Keith W. Wegmann

Endochin-like quinolones are highly efficacious against acute and latent experimental toxoplasmosis

Toxoplasma gondii is a widely distributed protozoan pathogen that causes devastating ocular and central nervous system disease. We show that the endochin-like quinolone (ELQ) class of compounds contains extremely potent inhibitors of T. gondii growth in vitro and is effective against acute and latent toxoplasmosis in mice. We screened 50 ELQs against T. gondii and selected two lead compounds, ELQ-271 and ELQ-316, for evaluation. ELQ-271 and ELQ-316, have in vitro IC50 values of 0.1 nM and 0.007 nM, respectively. ELQ-271 and ELQ-316 have ED50 values of 0.14 mg/kg and 0.08 mg/kg when administered orally to mice with acute toxoplasmosis. Moreover, ELQ-271 and ELQ-316 are highly active against the cyst form of T. gondii in mice at low doses, reducing cyst burden by 76–88% after 16 d of treatment. To investigate the ELQ mechanism of action against T. gondii, we demonstrate that endochin and ELQ-271 inhibit cytochrome c reduction by the T. gondii cytochrome bc1 complex at 8 nM and 31 nM, respectively. We also show that ELQ-271 inhibits the Saccharomyces cerevisiae cytochrome bc1 complex, and an M221Q amino acid substitution in the Qi site of the protein leads to >100-fold resistance. We conclude that ELQ-271 and ELQ-316 are orally bioavailable drugs that are effective against acute and latent toxoplasmosis, likely acting as inhibitors of the Qi site of the T. gondii cytochrome bc1 complex.

Specificity of regulatory CD4+CD25+ T cells for self-T cell receptor determinants.

Although the phenotypic and regulatory properties of the CD4+CD25+ T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4+CD25+ Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline‐encoded CDR3β sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR‐reactive T cells required cell–cell contact and involved CTLA‐4, GITR, IL‐10, and IL‐17. Thus, the T–T regulatory network includes Treg cells with specificity directed toward self‐TCR determinants.

Inflammatory Skin Disease in K5.hTGF-β1 Transgenic Mice Is Not Dependent on the IL-23/Th17 Inflammatory Pathway

In the presence of IL-6, transforming growth factor (TGF)-beta1 induces differentiation of T helper (Th) 17 cells in mice. Interleukin (IL)-23, a heterodimeric cytokine composed of IL-23p19 and IL-12/23p40 subunits, stimulates the growth and expansion of Th17 cells, and has been implicated in psoriasis pathogenesis. To study the associations between TGF-beta1, the IL-23/Th17 inflammatory pathway, and psoriasis, we investigated inflammatory skin disease in transgenic mice that constitutively overexpress human TGF-beta1 in basal keratinocytes (K5.hTGF-beta1 transgenic mice); these mice had previously been reported as having a psoriasis-like disease. K5.hTGF-beta1 transgenic mice had high levels of TGF-beta1 mRNA and protein in both skin and serum. Levels of cytokines involved in IL-23/Th17-mediated inflammation were not elevated in lesional skin compared with those in non-lesional and wild-type skin. It is noteworthy that IL-4 and IgE were markedly elevated in inflamed skin and serum, respectively, of transgenic mice. Monoclonal antibodies (mAbs) specifically directed against IL-23p19 or IL-12/23p40 had no clinical effect on established inflammatory skin disease in K5.hTGF-beta1 transgenic mice, whereas the same mAbs were able to block the development of murine experimental autoimmune encephalomyelitis, an IL-23/Th17-mediated disease. In summary, the IL-23/Th17 inflammatory pathway is not responsible for the maintenance of inflammatory skin disease in K5.hTGF-beta1 transgenic mice.

Synthetic Peptide Dendrimers Block the Development and Expression of Experimental Allergic Encephalomyelitis

Multiple Ag peptides (MAPs) containing eight proteolipid protein (PLP)139–151 peptides arranged around a dendrimeric branched lysine core were used to influence the expression and development of relapsing experimental allergic encephalomyelitis (EAE) in SJL mice. The PLP139–151 MAPs were very efficient agents in preventing the development of clinical disease when administered after immunization with the PLP139–151 monomeric encephalitogenic peptide in CFA. The treatment effect with these MAPs was peptide specific; irrelevant multimeric peptides such as guinea pig myelin basic protein GPBP72–84 MAP (a dendrimeric octamer composed of the 72–84 peptide) and PLP178–191 MAP (a dendrimeric octamer composed of the PLP178–191 peptide) had no treatment effect on PLP139–151-induced EAE. PLP139–151 MAP treatment initiated after clinical signs of paralysis also altered the subsequent course of EAE; it limited developing signs of paralysis and effectively limited the severity and number of disease relapses in MAP-treated mice over a 60-day observation period. PLP139–151 MAP therapy initiated before disease onset acts to limit the numbers of Th17 and IFN-γ-producing cells that enter into the CNS. However, Foxp3+ cells entered the CNS in numbers equivalent for nontreated and PLP139–151 MAP-treated animals. The net effect of PLP139–151 MAP treatment dramatically increases the ratio of Foxp3+ cells to Th17 and IFN-γ-producing cells in the CNS of PLP139–151 MAP-treated animals.

OX40 (CD134) expression in sentinel lymph nodes correlates with prognostic features of primary melanomas

BACKGROUND The expression of OX40 (CD134) on activated CD4+ T cells has been associated with favorable cancer patient outcomes. Because of recent reports that sentinel lymph nodes (SLNs) may represent an immunosuppressive environment, we investigated the expression of OX40 in SLNs from patients with primary cutaneous melanoma. METHODS Samples of peripheral blood lymphocytes and a section of 71 SLNs from 53 patients with clinically node negative melanoma were purified for CD4+ T cells, stained for OX40, and analyzed by flow cytometry. RESULTS The mean percentage of OX40 on CD4 T cells in the SLNs versus peripheral blood lymphocytes was related indirectly to the T stage of the primary tumor and was decreased in ulcerated primary tumors and positive sentinel nodes. CONCLUSIONS The expression of OX40 on CD4+ T cells in SLNs draining primary melanomas decreased with more advanced tumor features (higher T stage, ulceration) and nodal involvement, suggesting that such tumors may have an immunosuppressive effect on the SLN microenvironment.

Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone

Human babesiosis is a tick-borne multisystem disease, and current treatments have both adverse side effects and a significant rate of drug failure. Lawres et al. report that endochin-like quinolones, in combination with atovaquone, cure experimental babesiosis in immunodeficient mice.

Sontochin as a Guide to the Development of Drugs against Chloroquine-Resistant Malaria

ABSTRACT Sontochin was the original chloroquine replacement drug, arising from research by Hans Andersag 2 years after chloroquine (known as “resochin” at the time) had been shelved due to the mistaken perception that it was too toxic for human use. We were surprised to find that sontochin, i.e., 3-methyl-chloroquine, retains significant activity against chloroquine-resistant strains of Plasmodium falciparum in vitro. We prepared derivatives of sontochin, “pharmachins,” with alkyl or aryl substituents at the 3 position and with alterations to the 4-position side chain to enhance activity against drug-resistant strains. Modified with an aryl substituent in the 3 position of the 7-chloro-quinoline ring, Pharmachin 203 (PH-203) exhibits low-nanomolar 50% inhibitory concentrations (IC50s) against drug-sensitive and multidrug-resistant strains and in vivo efficacy against patent infections of Plasmodium yoelii in mice that is superior to chloroquine. Our findings suggest that novel 3-position aryl pharmachin derivatives have the potential for use in treating drug resistant malaria.

Treatment of relapsing autoimmune encephalomyelitis with T cell receptor Vβ-specific antibodies when proteolipid protein is the autoantigen

Monoclonal antibodies (mAbs) directed against the Vβ chain of the T cell receptor (TCR) of pathogenic T cells have been used to treat acute murine experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (BP). We evaluated anti‐Vβ mAb for the treatment of relapsing EAE (R‐EAE) induced in SJL/J mice by the myelin proteolipid protein (PLP) peptide 139–151. Spinal cord mononuclear cells isolated from mice immunized for R‐EAE with PLP 139–151 were shown to express a predominance of Vβ2 and Vβ17 during acute and relapsing disease. T cell lines specific for PLP 139–151 were magnetically sorted to express 80–90% Vβ2. These Vβ2‐enriched lines induced typical relapsing demyelinating EAE in naive recipient mice. SJL/J mice with R‐EAE induced by a PLP 139–151‐specific T cell line expressing 88% Vβ2 were treated with anti‐Vβ2 mAb. Anti‐Vβ2 mAb markedly reduced clinical and histological disease severity when given at the time of cell transfer or when given at clinical disease onset. In contrast, anti‐Vβ mAbs showed only a mild clinical effect on R‐EAE induced by immunization with PLP 139–151 or R‐EAE induced by immunization with PLP 139–151 or R‐EAE transferred by a PLP 139–151‐specific T cell line expressing multiple Vβs. A cocktail of mAbs directed against Vβ2, Vβ4, and Vβ17 significantly reduced the numbers of spinal cord T cells expressing these Vβs during acute EAE but had little effect on disease course, suggesting that pathogenic T cells expressing other Vβs were producing disease. These findings may have implications for the treatment of multiple sclerosis with Vβ‐selective therapy.

Eluding anaphylaxis allows peptide-specific prevention of the relapsing stage of experimental autoimmune encephalomyelitis.

We have used a peptide derived from Acanthamoeba castellanii (ACA) to treat the relapsing phase of EAE that develops in SJL mice following immunization with the PLP 139-151 peptide. The native sequence of the ACA 81-95 peptide that shares key residues with the PLP 139-151 peptide is weakly encephalitogenic in SJL mice but is not recognized by antiserum from SJL mice immunized with PLP 139-151. A single amino acid change to the ACA 81-95 peptide sequence significantly enhanced its encephalitogenicity. When administered to SJL mice as a nonlinear peptide octamer, the modified ACA peptide prevented relapsing episodes of EAE in SJL mice previously immunized with the PLP 139-151 encephalitogenic peptide.

Targeting T cells responsive to the priming epitope prevent the relapsing phase of experimental autoimmune encephalomyelitis

Upon recovery from the initial episode of experimental autoimmune encephalomyelitis (EAE), virtually all SJL mice develop relapsing/remitting episodes of disease. These relapses may occur due to the reactivation of memory T cells initially stimulated as part of the disease-inducing protocol or naïve T-cell populations stimulated by distinct encephalitogens derived from the inflammatory disease process (epitope spread). We have used encephalitogen-specific non-linear peptide octamers to modify the course of relapsing EAE (rEAE) in SJL mice immunized with an oliogodendrocyte-specific protein peptide (OSP 55-71). Our studies show that the peptide-octamers, which target the T cells stimulated by the priming encephalitogen, but not other candidate encephalitogens, prevent rEAE.