or Con A with IC 50s of 0.159, 0.26 and 0.73 µM, respectively. The IC 50 for dGuo in the presence of 1 µM BCX-4208 for the IL-2 stimulated lymphocytes was 3.12 µM. dGTP in human lymphocytes is elevated and a 3–5 fold increase in dGTP results in 50% inhibition after in vitro exposure to BCX-4208 and dGuo. Flow cytometric analyses of human lymphocytes using annexin V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis in T-cells (CD3+), B-cells (CD20+, CD19+) and NK (CD56+) cells. BCX-4208 is orally bioavailable in mice and elevates plasma dGuo levels to 3.7 µM (predose levels < 0.004 µM), similar to levels seen in PNP-deficient patients and levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of T-cell and B-cell mediated diseases. BCX-4208 is currently undergoing early clinical investigation in psoriasis and gout.

The CD226 Gly307Ser gene polymorphism is associated with severity of psoriasis

Psoriasis is a chronic inflammatory T-cell mediated disease of the skin with a complex pathogenesis involving both genetic and environmental factors [1]. Several regions in the genome including the major histocompatibility complex (MHC) [2], [3] have been reported to confer susceptibility to psoriasis risk; however, the genetic background of psoriasis is yet to be established...

Emerging from the shadows: Follicular helper T cells in autoimmunity

Emerging from the shadows: Follicular helper T cells in autoimmunity

The effect of a4β7 blockade on T-lymphocyte trafficking to intestinal compartments in the normal and inflamed intestine

Blockade of the integrin α4β7 has demonstrated promise as an organ specific therapy for the treatment of inflammatory bowel disease (IBD). It is believed that the therapeutic effect of α4β7 blockade is mediated by inhibiting T-cell trafficking into the intestine, however the specific T-lymphocyte compartment affected by α4β7 blockade and the effects of α4β7 blockade on normal T-lymphocyte trafficking and during acute injury are not well understood. In this study we investigated the effect of blocking α4β7 function on the presence of intestinal T-lymphocytes in the diffuse lamina propria and the lymphoid aggregates in the uninflamed intestine, in the acute injury model DSS colitis and the chronic splenocyte transfer model. The presence of intestinal T-lymphocytes in the diffuse lamina propria and lymphoid aggregates was evaluated in wildtype mice, β7-/- mice, and wildtype mice given blocking antibodies to α4β7 or control antibodies using flow cytometry and immunohistochemistry. To evaluate the effect of α4β7 blockade in acute injury, the onset and regression of inflammation, lymphoid aggregate formation and regression and the presence of T-lymphocytes were evaluated in the acute injury model of DSS colitis during α4β7 blockade. To evaluate the effect of α4β7 blockade on T-cell mediated colitis, the course of colitis and the presence of T-lymphocytes in the diffuse lamina propria and lymphoid aggregates was evaluated in the splenocyte transfer model of colitis. In the unchallenged animals, the intestinal T-lymphocyte population in the diffuse lamina propria was normal in the absence of α4β7 function. The T-lymphocyte population in the lymphoid aggregates was also normal with the exception that the Foxp-3 T-lymphocyte population was significantly decreased in the absence of β7. DSS-induced colitis peaked at day 3 after the removal of as DSS demonstrated by increased histological score, MPO activity, and began to regress at day 7 after removal of DSS. The number of B-cell containing lymphoid aggregates peaked at 7 days after the removal of DSS and remained elevated at 14 days after the removal of DSS even though clinical inflammation had regressed. Anti-α4β7 blockade or loss of β7 function dramatically decreased the number B-cell containing lymphoid aggregates, but did not show any other effects, including changes in T-lymphocyte trafficking into the aggregates, during DSS-induced colitis. In the T-cell mediated splenocyte transfer model of colitis, the population of T-lymphocytes in the diffuse lamina propria and the lymphoid aggregates were significantly decreased in β7-/- mice and the wildtype mice receiving α4β7 blockade. This reduction corresponded to the decreased inflammation as evidenced by significant lower expression of TNF-α, IFN-γ, IL-6, MPO and IL-17. The presence of Foxp3+ T-lymphocytes in lymphoid aggregates is dependent upon α4β7, however the trafficking of other T-lymphocytes into the diffuse lamina propria and lymphoid aggregates in the basal state and during acute injury is not dependent upon α4β7. α4β7 blockade can block pathogenic T-lymphocyte trafficking to the intestine in a T-cell mediated model of colitis and accordingly is effective at ameliorating inflammation in this model. These observations suggest that α4β7 blockade may have fewer effects than anticipated on normal T-lymphocyte trafficking and T-lymphocyte trafficking during acute injury and may be a more specific therapy for T-cell mediated inflammatory diseases of the intestine.

IL2-caspase3 chimeric protein controls lymphocyte reactivity by targeted apoptosis, leading to amelioration of experimental autoimmune encephalomyelitis

IL2-caspase3 chimeric protein was designed to target and kill cells expressing the high affinity IL-2 receptor. Its effects on lymphocyte reactivity and on experimental autoimmune encephalomyelitis (EAE), a T-cell mediated disease, were tested in this study. Our data show that IL2-caspase3 promoted cell specific apoptosis both in vitro and in vivo. Cell lines preferentially expressing the IL-2R α chain and encephalitogenic lymphocytes derived from EAE-induced mice were highly sensitive to the chimeras' activity. This was demonstrated by increased DNA fragmentation and annexin labeling together with reduced specific T-cell proliferation in response to IL2-casepase3 treatment. Furthermore, IL2-caspase3 treatment of EAE-induced mice caused a significant delay in disease onset together with a reduction in disease burden. The efficacy of IL2-caspase3 treatment was dependent on the time at which treatment begun, with the chimera ameliorating EAE only when administered at maximal activation of peripheral lymphocytes. According to our findings we suggest that the chimeric protein IL2-caspase3 may provide a novel approach for the treatment of a variety of autoimmune disorders, such as multiple sclerosis, as well as for other pathological conditions that involve uncontrolled expansion of activated T cells.

Treatment with bortezomib of human CD4+ T cells preserves natural regulatory T cells and allows the emergence of a distinct suppressor T-cell population

In vitro depletion of alloreactive T cells using the proteasome inhibitor bortezomib is a promising approach to prevent graft-versus-host disease after allogeneic stem cell transplantation. We have previously described the ability of bortezomib to selectively eliminate alloreactive T cells in a mixed leukocyte culture, preserving non-activated T cells. Due to the role of regulatory T cells in the control of graft versus host disease, in the current manuscript we have analyzed the effect of bortezomib in regulatory T cells. Conventional or regulatory CD4(+) T cells were isolated with immunomagnetic microbeads based on the expression of CD4 and CD25. The effect of bortezomib on T-cell viability was analyzed by flow cytometry using 7-amino-actinomycin D staining. To investigate the possibility of obtaining an enriched regulatory T-cell population in vitro with the use of bortezomib, CD4(+) T cells were cultured during four weeks in the presence of anti-CD3 and anti-CD28 antibodies, IL-2 and bortezomib. The phenotype of these long-term cultured cells was studied, analyzing the expression of CD25, CD127 and FOXP3 by flow cytometry, and mRNA levels were determined by RT-PCR. Their suppressive capacity was assessed in co-culture experiments, analyzing proliferation and IFN-gamma and CD40L expression of stimulated responder T cells by flow cytometry. We observed that naturally occurring CD4(+)CD25(+) regulatory T cells are resistant to the pro-apoptotic effect of bortezomib. Furthermore, we found that long-term culture of CD4(+) T cells in the presence of bortezomib promotes the emergence of a regulatory T-cell population that significantly inhibits proliferation, IFN-gamma production and CD40L expression among stimulated effector T cells. These results reinforce the proposal of using bortezomib in the prevention of graft versus host disease and, moreover, in the generation of regulatory T-cell populations, that could be used in the treatment of multiple T-cell mediated diseases.

Palmitoyl acyltransferase assays and inhibitors (Review)

Palmitoylated proteins have been implicated in several disease states including Huntington's, cardiovascular, T-cell mediated immune diseases, and cancer. To proceed with drug discovery efforts in this area, it is necessary to: identify the target enzymes, establish efficient assays for palmitoylation, and conduct high-throughput screening to identify inhibitors. The primary objectives of this review are to examine the types of assays used to study protein palmitoylation and to discuss the known inhibitors of palmitoylation. Six main palmitoylation assays are currently in use. Four assays, radiolabeled palmitate incorporation, fatty acyl exchange chemistry, MALDI-TOF MS and azido-fatty acid labeling are useful in the identification of palmitoylated proteins and palmitoyl acyltransferase (PAT) enzymes. Two other methods, the in vitro palmitoylation (IVP) assay and a cell-based peptide palmitoylation assay, are useful in the identification of PAT enzymes and are more amenable to screening for inhibitors of palmitoylation. To date, two general types of palmitoylation inhibitors have been identified. Lipid-based palmitoylation inhibitors broadly inhibit the palmitoylation of proteins; however, the mechanism of action of these compounds is unknown, and each also has effects on fatty acid biosynthesis. Conversely, several non-lipid palmitoylation inhibitors have been shown to selectively inhibit the palmitoylation of different PAT recognition motifs. The selective nature of these compounds suggests that they may act as protein substrate competitors, and may produce fewer non-specific effects. Therefore, these molecules may serve as lead compounds for the further development of selective inhibitors of palmitoylation, which may lead to new therapeutics for cancer and other diseases.

BCX-4208 (RO5092888), a Purine Nucleoside Phosphorylase (PNP) Inhibitor, Is a Novel, Potent Orally Active Anti-T-Cell and B-Cell Agent.

The profound suppression of T-cell immunity seen in patients with an inherited PNP deficiency supports the potential application of inhibitors of this purine salvage enzyme in the therapy of T-cell malignancies and T-cell mediated autoimmune diseases. About thirty percent of PNP deficient patients also show evidence of B-cell dysfunction. BCX-4208 is a novel potent transition state analog inhibitor of PNP (IC50 ~ 0.0005 μM) and in the presence of 10 μM deoxyguanosine (dGuo), inhibits human lymphocyte proliferation induced by MLR, IL-2 or Con-A with IC50s of 0.159, 0.26 and 0.73 μM, respectively. The IC50 for dGuo in the same assays in the presence of 1 μM BCX-4208 ranges from 1–3 μM. Neither BCX-4208 alone nor dGuo alone inhibits proliferation of lymphocytes. In the presence of PNP inhibitor, dGuo is converted to dGMP and then to dGTP. Accumulation of dGTP results in the alteration of deoxynucleotide (dNTP) pools, causing death of cells via a mechanism characteristic of apoptosis. In vitro data demonstrates that following exposure to BCX-4208 and dGuo, dGTP in human lymphocytes is elevated and a 5–8 fold increase in dGTP results in 50% inhibition of lymphocyte proliferation. Flow cytometric analyses of human lymphocytes using annexin-V staining reveal that BCX-4208 in the presence of dGuo induces cellular apoptosis not only in T cells (CD3+), but also in B cells (CD20+; CD19+) (Table 1). BCX-4208 is orally bioavailable in mice, can achieve maximal inhibition of PNP, and elevates plasma dGuo levels to 3–5 μM (predose levels < 0.004 μM), which is similar to levels seen in PNP deficient patients and to levels needed to cause apoptosis in T and B-cells. These data support the evaluation of BCX-4208 in the treatment of not only T-cell mediated diseases but also B-cell mediated diseases. BCX- 4208 is currently undergoing early clinical investigation in patients with psoriasis.Table 1.Cell subsets% Apoptotic cells mean ± SEM (n = 4–7)VehicleTreatment groupCD3+7.2 ± 0.918.3 ± 3.7*CD20+24.0 ± 3.641.8 ± 6.2*CD19+25.0 ± 3.151.6 ± 7.4**p ≤ 0.01 compared to vehicle

Anti-Moesin Antibodies Derived from Patients with Aplastic Anemia Stimulate Monocytes To Secrete TNF-α through an ERK1/2-Dependent Pathway.

Although aplastic anemia (AA) is a T-cell mediated disease, antibodies (Abs) specific to proteins derived from hematopoietic cells are often detected in the serum of patients with AA. It is not clear whether these auto-Abs play any role in the pathophysiology of AA. A previous study demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 40% of AA patients (Takamatsu H, et al. Blood, 2007) and that anti-moesin Abs derived from AA patients could bind immune cells such as T cells and monocytes and enhance TNF-α secretion from a monocytic leukemia cell line THP-1. To substantiate the ability of anti-moesin Abs to induce the expression of myelosuppressive cytokines through immune cells, the secretion of other cytokines from THP-1 cells in response to binding of anti-moesin Abs was analyzed. THP-1 cells were incubated in the presence of PMA and LPS with 10 μg/ml of anti-moesin Abs purified from AA patients' serum or the same amount of control IgG for 48 hr. Anti-moesin Abs enhanced the secretion of TNF-α and IL-1β 2 to 5 times more than the control Abs. However, anti-moesin Abs did not enhance secretion of IL-2, IL-6 and IL-10. When peripheral blood mononuclear cells (PBMCs) isolated from 10 healthy individuals were incubated in the presence of 5 μg/ml of anti-moesin Abs alone, the amount of TNF-α in the medium was 2 to 10 times more than those of control cultures and was almost comparable to that of the culture stimulated by 100 ng/ml of LPS. Next, in order to determine whether the cytokine secretion induced by anti-moesin Abs requires FcγR cross-linking of the Abs, Fab fragments were prepared from anti-moesin Abs of an AA patient with papain treatment and the effect of the Fab fragment on THP-1 cells was examined. Purified Fab fragments failed to induce TNF-α from THP-1 cells while they moderately inhibited TNF-α secretion induced by anti-moesin Abs. Finally, to determine which signaling pathway is activated by the treatment of THP-1 cells with anti-moesin Abs, the effects of specific inhibitors of kinases were examined, including extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), all of which are involved in the signal transduction required for TNF-α secretion. The inhibition of ERK 1/2 using PD98059 suppressed TNF-α secretion induced by anti-moesin Abs in a dose dependent manner. In contrast, the interruption of JNK or p38 kinase with specific inhibitors JNK I, (L) form or SB202190 significantly enhanced TNF-α secretion in response to anti-moesin Abs in association with the up-regulation of the ERK 1/2 activity. These results indicate that anti-moesin Abs may therefore be involved in the suppression of hematopoiesis in AA patients by stimulating pro-inflammatory cytokine secretion from monocytes and that the cross-linking of the Fc portion of anti-moesin Abs and the activation of the ERK1/2-dependent pathway thus play an essential role in the anti-moesin Abs-induced TNF-α secretion from monocytes.

I02 GVHD and extracorporeal photopheresis

Graft-versus-host disease (GVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal exposure of peripheral blood mononuclear cells to the photosensitizing agent 8-methoxypsoralen and ultraviolet A radiation has been shown to be effective in treatment of selected T-cell mediated diseases, including cutaneous T-cell lymphoma and rejection after organ transplantation. In a phase II study on patients with steroid-refractory acute GVHD grades II to IV given adjunct ECP as secondline therapy CR was achieved in 82% of patients with cutaneous involvement, 61% with liver involvement, and 61% with gut involvement. The probability of survival was 59% among patients who responded completely to ECP compared to 11% in patients not responding completely. Thus, intensified ECP was highly effective in acute GVHD and sustained responses are associated with over 50% long-term survival. Currently, a randomized controlled multicenter study of ECP is ongoing for the treatment of patients with newly diagnosed acute GVHD. ECP given in retrospective studies to heterogeneous patient populations is associated with high response rates in chronic extensive GVHD with skin, liver and oral involvement. In a recent multicenter, prospective, phase II study in steroidintolerant, steroid-dependent or steroid-refractory chronic GVHD patients, ECP was effective in skin manifestations and steroid-sparing could be demonstrated. ECP was well tolerated with a safety profile consistent with previous studies.

Cytokines, chemokines and soluble adhesion molecules in aqueous humor of children with uveitis

Uveitis in childhood is a visual threatening disease with a complication rate of more than 75%. Despite extensive research, the etiology of uveitis is still unclear although the general opinion is now that uveitis is a T-cell mediated disease. The purpose of this study was to investigate the profile of cytokines, chemotactic cytokines (chemokines) and soluble adhesion molecules in the aqueous humor (AqH) of children with uveitis in order to identify the factors that control the immune response in the eye. In this clinical laboratory investigation we analyzed, with a multiplex immunoassay, 16 immune mediators in the AqH of 25 children with uveitis and 6 children without uveitis. Increased levels of interleukin-2 (IL-2), IL-6, IL-10, IL-13, IL-18, interferon-γ, tumor necrosis factor-α, soluble intercellular adhesion molecule-1, RANTES, IL-8 and interferon-inducible 10-kDa protein were found in the AqH of children with uveitis compared with controls. No significant differences were found for IL-1β, IL-4, IL-12 p-70, soluble vascular cell adhesion molecule 1 and Eotaxin. Lower levels of IL-10 and IL-8 were found in quiet stage uveitis (surgical) samples compared with active uveitis (diagnostic) samples and in samples of patients treated with methotrexate (MTX) compared with samples of patients not treated with MTX. Lower levels of IL-10 were as well found in samples taken during the first 3 months after the diagnosis of uveitis than samples taken later during the disease process. No significant differences were found between patients treated with or without topical or systemic (perioperative and long term) corticosteroids. In conclusion, in children with uveitis, multiple intraocular cytokines, chemokines and soluble adhesion molecules are increased in the AqH regardless of active or inactive inflammation. Whether the IL-8 and IL-10 levels in AqH of children with uveitis are correlated with uveitis activity, early or late phase of the course of the disease and systemic treatment with MTX needs further investigation in a bigger study population.

A new device to study ex-vivo the effects of extracorporeal photochemotherapy on the immune system

Extracorporeal photochemotherapy (ECP) is a medical procedure effective in the treatment of several different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient’s blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes, which are then incubated with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused to the patient. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Although ECP has been shown to have an in-vivo immunomodulatory effect, the mechanisms through which ECP exerts its effect remain elusive. One of the reasons for this incomplete knowledge is the absence of a reliable model for ECP. In order to investigate the effect of ECP on the peripheral immune system, we developed a new device which mimics the complete ECP cycle including blood transit through the cell separator. Peripheral blood samples (50 ml) were obtained from volunteers and processed using a peristaltic pump. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and UV-A under the same conditions used for the patients’ therapy. Using this strategy we investigated 8-MOP, UV-A and their combined effect on the production of the pro-inflammatory cytokines interferon-γ (IFN-γ), interleukine-2 (IL-2) and tumor necrosis factor-α (TNF-α) in PBMC with and without polyclonal stimulation. We firstly demonstrated that our device does not affect total red and white blood cell counts. After 8-MOP and UV-A irradiation a significant decrease was observed in both activated CD4 + and CD8 + T lymphocytes producing IFN-γ, IL-2 and TNF-α. Our findings are in line with those previously obtained in humans after complete ECP treatment, thus suggesting that our newly developed device is suitable for investigating the mechanism of action of ECP ex-vivo.

Biological therapy of arthritis and systemic autoimmune diseases

The concept of biological therapy arises from the specific targeting of a factor, e.g. a cytokine, involved in the inflammatory cascade. Thus, biologicals disrupt the complex network of autoimmune-inflammatory events. Today, rheumatoid arthritis is a prototype disease in this context as most compounds have been tried in this disease. Recently, biological therapy has been introduced to the treatment of other diseases including various forms of arthritis, such as ankylosing spondylitis and psoriatic arthritis, as well as systemic autoimmune disorders, such as systemic lupus erythematosus, scleroderma, inflammatory myopathies and Sjogren's syndrome. Anti-tumor necrosis factor-alpha (TNF-alpha) agents play a central role in biological therapy as these agents have been successfully tried in most of these diseases. When seeking for specific targets for biologicals, pathogenic factors of the disease, such as Th1 or Th2 type responses, should be evaluated. Some mostly T-cell mediated diseases, such as rheumatoid arthritis, ankylosing spondylitis, psoriasis, polymyositis, polyarticular juvenile arthritis respond well to anti-TNF agents and T cell targeting, while others, such as lupus, Sjogren's syndrome, dermatomyositis may rather respond to anti-B cell biologicals. In this review, authors discuss the most recent advances in the biological therapy of arthritis and systemic autoimmune diseases including issues of efficacy and safety.

Unraveling the paradoxes of HIV-associated psoriasis: A review of T-cell subsets and cytokine profiles

HIV-associated psoriasis appears paradoxical, being a T-cell mediated disease in the face of decreasing T-cell counts. Furthermore, psoriasis is generally mediated by type-1 cytokines, whereas in HIV, type-2 cytokines tend to predominate. How can one have psoriasis in the essentially Th2 environment of HIV? The details and pertinent research regarding T cell subsets and cytokine profiles in psoriasis, HIV, and HIV-associated psoriasis were reviewed. It appears that both in the presence and absence of HIV infection, psoriasis is largely mediated by memory CD8 T cells, and that IFN-gamma secreted by these cells and others is of key importance. Studying psoriasis in a model such as HIV in which certain elements of the immune system are stripped away or altered may help us better understand the pathogenic mechanisms and potential treatment targets for psoriasis vulgaris.

Stimulation of TNF-α Production from Monocytes by Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia: A Novel Mechanism of Myelosuppression by Autoantibodies.

Although aplastic anemia (AA) is a T-cell mediated disease, recent studies have revealed the presence of antibodies (Abs) specific to proteins derived from hematopoietic progenitor cells in the serum of AA patients. It is as yet unclear whether these auto-Abs play some roles in the pathophysiology of AA. We previously demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 37% of AA patients. Some reports identified moesin-like molecules on the surface of blood cells such as T cells and macrophages. It is therefore conceivable that anti-moesin Ab in AA patients may bind these immune cells and modulate hematopoietic function of AA patients. To test these hypotheses, we first studied the expression of moesin on various types of blood cells using monoclonal Ab specific to moesin (clone 38/87). Flow cytometry detected the expression of the protein recognized by anti-moesin Ab on T cells and monocytes from healthy individuals, acute monocytic leukemia cells lines including U937 and THP-1, and an acute T-lymphoblastic leukemia cell line, Molt-4, but failed to detect the molecule on CD34+ cells from healthy individuals and myeloid leukemia cell lines as well as B-lymphoblastic leukemia cell lines. Treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS) augmented the expression level of moesin. To confirm the expression of the moesin-like protein on the surface of monocytic leukemia cell lines, Molt-4 and THP-1 were treated with sulfo-NHS-SS-biotin, and the cell surface proteins were isolated with avidin-fixed column, and were subjected to Western blotting and peptide mass fingerprinting. Western blotting with anti-moesin monoclonal Abs showed two clear bands of proteins (75 kD and 80 kD); an amino acid sequence compatible with moesin was confirmed in the protein eluted from the 80 kD band. Next, we purified anti-moesin Abs from AA patients' sera using affinity chromatography with recombinant moesin protein. Western blotting showed binding of the serum-derived Abs to a fraction of surface proteins of Molt-4, U937 and THP-1. When THP-1 cells were incubated in the presence of PMA and LPS with 5 αg/ml of control IgG or anti-moesin Abs derived from an AA patient's serum, TNF-α production from THP-1 cells stimulated by anti-moesin Abs was 1.9–2.3 times as much as that from the control culture depending on the concentration of LPS. Incubation of THP-1 cells in the presence of monoclonal anti-moesin Abs showed the similar augmentation of TNF-α production. These results indicate that anti-moesin Abs may be involved in the suppression of hematopoiesis of AA patients by stimulating TNF-α production from monocytes.

Recommendations for incorporating biologicals into management of moderate to severe plaque psoriasis: individualized patient approaches

Psoriasis is a T-cell mediated skin disease that affects approximately 2% of the population worldwide. Despite the prevalence of the disease and long-standing efforts to develop strategies to treat it, there is a need for safe and effective therapies to treat psoriasis, particularly the more severe forms. Biological agents such as alefacept, efalizumab, etanercept, and infliximab have been recognized as a class of treatment distinct from other forms of therapy in the treatment algorithm of psoriasis. Recent national and international consensus meetings have developed statements that position biological agents as an important addition to the treatment armamentarium for moderate to severe psoriasis, along with phototherapy and traditional systemic agents. There has been consensus that treatment should be individualized to each patient's needs and circumstances. Biological agents offer the hope of safe, effective, long-term management of moderate to severe psoriasis. As new agents receive approval from Health Canada, the available range of therapeutic options for treating this chronic disease will broaden. A Canadian Psoriasis Expert Panel recently convened in February 2005 to analyze, based on a series of clinical case scenarios, the indications, contraindications, and considerations for and against each of the four biological agents, derived from product labelling, where available, and from the efficacy and safety data from phase 3 and earlier clinical trials, as well as post-marketing reports. The Panel has formulated a set of recommendations for incorporating these biological agents into the current treatment paradigm of moderate to severe plaque psoriasis and has identified the preferred biological agents for each patient based on individual needs and circumstances.

Increased levels of bioactive IL-16 correlate with disease activity during relapsing experimental autoimmune encephalomyelitis (EAE)

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T-cell mediated disease, which resembles immunopathology of multiple sclerosis (MS). Interleukin (IL)-16 is a CD4+ cell-specific chemoattractant cytokine. In CD4+ T cells, production of bioactive IL-16 from constitutive pro-IL-16 requires cleavage by active caspase-3. We reported reversal of established relapsing disease by IL-16 neutralization. To better understand role(s) of IL-16 in regulation of relapsing EAE, we comparatively analyzed levels of IL-16, active caspase-3 and CD4 in mice with severe relapsing-remitting [(B6 × SJL) F1], and low-relapsing (B6), disease. Elevated levels of IL-16 along with an increase in active-caspase-3 and CD4 levels correlated with stages of clinically active disease in both strains. CNS levels of bioactive IL-16 were notably higher in F1 compared to B6 mice at all stages, being most prominent during relapse. Similar patterns of regulation for IL-16 and active caspase-3 were observed in peripheral lymphoid organs, and in T cells isolated from lymph nodes following T-cell activation in vitro. IL-16 was co-immunoprecipitated with CD4 from CNS of relapsing mice. Our data suggest that caspase-3 mediated production of IL-16 by infiltrating CD4+ T cells, contributes to ongoing neuroinflammation by chemoattraction of additional waves of CD4+ T cells.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.