Canine Retina Has a Primate Fovea-Like Bouquet of Cone Photoreceptors Which Is Affected by Inherited Macular Degenerations

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations

The TRPA1 activator AITC activates SmTRPA but not ShTRPA.

<p><b>A.</b> Traces of averaged GCaMP6f fluorescence intensity change in cells transfected with empty vector (pcDNA3.1/zeo⁽⁺⁾; orange), rat TRPA1 (green), SmTRPA (purple), or ShTRPA (blue). <b>B.</b> Normalized maximal GCaMP6f fluorescence intensity in response to 20 μM AITC in cells transfected with empty vector (Vector, n = 149, 3 independent transfections); SmTRPA (n = 64, 4 independent transfections); or ShTRPA (n = 72, 4 independent transfections). *, P < 0.05, ***, P < 0.0001, unpaired two-tailed t-test vs. empty vector data. <b>C.</b> <i>S</i>. <i>haematobium</i> adult females do not exhibit hyperactivity in response to 60 μM AITC (n = 20). Activity was measured as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006495#pntd.0006495.g002" target="_blank">Fig 2</a> and [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006495#pntd.0006495.ref039" target="_blank">39</a>]. Data are normalized to Control response for each individual worm. As a positive control, we also tested 40 μM 5-HT, which does produce significant hyperactivity in <i>S</i>. <i>haematobium</i> females (n = 26). *, P < 0.05, paired, two-tailed t-test vs. Control, prior to normalization.</p

Atypical pharmacology of schistosome TRPA1-like ion channels

<div><p>Parasitic flatworms of the genus <i>Schistosoma</i> cause schistosomiasis, a neglected tropical disease estimated to affect over 200 million people worldwide. Praziquantel is the only antischistosomal currently available for treatment, and there is an urgent need for new therapeutics. Ion channels play key roles in physiology and are targets for many anthelmintics, yet only a few representatives have been characterized in any detail in schistosomes and other parasitic helminths. The transient receptor potential (TRP) channel superfamily comprises a diverse family of non-selective cation channels that play key roles in sensory transduction and a wide range of other functions. TRP channels fall into several subfamilies. Members of both the TRPA and TRPV subfamilies transduce nociceptive and inflammatory signals in mammals, and often also respond to chemical and thermal signals. We previously showed that although schistosomes contain no genes predicted to encode TRPV channels, TRPV1-selective activators such as capsaicin and resiniferatoxin elicit dramatic hyperactivity in adult worms and schistosomula. Surprisingly, this response requires expression of a <i>S</i>. <i>mansoni</i> TRPA1-like orthologue (SmTRPA). Here, we show that capsaicin induces a rise in intracellular Ca²⁺ in mammalian cells expressing either SmTRPA or a <i>S</i>. <i>haematobium</i> TRPA1 orthologue (ShTRPA). We also test SmTRPA and ShTRPA responses to various TRPV1 and TRPA1 modulators. Interestingly, in contrast to SmTRPA, ShTRPA is not activated by the TRPA1 activator AITC (allyl isothiocyanate), nor do <i>S</i>. <i>haematobium</i> adult worms respond to this compound, a potentially intriguing species difference. Notably, 4-hydroxynonenal (4-HNE), a host-derived, inflammatory product that directly activates mammalian TRPA1, also activates both SmTRPA and ShTRPA. Our results point to parasite TRPA1-like channels which exhibit atypical, mixed TRPA1/TRPV1-like pharmacology, and which may also function to transduce endogenous host signals.</p></div

The TRPV1 activator olvanil stimulates an increase in intracellular Ca²⁺ in cells transfected with SmTRPA or ShTRPA and increases motor activity in adult <i>S</i>. <i>mansoni</i>.

<p><b>A.</b> Traces of averaged GCaMP6f fluorescence intensity change in response to 10 μM olvanil in cells transfected with empty pcDNA3.1/zeo⁽⁺⁾ (orange), SmTRPA (purple), or ShTRPA (blue). Olvanil was applied at 5 min. <b>B.</b> Normalized maximal GCaMP6f fluorescence intensity in response to 10 μM olvanil in cells transfected with: empty vector (n = 67, 3 independent transfections); SmTRPA (n = 23, 4 independent transfections); ShTRPA (n = 177, 4 independent transfections). *, P < 0.05, ***, P < 0.0001, unpaired, two-tailed t-test vs. empty vector data. <b>C, D.</b> Measurement of motor activity in adult (~7 weeks post infection) <i>S</i>. <i>mansoni</i> males (C) or females (D) that were exposed in culture to 10 μM olvanil. Motility was assayed before and after addition of compound. "Control" contained no added compounds, and the "DMSO" sample contained 0.1% DMSO. Motility was analyzed as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006495#pntd.0006495.ref039" target="_blank">39</a>], with each individual tested worm serving as its own control, and data normalized to Control response for each worm. *, P < 0.05, **, P < 0.01, paired, two-tailed t-test against Control, prior to normalization.</p

4-HNE, a host-derived, inflammatory activator of mammalian TRPA1, evokes hyperactivity in adult <i>S</i>. <i>mansoni</i> and activates SmTRPA and ShTRPA.

<p><b>A.</b> Response of male (left panel) and female (right panel) <i>S</i>. <i>mansoni</i> adults to 0.1% DMSO (n = 12) or 10 μM 4-HNE (n = 47, males; n = 24, females), measured as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006495#pntd.0006495.g002" target="_blank">Fig 2</a>. **, P < 0.01, paired, two-tailed t-test vs. Control. <b>B.</b> 4-HNE (50 μM) triggers an increase in intracellular GCaMP6f fluorescence in cells expressing either SmTRPA or ShTRPA. Traces of averaged GCaMP6f fluorescence intensity change in cells transfected with empty vector (pcDNA3.1/zeo⁽⁺⁾; orange), SmTRPA (purple), or ShTRPA (blue). <b>C.</b> Normalized maximal GCaMP6f fluorescence intensity in response to 50 μM 4-HNE in cells transfected with empty vector (Vector, n = 21, 3 independent transfections); SmTRPA (n = 18, 3 independent transfections); or ShTRPA (n = 95, 4 independent transfections). ***, P < 0.0001, unpaired, two-tailed t-test vs. empty vector data.</p

The TRPV1 activator capsaicin stimulates a significant Ca²⁺ influx in CHO cells expressing schistosome TRPA1-like channels.

<p><b>A.</b> Montage showing the response of CHO cells co-transfected with SmTRPA and pGP-CMV-GCaMP6f to 10 μM capsaicin over time. Number in upper left corner of each picture represents elapsed time (in minutes:seconds). Capsaicin was applied at frame 6 (0:19). Calibration bar = 85 μm. <b>B.</b> Traces of averaged GCaMP6f fluorescence intensity change in response to 10 μM capsaicin in cells transfected with empty vector (pcDNA3.1/zeo⁽⁺⁾; orange), rat TRPV1 (green), SmTRPA (purple), or ShTRPA (blue). The black trace is a positive control indicating the averaged response of randomly selected cells (n = 90, 5 independent transfections) from each group to 1 μM ionomycin, a Ca²⁺ ionophore. Capsaicin was applied at 5 min. <b>C.</b> Normalized maximal GCaMP6f fluorescence intensity in response to 10 μM capsaicin in cells transfected with: empty vector (Vector; n = 62, 3 independent transfections); SmTRPA (n = 131 cells, 6 independent transfections); ShTRPA (n = 112, 6 independent transfections); rat TRPA1 (rTRPA1, n = 99, 3 independent transfections); and rat TRPV1 (rTRPV1, n = 41, 3 independent transfections). ***, P < 0.0001, unpaired, two-tailed t-test vs. empty vector data. Responses of cells transfected with SmTRPA or ShTRPA to capsaicin are also significantly higher (P <0.0001) than responses to DMSO (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006495#pntd.0006495.s001" target="_blank">S1 Fig</a>). <b>D.</b> Dose response of expressed SmTRPA to capsaicin. Normalized maximal GCaMP6f fluorescence in response to different concentrations of capsaicin is shown for CHO cells transfected with SmTRPA. Capsaicin concentrations tested are 5 μM (n = 53), 10 μM (n = 34), 30 μM (n = 121), 70 μM (n = 90). Control (n = 39) is 0.01% DMSO. In this experiment, all cells tested were from a single transfection, which likely accounts for the differences from the maximal fluorescence value at 10 μM shown in panel C, which represents results from multiple transfections.</p

Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

<div><p>Staphylococcal enterotoxin B (SEB) exposure triggers an exaggerated pro-inflammatory cytokine response that often leads to toxic shock syndrome (TSS) associated with organ failure and death. MyD88 mediates pro-inflammatory cytokine signaling induced by SEB exposure and MyD88^−/− mice are resistant to SEB intoxication, suggesting that MyD88 may be a potential target for therapeutic intervention. We targeted the BB loop region of the Toll/IL-1 receptor (TIR) domain of MyD88 to develop small-molecule therapeutics. Here, we report that a synthetic compound (EM-163), mimic to dimeric form of BB-loop of MyD88 attenuated tumor necrosis factor (TNF)- α, interferon (IFN)-γ, interleukin (IL)-1β, IL-2 and IL-6 production in human primary cells, whether administered pre- or post-SEB exposure. Results from a direct binding assay, and from MyD88 co-transfection/co-immunoprecipitation experiments, suggest that EM-163 inhibits TIR-TIR domain interaction. Additional results indicate that EM-163 prevents MyD88 from mediating downstream signaling. In an NF-kB-driven reporter assay of lipopolysaccharide-stimulated MyD88 signaling, EM-163 demonstrated a dose-dependent inhibition of reporter activity as well as TNF-α and IL-1β production. Importantly, administration of EM-163 pre- or post exposure to a lethal dose of SEB abrogated pro-inflammatory cytokine responses and protected mice from toxic shock-induced death. Taken together, our results suggest that EM-163 exhibits a potential for therapeutic use against SEB intoxication.</p> </div

Perforin is required for CD8+ T cell induced pathology in <i>L. </i>braziliensis infection.

<p><i>Rag1−/−</i> mice were infected with <i>L. braziliensis</i> in the ear, and reconstituted with either WT or <i>Prf−/−</i> CD8+ T cells. (A) Ear thickness at the site of infection. (B) Representative images of leishmanial lesions at 5 weeks post infection. At 7 weeks post infection mice were euthanized and shown are: (C) parasite burden in the lesions; representative (D) contour plots and (E) bar graph of CD8+ T cell in the lesion (pregated on: Live/Singlets/CD45+/CD8β+ cells). Graphs are representative data from one of three independent experiments (n = 5). <i>Rag1−/−</i> mice were infected with <i>L. braziliensis</i> in the ear, and reconstituted with either WT, IFN-γ−/−, or IL-17−/− CD8+ T cells. (F,H) Ear thickness at the site of infection; (G,I) Parasite burden in the lesions at 7 weeks post infection. Data from one (F,G) experiment or one representative experiment of two (H,I) (n = 5) are presented. <i>*p<0.05.</i></p

Cytotoxic T Cells Mediate Pathology and Metastasis in Cutaneous Leishmaniasis

<div><p>Disease progression in response to infection can be strongly influenced by both pathogen burden and infection-induced immunopathology. While current therapeutics focus on augmenting protective immune responses, identifying therapeutics that reduce infection-induced immunopathology are clearly warranted. Despite the apparent protective role for murine CD8+ T cells following infection with the intracellular parasite <i>Leishmania</i>, CD8+ T cells have been paradoxically linked to immunopathological responses in human cutaneous leishmaniasis. Transcriptome analysis of lesions from <i>Leishmania braziliensis</i> patients revealed that genes associated with the cytolytic pathway are highly expressed and CD8+ T cells from lesions exhibited a cytolytic phenotype. To determine if CD8+ T cells play a causal role in disease, we turned to a murine model. These studies revealed that disease progression and metastasis in <i>L. braziliensis</i> infected mice was independent of parasite burden and was instead directly associated with the presence of CD8+ T cells. In mice with severe pathology, we visualized CD8+ T cell degranulation and lysis of <i>L. braziliensis</i> infected cells. Finally, in contrast to wild-type CD8+ T cells, perforin-deficient cells failed to induce disease. Thus, we show for the first time that cytolytic CD8+ T cells mediate immunopathology and drive the development of metastatic lesions in cutaneous leishmaniasis.</p></div

CD8+ T cell induced pathology requires specificity.

<p><i>Rag1−/−</i> mice were infected with <i>L. braziliensis</i> in the ear, and reconstituted with either WT or OT1 CD8+ T cells. (A) Ear thickness was evaluated at the site of infection. (B) Representative images of leishmanial lesions at 6 weeks post infection. At 7 weeks post infection mice were euthanized and shown are: (C) parasite burden in the lesions; (D) contour plots and (E) bar graph of CD107a expression by CD8+ T cells (pregated on: Live/Singlets/CD45+/CD8β+ cells). Graphs are representative data from one of two independent experiments (n = 3) with similar results. <i>*p<0.05.</i></p