[Early reduction of serum RANTES can predict HBsAg clearance in patients with chronic hepatitis B treated with nucleos(t)ide analogues combined with peginterferon alpha].

Objective: To observe the dynamic changes of serum RANTES during the treatment with nucleos(t)ide analogues combined with pegylated interferon alpha (peginterferon-α), and further analyze the predictive effect of RANTES on HBsAg clearance in patients with chronic hepatitis B. Methods: 98 cases of chronic hepatitis B with quantitative HBsAg < 3 000 IU/ml and HBV DNA < 20 IU/ml after≥1 year NAs treatment were enrolled. Among them, 26 cases continued to receive NAs monotherapy, 72 cases received NAs combined with pegylated interferon alpha therapy. The changes in RANTES during treatment were observed. The receiver operating characteristic curve was used to analyze the early changes of RANTES to predict the HBsAg clearance during 48 weeks. Results: During 48 weeks, 15 cases (20.83%) had achieved HBsAg clearance in combination group, while no patient had achieved HBsAg clearance in NAs group. The overall serum RANTES level had decreased from baseline in NAs and combination group. At week 48, in the combination group, the serum RANTES level was decreased more significantly in patients with HBsAg clearance than patients without. Further analysis showed that, in combination group, HBsAg clearance rate of patients with serum RANTES decreased at week 12 and 24 was higher than patients with elevated (29.17% vs. 4.17%, P = 0.014; 28.00% vs. 4.55%, P = 0.052), and quantitative HBsAg reduction was larger significantly [(1.49 ± 1.26) log(10)IU/ml vs. (0.73 ± 0.81) log(10)IU/ml, P = 0.017; (1.54 ± 1.27) log(10)IU/ml vs. (0.57 ± 0.56) log(10)IU/ml, P = 0.004]. Receiver operating characteristic curve analysis showed that the baseline quantitative HBsAg and the reduction in quantitative HBsAg and serum RANTES during the early period were predictors of HBsAg clearance after 48-week combination therapy. Furthermore, the combination of baseline quantitative HBsAg and 12 - or 24-week reduction of serum RANTES were better predictors of HBsAg clearance than that of baseline quantitative HBsAg combined with HBsAg decrease at week 12 or 24. The area under the receiver operating characteristic curve of the former was 0.925 and 0.939, while that of the latter was 0.909 and 0.929, respectively. Conclusion: Early reduction of serum RANTES at week 12 and 24 can predict HBsAg loss in CHB patients receiving addition of peginterferon-α to ongoing NAs Therapy, so serum RANTES could be one of the key immunological markers for predicting HBsAg clearance.

Multifaceted mechanisms of HIV inhibition and resistance to CCR5 inhibitors PSC-RANTES and Maraviroc.

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated. In single-cycle assays, these HIV-1 clones displayed equal sensitivity to PSC-RANTES inhibition, suggesting effective receptor downregulation. Prolonged PSC-RANTES exposure resulted in desensitization of the receptor to internalization such that increasing virus concentration (substrate) could saturate the receptors and overcome PSC-RANTES inhibition. In contrast, resistance to MVC was observed with the MVC-resistant HIV-1 (R3 versus S2) in both multiple- and single-cycle assays and with altered virus concentrations, which is indicative of allosteric inhibition. MVC could also mediate inhibition and possibly resistance through competitive mechanisms.

Deep brain stimulation improves central nervous system inflammation in Parkinson's disease: Evidence and perspectives.

BACKGROUND: In Parkinson's disease (PD), inflammation may lead to the degeneration of dopaminergic (DAergic) neurons. Previous studies showed that inflammatory mediators mainly contributed to this phenomenon. On the other hand, invasive neuromodulation methods such as deep brain stimulation (DBS) have better therapeutic effects for PD. One possibility is that DBS improves PD by influencing inflammation. Therefore, we further explored the mechanisms underlying inflammatory mediators and DBS in the pathogenesis of PD. METHODS: We measured serum levels of two inflammatory markers, namely RANTES (regulated on activation, normal T cell expressed and secreted) and tumor necrosis factor-alpha (TNF-α), using Luminex assays in 109 preoperative DBS PD patients, 49 postoperative DBS PD patients, and 113 age- and sex-matched controls. The plasma protein data of the different groups were then statistically analyzed. RESULTS: RANTES (p < 0.001) and TNF-α (p = 0.005) levels differed significantly between the three groups. A strong and significant correlation between RANTES levels and Hoehn-Yahr (H-Y) stage was observed in preoperative PD patients (rs  = 0.567, p < 0.001). Significant correlations between RANTES levels and Unified Parkinson's Disease Rating Scale III (UPDRS III) score (rs1  = 0.644, p = 0.033 and rs2  = 0.620, p = 0.042) were observed in matched patients. No correlation was observed for TNF-α levels. CONCLUSION: The results of this study indicate that PD patients have a persistent inflammatory profile, possibly via recruitment of activated monocytes, macrophages, and T lymphocytes to the central nervous system (CNS). DBS was shown to have a significant therapeutic effect on PD, which may arise by improving the inflammatory environment of the central nervous system.

The therapeutic function of the chemokine RANTES on the H22 hepatoma ascites model.

This study aimed at analyzing the therapeutic function of the chemokine RANTES on the H22 hepatoma ascites model and preliminarily explore the mechanism of RANTES in malignant ascites to provide an important reference for applying chemokines in anti-tumor therapy. The murine H22 hepatoma ascites model was used. Three treatment groups were analyzed: a RANTES treatment group, an IL-2 control group, and an NS control group. Two regimens of early treatment and late treatment were designed, and the therapeutic effect of RANTES on malignant ascites was studied by measuring changes in mouse body weight and abdominal circumference and observing the survival time. The expression of TNF-α, IFN-γ, TGF-ß1, and MCP-1 in mouse ascites was detected by ELISA, and the chemotactic function of RANTES on B lymphocytes and T lymphocytes was analyzed by flow cytometry. In the early and late treatment regimens, RANTES could effectively inhibit the increase in mouse body weight and abdominal circumference in the murine H22 hepatoma ascites model. The secretion of TNF-α and IFN-γ, which had anti-tumor effects, was higher in the RANTES treatment group than in the control groups (P < 0.05), whereas the secretion of TGF-ß1 and MCP-1, which promoted tumor growth, invasion, and metastasis, was lower than in the control groups (P < 0.05). RANTES had chemotactic effects on CD4(+) and CD8(+) T lymphocytes; therefore, the percentage of CD3, CD4, and CD8 in the mouse ascites in the RANTES treatment group was significantly higher than in the NS control and IL-2 treatment groups, and the CD4/CD8 ratio was also significantly higher. RANTES can effectively inhibit the increase in body weight and abdominal circumference and significantly extend survival time in mice in the H22 hepatoma ascites model.

Chemokine level predicts the therapeutic effect of anti-PD-1 antibody (nivolumab) therapy for malignant melanoma.

Anti-programmed cell death protein 1 (PD-1) antibody drugs, nivolumab and pembrolizumab, are regarded as first-line therapies for advanced malignant melanoma. Anti-PD-1 therapy suppresses tumor immunity, and the therapeutic effect is frequently correlated with the number of tumor-infiltrating lymphocytes (TIL) and tumor mutation burden (TMB). However, sampling tumor tissues from the metastatic sites to examine the number of TILs and TMB level is often challenging. Herein, we focused on chemokines in blood to determine whether they can predict the therapeutic effect of anti-PD-1 (nivolumab) therapy. First, we measured 44 types of chemokines and cytokines in the blood of 8 advanced malignant melanomas before anti-PD-1 (nivolumab) treatment and examined the relationship between the levels of these proteins and therapeutic effect of the drug treatment, which suggested that C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine ligand 12 (CXCL12) were candidates for biomarkers to predict the therapeutic effect of anti-PD-1 therapy. Next, we measured the blood levels of CCL5 and CXCL12 in 22 patients with advanced malignant melanomas before the administration of anti-PD-1 antibody. We evaluated tumor infiltration of CD8-positive T cells by immunostaining in nine patients in whom the metastatic site could be sampled at the beginning of the treatment. The patients with lower than average levels of CCL5 and CXCL12 had a large number of TILs (P = 0.04) and good disease-specific survival rate (P = 0.04). Therefore, CCL5 and CXCL12 could likely be used as biomarkers to predict the therapeutic effect of anti-PD-1 (nivolumab) therapy.

Terminalia chebula Retz. extract ameliorates the symptoms of atopic dermatitis by regulating anti-inflammatory factors in vivo and suppressing STAT1/3 and NF-ĸB signaling in vitro.

BACKGROUND: Terminalia chebula (TC) is a traditional medicinal plant used for treating various diseases in humans. However, pharmacological mechanisms underlying the effects of TC in atopic treatment remain unelucidated. HYPOTHESIS/PURPOSE: We investigated the therapeutic effects of TC extract in a mouse model of atopic dermatitis (AD) in vivo and the anti-inflammatory mechanism in vitro. STUDY DESIGN/METHODS: For the in vivo study, AD was induced by Dermatophagoides farinae extract (Dfe) in NC/Nga mice. After 14 days of oral administration, the effects of TC concentrations of 30, 100, and 300 mg/kg were analyzed by assessing morphological changes visually; measuring serum levels of inflammatory chemokines/cytokines, IgE, histamine, MDC, TARC, RANTES, and TSLP using ELISA kits; and counting infiltrated mast cells. For in vitro analyses, we used IFNγ/TNF-α-stimulated human keratinocyte cell lines to study the mechanism of action. The production of chemokines/cytokines in the IFNγ/TNF-α-stimulated HaCaT cells was measured using ELISA and a bead array kit. The signaling pathways were analyzed by western blotting and the expression of the transcriptional factors using RT-PCR and luciferase assay. RESULTS: Administration of TC significantly alleviated AD-like symptoms in vivo and decreased the ear thickness, dermatitis score, keratinization, and mast cell infiltration. It also resulted in decreased serum levels of IgE, histamine, and inflammation-related mediators MDC, TARC, RANTES, and TSLP compared with those in the Dfe treatment group. Moreover, TC downregulated the expression of the inflammatory chemokines RANTES and MDC in IFNγ/TNF-α-stimulated HaCaT cells. TC inhibited phosphorylated STAT1/3 and NK-κB subunits and nuclear translocation of NF-κB. It also suppressed the transcription of IFNγ, IL-6, IL-8 and MCP-1 in the IFNγ/TNF-α-stimulated HaCaT cells. TC and its constituents, chebulic acid, gallic acid, corlagin, chebulanin, chbulagic acid, ellagic acid, and chebulinic acid, strongly inhibited the nuclear translocation of NF-κB, STAT1, and STAT3 and decreased the expression of inflammatory cytokines at the mRNA level. CONCLUSIONS: Overall, TC extract alleviated AD-like symptoms by regulating anti-inflammatory factors in vivo and suppressing STAT1/3 and NF-κB signaling in vitro. In addition, our results show the in vivo effect of partial improvements in AD, as well as the in vitro effect on inflammatory factors by the constituents of TC. This finding provides that TC extract and its components could be potential therapeutic drugs for AD.

Engineering of Lactobacillus jensenii to secrete RANTES and a CCR5 antagonist analogue as live HIV-1 blockers.

The development of effective microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of AIDS epidemics worldwide. A promising strategy is the use of bacteria belonging to the vaginal microbiota as live microbicides for the topical production of HIV-1 inhibitors. We have engineered a human vaginal isolate of Lactobacillus jensenii to secrete the anti-HIV-1 chemokine RANTES, as well as C1C5 RANTES, a mutated analogue that acts as a CCR5 antagonist and therefore is devoid of proinflammatory activity. Full-length wild-type RANTES and C1C5 RANTES secreted by L. jensenii were purified to homogeneity and shown to adopt a correctly folded conformation. Both RANTES variants were shown to inhibit HIV-1 infection in CD4(+) T cells and macrophages, displaying strong activity against HIV-1 isolates of different genetic subtypes. This work provides proof of principle for the use of L. jensenii-produced C1C5 RANTES to block HIV-1 infection of CD4(+) T cells and macrophages, setting the basis for the development of a live anti-HIV-1 microbicide targeting CCR5 in an antagonistic manner.

CCR1 Activation Promotes Neuroinflammation Through CCR1/TPR1/ERK1/2 Signaling Pathway After Intracerebral Hemorrhage in Mice.

The activation of C-C chemokine receptor type 1 (CCR1) has been shown to be pro-inflammatory in several animal models of neurological diseases. The objective of this study was to investigate the activation of CCR1 on neuroinflammation in a mouse model of intracerebral hemorrhage (ICH) and the mechanism of CCR1/tetratricopeptide repeat 1 (TPR1)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in CCR1-mediated neuroinflammation. Adult male CD1 mice (n = 210) were used in the study. The selective CCR1 antagonist Met-RANTES was administered intranasally at 1 h after autologous blood injection. To elucidate potential mechanism, a specific ERK1/2 activator (ceramide C6) was administered prior to Met-RANTES treatment; CCR1 activator (recombinant CCL5, rCCL5) and TPR1 CRISPR were administered in naïve mouse. Neurobehavioral assessments, brain water content, immunofluorescence staining, and western blot were performed. The endogenous expressions of CCR1, CCL5, TPR1, and p-ERK1/2 were increased in the brain after ICH. CCR1 were expressed on microglia, neurons, and astrocytes. The inhibition of CCR1 with Met-RANTES improved neurologic function, decreased brain edema, and suppressed microglia/macrophage activations and neutrophil infiltration after ICH. Met-RANTES treatment decreased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1ß, which was reversed by ceramide C6. The brain CCR1 activation by rCCL5 injection in naïve mouse resulted in neurological deficits and increased expressions of CCR1, TPR1, p-ERK, TNF-α, and IL-1ß. These detrimental effects of rCCL5 were reversed by TPR1 knockdown using TPR1 CRISPR. Our study demonstrated that CCR1 activation promoted neuroinflammation through CCR1/TPR1/ERK1/2 signaling pathway after ICH in mice. CCR1 inhibition with Met-RANTES attenuated neuroinflammation, thereby reducing brain edema and improving neurobehavioral functions. Targeting CCR1 activation may provide a promising therapeutic approach in the management of ICH patients.

CCL3/Macrophage Inflammatory Protein-1α Is Dually Involved in Parasite Persistence and Induction of a TNF- and IFNγ-Enriched Inflammatory Milieu in Trypanosoma cruzi-Induced Chronic Cardiomyopathy.

CCL3, a member of the CC-chemokine family, has been associated with macrophage recruitment to heart tissue and parasite control in the acute infection of mouse with Trypanosoma cruzi, the causative agent of Chagas disease. Here, we approached the participation of CCL3 in chronic chagasic cardiomyopathy (CCC), the main clinical form of Chagas disease. We induced CCC in C57BL/6 (ccl3+/+) and CCL3-deficient (ccl3-/-) mice by infection with the Colombian Type I strain. In ccl3+/+ mice, high levels of CCL3 mRNA and protein were detected in the heart tissue during the acute and chronic infection. Survival was not affected by CCL3 deficiency. In comparison with ccl3+/+, chronically infected ccl3-/- mice presented reduced cardiac parasitism and inflammation due to CD8+ cells and macrophages. Leukocytosis was decreased in infected ccl3-/- mice, paralleling the accumulation of CD8+ T cells devoid of activated CCR5+ LFA-1+ cells in the spleen. Further, T. cruzi-infected ccl3-/-mice presented reduced frequency of interferon-gamma (IFNγ)+ cells and numbers of parasite-specific IFNγ-producing cells, while the T. cruzi antigen-specific cytotoxic activity was increased. Stimulation of CCL3-deficient macrophages with IFNγ improved parasite control, in a milieu with reduced nitric oxide (NOx) and tumor necrosis factor (TNF), but similar interleukin-10 (IL-10), concentrations. In comparison with chronically T. cruzi-infected ccl3+/+ counterparts, ccl3-/- mice did not show enlarged heart, loss of left ventricular ejection fraction, QTc prolongation and elevated CK-MB activity. Compared with ccl3+/+, infected ccl3-/- mice showed reduced concentrations of TNF, while IL-10 levels were not affected, in the heart milieu. In spleen of ccl3+/+ NI controls, most of the CD8+ T-cells expressing the CCL3 receptors CCR1 or CCR5 were IL-10+, while in infected mice these cells were mainly TNF+. Lastly, selective blockage of CCR1/CCR5 (Met-RANTES therapy) in chronically infected ccl3+/+ mice reversed pivotal electrical abnormalities (bradycardia, prolonged PR, and QTc interval), in correlation with reduced TNF and, mainly, CCL3 levels in the heart tissue. Therefore, in the chronic T. cruzi infection CCL3 takes part in parasite persistence and contributes to form a CD8+ T-cell and macrophage-enriched cardiac inflammation. Further, increased levels of CCL3 create a scenario with abundant IFNγ and TNF, associated with cardiomyocyte injury, heart dysfunction and QTc prolongation, biomarkers of severity of Chagas' heart disease.

Highly potent HIV inhibition: engineering a key anti-HIV structure from PSC-RANTES into MIP-1 beta/CCL4.

The HIV coreceptor CCR5 is a validated target for both the prevention and therapy of HIV infection. PSC-RANTES, an N-terminally modified analogue of one of the natural chemokine ligands of CCR5 (RANTES/CCL5), is a potent inhibitor of HIV entry into target cells. Here, we set out to engineer the anti-HIV activity of PSC-RANTES into another natural CCR5 ligand (MIP-1beta/CCL4), by grafting into it the key N-terminal pharmacophore region from PSC-RANTES. We were able to identify MIP-1beta/CCL4 analogues that retain the receptor binding profile of MIP-1beta/CCL4, but acquire the very high anti-HIV potency and characteristic inhibitory mechanism of PSC-RANTES. Unexpectedly, we discovered that in addition to N-terminal structures from PSC-RANTES, the side chain of Lys33 is also necessary for full anti-HIV potency.

N-terminal proteolytic processing by cathepsin G converts RANTES/CCL5 and related analogs into a truncated 4-68 variant.

N-terminal proteolytic processing modulates the biological activity and receptor specificity of RANTES/CCL5. Previously, we showed that an unidentified protease associated with monocytes and neutrophils digests RANTES into a variant lacking three N-terminal residues (4-68 RANTES). This variant binds CCR5 but exhibits lower chemotactic and antiviral activities than unprocessed RANTES. In this study, we characterize cathepsin G as the enzyme responsible for this processing. Cell-mediated production of the 4-68 variant was abrogated by Eglin C, a leukocyte elastase and cathepsin G inhibitor, but not by the elastase inhibitor elastatinal. Further, anti-cathepsin G antibodies abrogated RANTES digestion in neutrophil cultures. In accordance, reagent cathepsin G specifically digested recombinant RANTES into the 4-68 variant. AOP-RANTES and Met-RANTES were also converted into the 4-68 variant upon exposure to cathepsin G or neutrophils, while PSC-RANTES was resistant to such cleavage. Similarly, macaque cervicovaginal lavage samples digested Met-RANTES and AOP-RANTES, but not PSC-RANTES, into the 4-68 variant and this processing was also inhibited by anti-cathepsin G antibodies. These findings suggest that cathepsin G mediates a novel pathway for regulating RANTES activity and may be relevant to the role of RANTES and its analogs in preventing HIV infection.

Lentiviral gene transfer of the chemokine antagonist RANTES 9-68 prolongs heart graft survival.

BACKGROUND: Allograft tolerance might be achieved by expressing immunomodulatory proteins through gene therapy. We have evaluated the possibility of promoting significantly allograft survival in a vascularized cardiac allograft model by performing ex vivo gene transfer. We used a lentiviral vector encoding the chemokine antagonist RANTES 9-68 that is capable of competing with native RANTES. METHODS: The Fisher donor/Lewis recipient rat strain combinations were used and all animals received for the first 5 days posttransplantation a subtherapeutic dose of cyclosporine A (1.5 mg/kg). Ex vivo gene transfer into heart allograft was performed by multiple injections of the SIN.cPPT lentiviral vector, which corresponds to the multiply attenuated, self-inactivating lentivector derived from the human immunodeficiency virus (HIV)-1. RESULTS: About 6% of the cardiac tissue had integrated lentiviral vector, which closely matches the mean in vivo RANTES antagonist expression of 5% obtained by immunohistochemistry. In vivo RANTES 9-68 expression has significantly prolonged graft survival (median [25%-75%]: 20 [17-26] days), compared to the control 15 ([14-15] days; P=0.0007). Furthermore, hearts transduced with RANTES 9-68 showed a significant (P<0.05) reduction in cell infiltration and intragraft expression of TNF-alpha, IFN-gamma, endogenous RANTES, and TGF-beta. CONCLUSION: Lentiviral gene transfer of RANTES 9-68 antagonist attenuates significantly the inflammatory response and delays allograft rejection, despite low levels of transduction. Future improvement of heart transduction by lentiviral vectors, as it has been achieved with other vectors, might become an attractive alternative therapy for treating allografts that require sustained gene expression for better organ preservation.

Combined blockade of the chemokine receptors CCR1 and CCR5 attenuates chronic rejection.

BACKGROUND: Chemokine-chemokine receptor interaction and the subsequent recruitment of T-lymphocytes to the graft are early events in the development of chronic rejection of transplanted hearts or cardiac allograft vasculopathy (CAV). In this study, we sought to determine whether blockade of chemokine receptors CCR1 and CCR5 with Met-RANTES affects the development of CAV in a murine model. METHODS AND RESULTS: B6.CH-2(bm12) strain donor hearts were transplanted heterotopically into wild-type C57BL/6 mice (myosin heavy chain II mismatch). Recipients were treated daily with either Met-RANTES or vehicle starting on postoperative day 4 and were euthanized on postoperative days 24 and 56. We found that Met-RANTES significantly reduced intimal thickening in this model of chronic rejection and that Met-RANTES markedly decreased the infiltration of CD4 and CD8 T lymphocytes and MOMA-2+ monocytes/macrophages into transplanted hearts. Met-RANTES also suppressed the ex vivo and in vitro proliferative responses of recipient splenocytes to donor antigens. Finally, Met-RANTES treatment was associated with a marked reduction in RANTES/CCL5 and monocyte chemoattractant protein-1 gene transcript levels in the donor hearts. CONCLUSIONS: Antagonism of the chemokine receptors CCR1 and CCR5 with Met-RANTES attenuates CAV development in vivo by reducing mononuclear cell recruitment to the transplanted heart, proliferative responses to donor antigens, and intragraft RANTES/CCL5 and monocyte chemoattractant protein-1 gene transcript levels. These findings suggest that chemokine receptors CCR1 and CCR5 play significant roles in the development of chronic rejection and may serve as potential therapeutic targets.

Regulated on activation, normal T cell expressed and secreted (RANTES) antagonist (Met-RANTES) controls the early phase of Trypanosoma cruzi-elicited myocarditis.

BACKGROUND: Comprehension of the pathogenesis of Trypanosoma cruzi-elicited myocarditis is crucial to delineate strategies aimed at ameliorating the inflammation associated with heart dysfunction. The augmented expression of CC chemokines, especially CCL5/RANTES and CCL3/MIP-1alpha, in the hearts of infected mice suggests a role for CC chemokines and their receptors in the pathogenesis of T cruzi-elicited myocarditis. METHODS AND RESULTS: We report that during the early phase of infection in C3H/HeJ mice infected with 100 blood trypomastigotes of T cruzi, most of the inflammatory cells invading the heart tissue were CD8+ cells and expressed CCR5, a CCL5/RANTES, and CCL3/MIP1-alpha receptor. Furthermore, peripheral blood CD8+ T lymphocytes displayed increased expression of CCR5. These findings led us to use Met-RANTES, a selective CCR1 and CCR5 antagonist, to modulate the acute T cruzi-elicited myocarditis. Met-RANTES treatment did not interfere with parasitism but significantly decreased the numbers of CD4+ and CD8+ T cells, CCR5+, and interleukin-4+ cells invading the heart, paralleling the diminished deposition of fibronectin. Moreover, Met-RANTES treatment resulted in increased survival of infected animals, compared with saline treatment. CONCLUSIONS: These results indicate that the massive influx of CCR5+ cells into cardiac tissue is not crucial for cell-mediated anti-T cruzi immunity but appears to be critical for pathogenesis of T cruzi-elicited myocarditis. Thus, CC chemokine receptors might become an attractive therapeutic target for further evaluation during T cruzi infection.

The effect of the CC chemokine receptor antagonist Met-RANTES on experimental autoimmune uveitis and oral tolerance.

Lymphocyte trafficking is controlled in part by the actions of chemokines. In rat experimental autoimmune uveitis (EAU) we observed differential therapeutic effects of Met-RANTES, a CCR1/CCR5 receptor antagonist, depending on the retinal antigen peptides inducing the disease and the time of application during the afferent or efferent immune response. CCR1 and/or CCR5 blockade may have inhibitory effects on different phases of the autoimmune response, depending on the antigen specificity of T cells in EAU. In contrast, Met-RANTES enhanced therapeutic oral tolerance independently of orally applied antigen.

RANTES stimulates cell-mediated transmission of HIV-1 infection.

We wished to determine the effects of the beta-chemokine RANTES in an established system of cell-mediated transmission of HIV-1, that is, normal human umbilical vein endothelial cells (HUVEC) nonproductively infected with HIV-1, cocultivated with CD4+ T cells to rescue productive infection. The results indicate that the addition of RANTES to HUVEC, either before or after HIV-1 infection, stimulates HIV-1 rescue by CD4+ T cells. However, viral DNA is not increased in HUVEC, suggesting that the stimulation exerted by RANTES could be mediated by events following HUVEC infection. The mechanisms of increase seem to be related to the rescue phase, involving membrane interaction of abortively infected HUVEC with permissive T cells. In fact, a strong upregulation and polarization of intercellular adhesion molecule-1 (ICAM-1) is induced in HUVEC by RANTES, and antibodies against ICAM-1 inhibit HIV-1 rescue by T cells. These results indicate that RANTES, similarly to other inflammatory cytokines, may favor HIV-1 spreading and crossing of blood-tissue barriers by indirect mechanisms involving membrane interactions between nonproductively infected and permissive cells.

Treatment of experimental autoimmune encephalomyelitis with the chemokine receptor antagonist Met-RANTES.

Specific chemokines and chemokine receptors have been implicated in inflammatory demyelinating diseases of the central nervous system (CNS), including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Amino-terminal modifications of chemokines can alter receptor interactions, converting agonists to specific antagonists. To examine the function in EAE of murine types 1 and 5 CC chemokine receptors (CCR1 and CCR5), we used Met-RANTES, a peptide that blocks both receptors; controls received heat-inactivated peptide. There was no effect of active treatment on acute-monophasic EAE, regardless whether compound was given at onset or in a pre-treatment regimen. Administered at disease onset, Met-RANTES modestly but significantly ameliorated fixed neurological disability at the endpoint of chronic-relapsing EAE. Met-RANTES treatment did not reduce CNS cellular infiltrates or up-regulation of CCR1 and CCR5 in affected CNS tissues. Analysis of a subset of mice suggested a trend towards reduced axonal pathology in those receiving active treatment. These data indicate that chemokine receptor blockade with Met-RANTES does not affect leukocyte trafficking in chronic-relapsing EAE. Further analysis of the effects of chemokine receptor blockade may need to focus on leukocyte activation within the affected CNS as well as trafficking events.

Chemokine receptors and their role in leukocyte activation.

Chemokines were originally isolated based on their abilities to selectively attract and recruit leukocyte populations. Over the last few years there has been an explosion in the number of new chemokines identified, and as a result many receptors previously considered to be orphans have now been paired up with their ligands. Here we review some of the latest results in this area, illustrating with data from our laboratory. The central question from a drug discovery perspective, is to show whether inhibiting chemokine receptors leads to a change in disease status. Although we are still a long way from having candidate molecules to take into the clinic, a flavour of what may be possible can be inferred from mutant chemokines with antagonistic properties. We discuss recent data using two of these proteins, Met-RANTES which has anti-inflammatory properties, and AOP-RANTES which has been shown to prevent infection of macrophages and T-cells by M-tropic HIV strains.

C-C chemokine RANTES and HIV long terminal repeat-driven gene expression.

The C-C chemokines RANTES, MIP-1alpha, and MIP-1beta have been characterized as constituents of an HIV- and SIV-suppressive factor released by CD8+ cells. Furthermore, it has been demonstrated that chemokine receptors cooperate in HIV entry. However, these proteins are also known to have an effect on multiple intracellular signaling cascades that may affect the process of transcription. In the present study we demonstrate that treatment of CD4+ T cells with these chemokines or with cell supernatants from HTLV-I-immortalized CD8+ T cells results in significant reduction in the abundance of HIV-1-specific RNA as analyzed by Northern blot hybridization. To examine the possibility that such suppressive factors may inhibit HIV RNA transcription, we studied the effect of RANTES, the most effective HIV-suppressive chemokine, on basal and Tat-induced HIV-directed LTR expression of a reporter gene. Neither recombinant RANTES nor conditioned medium from CD8+ cells significantly altered HIV-1 LTR-directed chloramphenicol acetyltransferase expression in either transiently or stably transfected CD4+ T cell lines, either in the presence or in the absence of Tat. These results suggest that C-C chemokines do not inhibit viral RNA transcription.

New therapies and vaccines for meningococcal disease.

Meningococcal disease (MCD) is an important cause of morbidity and mortality. The pathophysiology consists of a complex interaction of bacterial and host factors, triggered by the release of endotoxin which initiates the inflammatory cascade, resulting in multi-organ failure, coagulopathy, capillary leak, metabolic derangement and eventually death. Prompt recognition and aggressive management are essential in reducing mortality. Over the past decade, there has been intense research into novel therapies and vaccines, with largely disappointing results. Therapies have been broadly divided into anti-endotoxin and anti-TNF-alpha therapies, treatment aimed at correcting coagulopathy and at blood purification and anti-inflammatory cytokine therapy. The reasons for the disappointing results in the search for new therapeutic strategies are difficult to identify. The disordered physiology in MCD results from a complex interaction of several mediators; therefore attempts to correct this by altering just one step represents a gross oversimplification of the process. In addition, the experimental model of endotoxaemia, which is often used, is a poor representation of an acutely ill patient with rapidly progressive shock. There have been several small or poorly designed trials, which have failed to reach definite conclusions. In order to yield conclusive results any future trials must be multicentre, randomised, controlled trials, but these are expensive and, in practice, difficult to conduct. The BPI trial (vide infra) was a significant step forward in this regard and demonstrated the ability to organise a large multicentred trial which can act as a template for future trials. Although the results were not significant there was an overall trend towards improved outcome in the treatment arm. Whilst the development of effective therapies and vaccines are awaited, the priorities at present must be the prompt recognition and aggressive management of disease.