Small-molecule costimulatory blockade: organic dye inhibitors of the CD40–CD154 interaction

Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40–CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC₅₀ values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-α or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family. They specifically inhibited CD154-induced cell responses in human B cells as well as in THP-1 myeloid cells, which can serve as surrogate dendritic cells, at concentrations well below their cytotoxic concentrations determined in the same cells. Flow cytometry experiments confirmed their ability to inhibit the CD154-induced, but not the Staphylococcus aureus Cowan I- or phorbol 12-myristate 13-acetate-induced increase in the surface expression of CD54, CD40, and major histocompatibility complex class II. Accordingly, these compounds can be useful not only for experimental investigations involving the inhibition of the CD40–CD154 costimulatory interaction but can also provide important structure–activity relationship information and can serve as the starting point of a targeted drug discovery program.     

Phoenix from the flames: Rediscovering the role of the CD40–CD40L pathway in systemic lupus erythematosus and lupus nephritis

Lupus nephritis (LN) is a significant complication of systemic lupus erythematosus (SLE), increasing its morbidity and mortality. Although the current standard of care helps suppress disease activity, it is associated with toxicity and ultimately does not cure SLE. At present, there are no therapies specifically indicated for the treatment of LN and there is an unmet need in this disease where treatment remains a challenge.The CD40–CD40L pathway is central to SLE pathogenesis and the generation of autoantibodies and their deposition in the kidneys, resulting in renal injury in patients with LN. CD40 is expressed on immune cells (including B cells, monocytes and dendritic cells) and also non-haematopoietic cells. Interactions between CD40L on T cells and CD40 on B cells in the renal interstitium are critical for the local expansion of naive B cells and autoantibody-producing B cells in LN. CD40L-mediated activation of myeloid cells and resident kidney cells, including endothelial cells, proximal tubular epithelial cells, podocytes and mesangial cells, further amplifies the inflammatory milieu in the interstitium and the glomeruli. Several studies have highlighted the upregulated expression of CD40 in LN kidney biopsies, and preclinical data have demonstrated the importance of the CD40–CD40L pathway in murine SLE and LN. Blocking this pathway is expected to ameliorate inflammation driven by infiltrating immune cells and resident kidney cells.Initial experimental therapeutic interventions targeting the CD40–CD40L pathway, based on CD40L antibodies, were associated with an increased incidence of thrombosis. However, this safety issue has not been observed with second-generation CD40/CD40L antibodies that have been engineered to prevent platelet activation.With these advancements, together with recent preclinical and clinical findings, it is anticipated that selective blockade of the CD40–CD40L pathway may address the unmet treatment needs in SLE, LN and other autoimmune diseases.     

Is IgA nephropathy induced by abnormalities of CD4CD25Treg cells in the tonsils?

IgA nephropathy (IgAN) is characterized by the accumulation of IgA deposits, predominantly in the glomerular mesangium, and represents the most common form of glomerulonephritis. Much evidence showed that tonsils were closely related to IgAN. Urinary findings were deteriorated after tonsil stimulation in patients with IgAN. Tonsillectomy can improve the urinary findings, keep stable renal function, improve mesangial proliferation, decrease IgA deposits and have a favorable effect on long-term renal survival in some IgAN patients. Recent studies indicate that CD4(+)CD25(+) regulatory T cells (Treg) are of critical importance to the maintenance of tolerance by inhibiting the activation and proliferation of autoreactive T cells. Depletion of the minor CD4(+)CD25(+)Treg cells results in the development of organ-specific autoimmunity. Autoimmune diseases can be prevented by reconstitution of the animals with CD4(+)CD25(+)Treg cells. CD4(+)CD25(+)Treg cells are regulators in almost all of the animal models of human organ-specific diseases, transplant rejection and allergic diseases. Some patients with recurrent chronic tonsillitis have not suffered from renal disease, implying that it is possible to find a balance between immunity and tolerance. Some patients, however, suffered from IgAN along with recurrent chronic tonsillitis. It could be hypothesized that a numerical and/or functional deficit of CD4(+)CD25(+)Treg cells in the tonsils of IgAN patients might trigger the development of the diseases. If the hypothesis is correct, altering CD4(+)CD25(+)Treg cell numbers and/or enhancing CD4(+)CD25(+)Treg responses might be useful in the prevention and treatment of IgAN.     

Thiol-oxidation reduces the release of amylase induced by β-adrenergic receptor activation in rat parotid acinar cells

In parotid acinar cells, the activation of β-adrenergic receptors induces the accumulation of intracellular cAMP, and consequently provokes the exocytotic release of amylase, a digestive enzyme. The cellular redox status plays a pivotal role in regulating various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damage. In this study, we examined the effects of diamide, a thiol-oxidizing reagent, on amylase release by rat parotid acinar cells. In cells treated with diamide, the formation of cAMP and the release of amylase induced by the β-agonist isoproterenol (IPR) were partially reduced. The inhibitory effect of diamide on the IPR-induced release of amylase could be abrogated by reduced glutathione or dithiothreitol. Diamide had no effect on the amylase release induced by forskolin, an adenylate cyclase activator, or by mastoparan, a heterotrimeric GTPbinding protein activator. In cells treated with diamide, the binding affinity for [(3)H]DHA, but not the number of binding sites, was reduced. These results suggest that β-adrenergic receptor function is reduced by thiol-oxidation, which inhibits amylase secretion by parotid acinar cells.     

Histamine via the histamine H?-receptor reduces α-CD3-induced interferon-γ synthesis in murine CD4+ T cells in an indirect manner

Histamine is involved in the execution of an immune reaction. Receptors for histamine, of which four different subtypes are known so far, are found on dendritic cells and on T cells. Via these receptors, histamine either indirectly or directly affects the activation of T cells. Data in the literature regarding the involved receptor subtypes and the mode of action of histamine on T cells are somewhat contradictory and depend on the type of cells analyzed, polarized T cells, or freshly prepared T cells within the context of the whole splenocyte population. Therefore, we analyzed the effect of histamine on murine T cells within splenocytes in a detailed manner. We stimulated freshly prepared splenocytes in the presence or absence of histamine with α-CD3 in vitro and analyzed the induced cytokine production. We show that histamine reduced the α-CD3-induced interferon-γ (IFN-γ) production of CD4? cells via the histamine H?-receptor. Moreover, the effect of histamine on the α-CD3-induced IFN-γ production could be transferred within conditioned splenocyte supernatants induced by histamine (in the absence of α-CD3). Thus, the histamine effect is mediated by a soluble factor, which, however, is neither of the classical anti-inflammatory mediators, interleukin-10, or transforming growth factor-β.     

Murine AIDS requires CD154/CD40L expression by the CD4 T cells that mediate retrovirus-induced disease: Is CD4 T cell receptor ligation needed?

LP-BM5, a retroviral isolate, induces a disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS). Many of the features of the LP-BM5-initiated disease are shared with HIV/AIDS. Our lab has shown that the interaction of B and CD4 T cells that is central to MAIDS pathogenesis requires ligation of CD40 on B cells by CD154 on CD4 T cells. Despite this strict requirement for CD154 expression, whether CD4 T cell receptor (TCR) occupancy is essential for the induction of MAIDS is unknown. To block TCR engagement, Tg mouse strains with monoclonal TCR of irrelevant peptide/MHC specificities, all on MAIDS-susceptible genetic backgrounds, were tested: the study of a panel of TCR Tg CD4 T cells controlled for the possibility of serendipitous crossreactive recognition of virus-associated or induced-self peptide, or superantigen, MHC complexes by a given TCR. The results argue that TCR engagement is not necessary for the induction of MAIDS.     

Intra-articular administration of gelatin hydrogels incorporating rapamycin–micelles reduces the development of experimental osteoarthritis in a murine model

Autophagy is a cellular homeostasis mechanism that may have a protective role against osteoarthritis (OA). The present study investigated the therapeutic effect of local administration of rapamycin, a potent activator of autophagy, against OA. To achieve controlled intra-articular administration of rapamycin, gelatin hydrogels incorporating rapamycin-micelles were created and the release profile was evaluated in vitro. The therapeutic effects of gelatin hydrogels incorporating rapamycin-micelles were then tested in a murine OA model. Mice were divided into four groups: Group 1, gelatin hydrogels alone; Group 2, single injection of 1 μg rapamycin; and Groups 3 and 4, gelatin hydrogels incorporating 100 ng or 1 μg rapamycin-micelles, respectively. Immunohistochemical analysis revealed that autophagic marker-positive chondrocytes were increased in the rapamycin-treated mice at 10 weeks after surgery. The histologic score was better in Groups 3 and 4 than in Groups 1 and 2, and Group 2 had a better score than Group 1. Delayed OA progression was maintained even at 16 weeks after surgery in Group 4. Microarray and real-time polymerase chain reaction analysis indicated that OA mediator genes were downregulated in the rapamycin-treated mice. Our novel system for intra-articular administration of rapamycin could be a novel therapeutic approach for treating patients with OA.     

Anti-viral and anti-tumor effects induced by an attenuated Marek’s disease virus in CD4- or CD8-deficient chickens

Summary.  By vaccination with an attenuated Marek’s disease virus (MDV), strain CVI988, chickens are protected from the development of T cell lymphoma caused by an oncogenic MDV. To clarify the role of T lymphocyte subsets in the protection mechanisms of this vaccine, vaccinated chickens were depleted of T cell subsets by neonatal thymectomy and injections of monoclonal antibodies specific to chicken CD4 and CD8 molecules, and then challenged with an oncogenic MDV, strain Md5. The MDV titers rescued from CD8⁺ T cells, which are the main targets for latent infection and subsequent transformation by MDV, was much higher in the CD8-deficient vaccinated chickens than in untreated vaccinated chickens at the early stage of the latent phase. However, the neonatal vaccination prevented lymphoma formation by strain Md5 even in either CD4⁺ or CD8⁺ T cell-depleted chickens. These results suggest that specific CD8⁺ T cell responses induced by the MD vaccine play a crucial role in the prevention of MDV infection during the latent phase, but may not be essential for the prevention of lymphoma formation by an oncogenic MDV. Received Februray 15, 1999 Accepted May 7, 1999     

The Role of CD40–CD154 Interactions in Autoimmunity and the Benefit of Disrupting this Pathway

Many tissue injuries and immune mediated pathologies such as graft allo-rejections were found to involve CD40–CD40 ligand (CD40L, CD154) signaling pathway. The disruption of this pathway in many animal models led to the improvement of graft survival in these models. CD40–CD154 interactions were also shown to play a significant role in the maintenance of autoimmunity, and the production of auto-antibodies in systemic lupus erythematosus (SLE). High-level expression of CD154 has been detected on T cells from patients with active SLE, rheumatoid arthritis (RA) and other autoimmune diseases, indicating that such cells could account for the high-level expression of immune accessory molecules on B cells of patients with active disease. An increased serum level of soluble CD154 was also reported in SLE, RA, and Sjogren's disease in correlation with the relevant auto-antibodies and with the clinical disease activity.

Anti-CD154 antibody therapy prevents auto-antibody production and renal immune complex deposition in lupus nephritis, indicating that disruption of this pathway could be a beneficial treatment in SLE. However, the etiology of the higher than expected number of thromboembolic events in anti-CD154 treated SLE patients should be investigated and preventive measures should be considered.     

The Involvement of the CD40-CD40L Pathway in Activated Platelet-Induced Changes in HUVEC COX-2 and PPARα Expression

We aim to determine the extent of the CD40-CD40L pathway involvement in activated platelet-induced changes in human umbilical endothelial cells (HUVECs). Activated platelets were co-incubated with HUVECs in the presence or absence of CD40LmAb. HUVECs were also directly stimulated with rhCD40L. HUVEC endothelial cyclooxygenase 2 (COX-2) and peroxisome proliferator-activated receptor alpha (PPARα) expression was then assessed. To estimate COX-2 activity, PGE2 concentration was determined. PPARα activity was assessed using a nuclear factor activity kit. Co-incubation with activated platelets increased HUVEC COX-2 and PPARα mRNA expression (P < 0.01). The addition of CD40L mAb significantly attenuated these increases in mRNA and protein (both P < 0.01). Direct stimulation by rhCD40L increased HUVEC COX-2 mRNA and protein (P < 0.05) but did not significantly change the expression of PPARα mRNA and protein. CD40LmAb significantly decreased (P < 0.05) and rhCD40L significantly (P < 0.01) increased COX-2 enzymatic activity, but had almost no effects on PPARα binding activity. Activated platelets may increase HUVEC COX-2 expression and activity partly through the CD40-CD40L pathway.     

Soluble MICA-NKG2D interaction upregulates IFN-γ production by activated CD3CD56 NK cells: Potential impact on chronic graft versus host disease

A soluble isoform of MHC class I chain-related molecule A (soluble MICA), generated by proteolytic shedding from the membrane-bound MICA of various tumor cells, has been shown to downregulate both the expression of natural killer group 2-member D receptor and the cytotoxic function of effectors cells and was postulated as a mechanism for tumor immune evasion. Its effect on the expression of cytokines by the effector cells remained unexplored. Here we demonstrate that the sMICA molecules upregulate interferon gamma expression by interleukin-12/interleukin-18-activated CD3⁻CD56⁺ natural killer cells, witnessing the pro-inflammatory effect of soluble MICA. Overall, these data are in line with our previous observations that the raised serum levels of soluble MICA, following allogeneic hematopoietic stem cell transplantation, confer susceptibility to and the presence of pre-transplantation anti-MICA antibodies in the patient’s serum confer protection against chronic graft versus host disease.     

Copy number variation in chemokine superfamily: the complex scene of CCL3L�CCCL4L genes in health and disease

Genome copy number changes (copy number variations: CNVs) include inherited, de novo and somatically acquired deviations from a diploid state within a particular chromosomal segment. CNVs are frequent in higher eukaryotes and associated with a substantial portion of inherited and acquired risk for various human diseases. CNVs are distributed widely in the genomes of apparently healthy individuals and thus constitute significant amounts of population‐based genomic variation. Human CNV loci are enriched for immune genes and one of the most striking examples of CNV in humans involves a genomic region containing the chemokine genes CCL3L and CCL4L. The CCL3L–CCL4L copy number variable region (CNVR) shows extensive architectural complexity, with smaller CNVs within the larger ones and with interindividual variation in breakpoints. Furthermore, the individual genes embedded in this CNVR account for an additional level of genetic and mRNA complexity: CCL4L1 and CCL4L2 have identical exonic sequences but produce a different pattern of mRNAs. CCL3L2 was considered previously as a CCL3L1 pseudogene, but is actually transcribed. Since 2005, CCL3L‐CCL4L CNV has been associated extensively with various human immunodeficiency virus‐related outcomes, but some recent studies called these associations into question. This controversy may be due in part to the differences in alternative methods for quantifying gene copy number and differentiating the individual genes. This review summarizes and discusses the current knowledge about CCL3L–CCL4L CNV and points out that elucidating their complete phenotypic impact requires dissecting the combinatorial genomic complexity posed by various proportions of distinct CCL3L and CCL4L genes among individuals.     

Effect of nigrostriatal damage induced by 6-hydroxydopamine on the expression of glial cell line–derived neurotrophic factor in the striatum of the rat

Several types of brain injuries have been associated with alterations in the striatal expression of neurotrophic factors, including glial cell line–derived neurotrophic factor (GDNF). However, contradictory results on the striatal expression of GDNF have been reported in different animal models of Parkinson's disease. For this reason, we examined the effect of nigrostriatal damage on the mRNA and protein expression levels of GDNF in the striatum as a function of time following a striatal or medial forebrain bundle 6-hydroxydopamine lesion. At different time points after the administration of 6-hydroxydopamine, striatal expression levels of GDNF were analyzed with semi-quantitative Western blotting. No significant changes in GDNF expression levels were observed within the 35-day observation period, either between the denervated and the intact striatum of medial forebrain bundle and striatally lesioned rats or between the striata of lesioned animals and those of control animals. In order to reinforce these results, striata of lesioned rats, sacrificed 18 days after lesioning, were analyzed with enzyme-linked immunosorbent assay and real-time polymerase chain reaction. At this time point, both techniques confirmed the results of the Western blot analysis, detecting no changes in striatal expression of GDNF, either at the protein level, or at the mRNA level. These data show that nigrostriatal damage induced by 6-hydroxydopamine has no effect on the striatal expression of GDNF.     

Effects of OX40–OX40L Interaction on the Nuclear Factor of Activated T Cells c1 in ApoE-Deficient Mice

We previously reported the emerging role of OX40–OX40L interaction in inflammation and atherosclerosis. However, the mechanism by which OX40–OX40L interaction contributes to pathogenesis is poorly understood. This study investigated the effects of OX40–OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE^?/? mice. Atherosclerotic plaque was induced via rapid perivascular carotid collar placement in ApoE^?/? mice. The expression levels of OX40, OX40L, and NFATc1 in the lymphocytes were measured via real-time polymerase chain reaction and flow cytometry. The presence of NFATc1 in the atherosclerotic plaque was detected via immunohistochemistry, and the level of IL-4 was measured via enzyme-linked immunosorbent assay. The expression level of NFATc1 significantly increased in atherosclerotic lesion and in the leukocytes from the ApoE^?/? mice. After stimulating OX40–OX40L interaction, the mRNA and protein expression levels of NFATc1 in the lymphocytes significantly increased. Meanwhile, anti-OX40LmAb significantly suppressed the expression of NFATc1 in the leukocytes and substantially elevated the level of IL-4. NFATc1 inhibitor markedly suppressed IL-4 production. This study suggests that OX40–OX40L interaction regulates the expression of NFATc1, which may play a critical role in atherosclerotic plaque formation, and may therefore have implications with pathophysiology of atherosclerosis.     

Relation between vibrotactile perception thresholds and reductions in finger blood flow induced by vibration of the hand at frequencies in the range 8–250 Hz

Purpose

This study investigated how the vasoconstriction induced by vibration depends on the frequency of vibration when the vibration magnitude is defined by individual thresholds for perceiving vibration [i.e. sensation levels (SL)].

Methods

Fourteen healthy subjects attended the laboratory on seven occasions: for six vibration frequencies (8, 16, 31.5, 63, 125, or 250 Hz) and a static control condition. Finger blood flow (FBF) was measured in the middle fingers of both hands at 30-second intervals during five successive periods: (i) no force or vibration, (ii) 2-N force, no vibration, (iii) 2-N force, vibration, (iv) 2-N force, no vibration, (v) no force or vibration. During period (iii), vibration was applied to the right thenar eminence via a 6-mm diameter probe during ten successive 3-min periods as the vibration magnitude increased in ten steps (?10 to +40 dB SL).

Results

With vibration at 63, 125, and 250 Hz, there was vasoconstriction on both hands when the vibration magnitude reached 10 dB SL. With vibration at 8, 16, and 31.5 Hz, there was no significant vasoconstriction until the vibration reached 25 dB SL. At all frequencies, there was greater vasoconstriction with greater magnitudes of vibration.

Conclusions

It is concluded that at the higher frequencies (63, 125, and 250 Hz), the Pacinian channel mediates vibrotactile sensations near threshold and vasoconstriction occurs when vibration is perceptible. At lower frequencies (8, 16, and 31.5 Hz), the Pacinian channel does not mediate sensations near threshold and vasoconstriction commences at greater magnitudes when the Pacinian channel is activated.     

Involvement of inositol-1,4,5-trisphosphate receptors in the bidirectional synaptic plasticity induced in hippocampal CA1 neurons by 1–10 Hz low-frequency stimulation

In the present study, both potentiation and depression of the synaptic response were induced in hippocampal CA1 neurons by systematically varying the frequency of low frequency afferent stimulation (LFS) between 0.5 and 25 Hz and the pulse number between 40 and 1000. The input–response relationship for CA1 synapses showed that LFS at a higher frequency or with a smaller pulse number increased the magnitude of potentiation of the synaptic response by increasing the contribution of N-methyl-d-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs) to induction of potentiation. One possible mechanism for this bidirectional plasticity was that specific patterns of LFS differentially activate a uniform receptor population in producing depression or potentiation of synaptic responses. However, a pharmacological study indicated that, despite their opposite effects, both the synaptic depression induced by LFS at 1 Hz and the synaptic potentiation induced by LFS at 10 Hz were triggered by co-activation of NMDARs and mGluRs at CA1 synapses. We suggest that activation of protein kinase C or inositol-1,4,5-trisphosphate receptors, both coupled to group 1 mGluRs, is involved in the bidirectional synaptic plasticity induced in hippocampal CA1 neurons by 1–10 Hz LFS.     

Infectious bursal disease virus activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by interaction of VP5 protein with the p85α subunit of PI3K

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is commonly activated upon virus infection and has been implicated in the regulation of diverse cellular functions such as proliferation and apoptosis. The present study demonstrated for the first time that infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, can induce Akt phosphorylation in cultured cells, by a mechanism that is dependent on PI3K. Inhibition of PI3K activation greatly enhanced virus-induced cytopathic effect and apoptotic cell death as evidenced by cleavage of poly-ADP ribose polymerase and activation of caspase-3. Investigations into the mechanism of PI3K/Akt activation revealed that IBDV activates PI3K/Akt signaling through binding of the non-structural protein VP5 to regulatory subunit p85α of PI3K resulting in the suppression of premature apoptosis and improved virus growth after infection. The results presented here provide a basis for understanding molecular mechanism of IBDV infection.