Pixatimod (PG545) is a novel clinical-stage immunomodulatory agent capable of inhibiting the infiltration of tumor-associated macrophages (TAMs) yet also stimulate dendritic cells (DCs), leading to activation of natural killer (NK) cells. Preclinically, pixatimod inhibits heparanase (HPSE) which may be associated with its inhibitory effect on TAMs whereas its immunostimulatory activity on DCs is through the MyD88-dependent TLR9 pathway. Pixatimod recently completed a Phase Ia monotherapy trial in advanced cancer patients.
Methods
To characterize the safety of pixatimod administered by intravenous (IV) infusion, a one month toxicology study was conducted to support a Phase Ia monotherapy clinical trial. The relative exposure (AUC) of pixatimod across relevant species was determined and the influence of route of administration on the immunomodulatory activity was also evaluated. Finally, the potential utility of pixatimod in combination with PD-1 inhibition was also investigated using the syngeneic 4T1.2 breast cancer model.
Results
The nonclinical safety profile revealed that the main toxicities associated with pixatimod are elevated cholesterol, triglycerides, APTT, decreased platelets and other changes symptomatic of modulating the immune system such as pyrexia, changes in WBC subsets, inflammatory changes in liver, spleen and kidney. Though adverse events such as fever, elevated cholesterol and triglycerides were reported in the Phase Ia trial, none were considered dose limiting toxicities and the compound was well tolerated up to 100 mg via IV infusion. Exposure (AUC) up to 100 mg was considered proportional with some accumulation upon repeated dosing, a phenomenon also noted in the toxicology study. The immunomodulatory activity of pixatimod was independent of the route of administration and it enhanced the effectiveness of PD-1 inhibition in a poorly immunogenic tumor model.
Conclusions
Pixatimod modulates innate immune cells but also enhances T cell infiltration in combination with anti-PD-1 therapy. The safety and PK profile of the compound supports its ongoing development in a Phase Ib study for advanced cancer/pancreatic adenocarcinoma with the checkpoint inhibitor nivolumab (Opdivo®).
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Whether complement dysregulation directly contributes to the pathogenesis of peripheral nervous system diseases, including sensory neuropathies, is unclear. We addressed this important question in a mouse model of ocular HSV-1 infection, where sensory nerve damage is a common clinical problem. Through genetic and pharmacologic targeting, we uncovered a central role for C3 in sensory nerve damage at the morphological and functional levels. Interestingly, CD4 T cells were central in facilitating this complement-mediated damage. This same C3/CD4 T cell axis triggered corneal sensory nerve damage in a mouse model of ocular graft-versus-host disease (GVHD). However, this was not the case in a T-dependent allergic eye disease (AED) model, suggesting that this inflammatory neuroimmune pathology is specific to certain disease etiologies. Collectively, these findings uncover a central role for complement in CD4 T cell-dependent corneal nerve damage in multiple disease settings and indicate the possibility for complement-targeted therapeutics to mitigate sensory neuropathies. https://doi.org/10.7554/eLife.48378.001 eLife digest Most people have likely experienced the discomfort of an eyelash falling onto the surface of their eye. Or that gritty sensation when dust blows into the eye and irritates the surface. These sensations are warnings from sensory nerves in the cornea, the transparent tissue that covers the iris and pupil. Corneal nerves help regulate blinking, and control production of the tear fluid that protects and lubricates the eye. But if the cornea suffers damage or infection, it can become inflamed. Long-lasting inflammation can damage the corneal nerves, leading to pain and vision loss. If scientists can identify how this happens, they may ultimately be able to prevent it. To this end, Royer et al. have used mice to study three causes of hard-to-treat corneal inflammation. The first is infection with herpes simplex virus (HSV-1), which also causes cold sores. The second is eye allergy, where the immune system overreacts to substances like pollen or pet dander. And the third is graft-versus-host disease (GVHD), an immune disorder that can affect people who receive a bone marrow transplant. Royer et al. showed that HSV-1 infection and GVHD – but not allergies – made the mouse cornea less sensitive to touch. Consistent with this, microscopy revealed damage to corneal nerves in the mice with HSV-1 infection and those with GVHD. Further experiments showed that immune cells called CD4 T cells and a protein called complement C3 were contributing to this nerve damage. Treating the mice with an experimental drug derived from cobra venom protected the cornea from the harmful effects of inflammation. It did so by blocking activation of complement C3 at the eye surface. Identifying factors such as complement C3 that are responsible for corneal nerve damage is an important first step in helping patients with inflammatory eye diseases. Many drugs that target the complement pathway are currently under development. Some of these drugs could potentially be adapted for delivery as eye drops. But first, experiments must test whether complement also contributes to corneal nerve damage in humans. If it does, work can then begin on testing these drugs for safety and efficacy in patients. https://doi.org/10.7554/eLife.48378.002 Introduction Dysregulated complement activation is increasingly recognized as a significant pathological event in a variety of neurodegenerative and neuroinflammatory diseases of the central nervous system (Tenner et al., 2018). These include conditions from Alzheimer's disease and age-related macular degeneration (AMD) to amyotrophic lateral sclerosis and multiple sclerosis (Hong et al., 2016; Knickelbein et al., 2015; Lee et al., 2018; Loveless et al., 2018). Complement may orchestrate peripheral nervous system (PNS) disorders as well. For instance, nociceptive hypersensitivities, sensory neuropathies, and Guillain-Barré syndrome have been linked to aberrant complement activation (Rosoklija et al., 2000; Ramaglia et al., 2008; Jang et al., 2010; Fritzinger and Benjamin, 2016; Xu et al., 2018; Susuki et al., 2007). However, the mechanistic role of complement is not well understood in neuroinflammatory pathologies impacting the PNS. Moreover, the pathophysiological trigger of sensory fiber retraction which contributes to sensation loss and in some instances chronic pain is unclear (Stepp et al., 2017). Available treatments for peripheral neuropathies primarily focus on clinical symptoms without addressing the underlying pathomechanisms (Colloca et al., 2017). Accordingly, elucidation of relevant pathomechanisms underlying sensory neuropathies may translate into efficacious mechanism-based therapeutics. The complement cascade is comprised of dozens of soluble and membrane-bound factors essential for host defense and efficient clearance of cellular debris. However, proper regulation of complement activation is necessary to balance immune responses and prevent collateral tissue damage (Ricklin et al., 2016). The complement cascade pivots upon activation of complement component 3 (C3) for its downstream proinflammatory effects. The pathogenic contributions of complement in systemic disease have been reviewed extensively in recent years (Dobó et al., 2018; Hajishengallis et al., 2017; McGeer et al., 2017; Ricklin et al., 2016). Nonetheless, emerging concepts centering on complement effector synthesis within inflamed tissue microenvironments have ushered in a 'renaissance' of novel complement-targeted drug development strategies (Ricklin et al., 2018; Tomlinson and Thurman, 2018). Therefore, elucidating whether the complement cascade is a viable therapeutic target in inflammation-associated peripheral nerve impairment remains medically important. The eye has been labeled a 'complement dysregulation hotspot' due to complement's contributions to many ophthalmic diseases, but complement activation is restricted in the healthy cornea (Clark and Bishop, 2018). As observed in AMD, subtle inflammatory reactions in the eye can mediate significant visual morbidity. Accordingly, ocular inflammation is tightly regulated by various anatomic, physiologic, and immunologic mechanisms in order to maintain visual acuity (Amouzegar et al., 2016; Streilein, 2003; Taylor et al., 2018). These homeostatic mechanisms, collectively dubbed 'ocular immune privilege,' help preserve the ocular surface and enable the cornea to properly focus incoming light onto the retina. Furthermore, corneal nerves are increasingly recognized as central regulators of immune privilege at the ocular surface (Guzmán et al., 2018; Neelam et al., 2018; Paunicka et al., 2015). Consequently, inflammatory events that damage corneal nerves can have insidious consequences in terms of ocular surface health and transparency (Müller et al., 2003; Shaheen et al., 2014; Stepp et al., 2017). One such pathway that can rapidly initiate inflammation following activation is the complement cascade. Corneal nerves originate predominantly from the ophthalmic branch of the trigeminal ganglion (TG), and these peripheral nerves provide the cornea with the highest density of sensory fibers in the human body. While the cornea and peripheral nerves constitutively synthesize complement proteins (Bora et al., 2008; de Jonge et al., 2004), the role of the complement cascade in corneal nerve damage has not been explored. Nonetheless, the complement pathway is poised for vigorous activation in the cornea in response to noxious or inflammatory stimuli (Bora et al., 2008). Moreover, C3 activation has been reported in the cornea soon after infection with herpes simplex virus type 1 (HSV-1), which is a common cause of corneal sensory nerve damage in patients (Royer et al., 2017; Sacchetti and Lambiase, 2014). Given the cornea's high density of sensory nerves, it is particularly amenable for investigating the mechanistic role of complement in peripheral nerve damage. To this end, we evaluated corneal nerve integrity and mechano-sensory function using a murine model of ocular HSV-1 infection to test the hypothesis that local complement activation and T cell engagement coordinate corneal nerve damage. The pathobiology underlying non-penetrating corneal nerve damage is not well understood, although inflammation is generally recognized as an important feature (Neelam et al., 2018; Shaheen et al., 2014; Cruzat et al., 2015; Chucair-Elliott et al., 2016; Chucair-Elliott et al., 2017b; Chucair-Elliott et al., 2017a). While HSV-1 is a clinically prominent cause of corneal nerve damage and sensation loss, a variety of pathogenic microbes impair the corneal nerve architecture upon ocular infection (Cruzat et al., 2015). This observation adds to evidence that the neurotropism of HSV-1 is not directly responsible for nerve damage (Chucair-Elliott et al., 2016). In addition to sensation loss evoked by HSV-1, corneal nerve alterations in other contexts such as dry eye disease are associated with a broad array of neuropathic clinical symptoms including dryness, itch, and pain (Andersen et al., 2017). To further qualify the possible role of complement in corneal nerve damage independent of infection, we evaluated corneal mechanosensory function in two noninfectious T cell-dependent ocular surface inflammatory diseases. For this purpose, we utilized established murine models of allergic eye disease (AED) and ocular graft-versus-host disease (GVHD) (Herretes et al., 2015; Lee et al., 2015). Our rationale for this is that complement has been implicated in the etiology of systemic GVHD and allergic inflammation (Gour et al., 2018; Kwan et al., 2012; Ma et al., 2014; Nguyen et al., 2018; Zhang and Köhl, 2010). However, a neuroinflammatory role of complement has not been described for either disease within the eye. The translational relevance of this study is underscored by in vivo confocal microscopy data from multiple clinical studies showing architectural changes in the corneal nerves of patients with herpetic keratitis, chronic ocular allergy, and ocular GVHD (Hamrah et al., 2010; Müller et al., 2015; Moein et al., 2018; Hu et al., 2008; Le et al., 2011; Leonardi et al., 2012; Tepelus et al., 2017; He et al., 2017a). Elucidating the pathomechanisms underlying corneal nerve damage may enable development of more effective therapeutics to mitigate progression of such ocular surface inflammatory diseases. Each of the animal models utilized herein mimic clinically important, chronic ocular surface morbidities. Herpes simplex virus type 1 (HSV-1) is a common cause of neurotropic keratitis and remains a leading cause of infectious corneal blindness (Sacchetti and Lambiase, 2014; Farooq and Shukla, 2012). The incidence of ocular allergy exceeds twenty percent of the population with varying degrees of neurogenic ocular surface discomfort that can severely diminish quality of life (Craig et al., 2017; Patel et al., 2017; Saban et al., 2013). Finally, GVHD is the greatest cause of non-relapse morbidity following hematopoietic stem cell transplantation (HSCT) used to treat life-threatening malignancies and immunologic diseases (MacDonald et al., 2017). A majority of patients with chronic presentations of GVHD suffer from ocular surface involvement (Shikari et al., 2013). Results C3 facilitates corneal sensation loss in herpetic keratitis Adaptive immunity has been shown to prevent recovery of corneal sensory function following ocular HSV-1 infection (Yun et al., 2014), but the initial pathophysiological triggers of denervation and sensation loss are not definitively characterized. Ocular HSV-1 infection provokes corneal denervation and sensation loss between days 5 to 8 post-infection (p.i.) in immunologically naive C57BL/6 mice (Chucair-Elliott et al., 2015). This tempo appears to be synchronized with the host transition from innate to adaptive immunity following infection. Indeed, corneal nerve fiber retraction was evident upon T cell infiltration in the cornea (Figure 1A). Accordingly, we hypothesized that local complement activation and T cell engagement coordinate corneal nerve damage during HSV-1 infection. Figure 1 with 1 supplement see all Download asset Open asset Complement C3 contributes to corneal denervation. (A) Representative confocal images of cornea flat-mounts showing corneal nerves (βIII Tubulin, white) and T cells (CD3, green) in healthy uninfected (UI) and HSV-1-infected corneas 8 days post infection (p.i.). (B) Corneal mechanosensory function in WT and C3-/- mice following ocular HSV-1 infection (n = 6–8 mice/group; three independent experiments). (C) Viral titers shed in the tear film of WT and C3-/- mice at the indicated times p.i. (n = 5 mice per group; two independent experiments). Viral titers in the corneas and trigeminal ganglia (TG) of WT and C3-/- mice are shown at days 3 and 7 p.i. in (D) and (E), respectively. (F) Representative confocal images of stromal nerve fibers and T cells in healthy and HSV-infected corneas of WT and C3-/- mice as in (A). (G) Flow cytometry-based quantification of CD4+ and CD8+ T cells in HSV-infected corneas at day 7 p.i. (n = 7–8 mice/group; four independent experiments). Statistical differences were determined using two-way ANOVAs with Bonferroni posttests (B, C) or Student's T tests (D, E, G). https://doi.org/10.7554/eLife.48378.003 To address our hypothesis, corneal sensation and pathogen burden were evaluated in C57BL/6 wildtype (WT) and complement C3-deficient (C3-/-) mice following ocular HSV-1 infection. Progressive loss of corneal mechano-sensitivity was evident in WT mice by days 5 to 8 p.i., but corneal sensation was conserved in the C3-/- cohort (Figure 1B). The same trend revealing preservation of corneal sensation in C3-/- animals was also observed with an increased HSV-1 challenge inoculum (Figure 1—figure supplement 1). Viral titers were measured at time points before and after the onset of corneal sensation loss to determine whether the divergence in sensation stemmed from a differential susceptibility to infection. On day 3 p.i., the HSV-1 burden was similar in the tear film, corneas, and TG of WT and C3-/- animals (Figure 1C–D). By day 7 p.i., HSV-1 titers were elevated in the tear film and TG of C3-/- mice relative to WT (Figure 1C,E). Corneal buttons from WT mice exhibited extensive denervation with CD3+ T cell infiltration at day 8 p.i. (Figure 1F). However, T cell infiltration was observed without widespread denervation in C3-/- corneas (Figure 1F). The number of cornea-infiltrating CD4+ or CD8+ T cells were similar among WT and C3-/- mice (Figure 1G). Together, these data indicate that corneal sensation loss in herpetic keratitis involves a C3-dependent inflammatory process independent of viral burden. Corneal sensation loss during HSV-1 infection requires T cell coordination Although T cells successfully extravasate into the corneas of WT and C3-/- mice (Figure 1F,G), C3-deficiency can have broad impacts on T cell priming, clonal expansion, and recruitment (Clarke and Tenner, 2014; West et al., 2018). To investigate this possibility, corneas from HSV-1-infected WT and C3-/- mice were evaluated for chemokines associated with T cell recruitment, and the eye-draining mandibular lymph nodes (MLN) were harvested to evaluate T cell responses. Chemokines associated with T cell recruitment were elevated in corneas from both WT and C3-/- mice at day 5 p.i.—a time consistent with onset of sensation loss (Figure 2A). Moreover, CD4+ and CD8+ T cell expansion was comparable within the eye-draining mandibular lymph nodes (MLN) from WT and C3-/- mice (Figure 2B). Similarly, T cells harvested from WT and C3-/- mice at day 8 p.i. responded equally to in vitro stimulation in terms of IFNγ production (Figure 2C). Collectively, these data show that C3-/- T cells exhibit normal expansion and recruitment to the cornea during ocular HSV-1 infection. Figure 2 with 1 supplement see all Download asset Open asset T cells facilitate corneal sensation loss. (A) Chemokine concentrations in HSV-1-infected corneas from WT and C3-/- mice at day five post infection (p.i.). (B) T cell expansion in the eye-draining mandibular lymph nodes of WT and C3-/- mice. For panels (A) and (B), dashed lines reflect the average value for uninfected WT controls (n = 5 mice per group; two independent experiments; Student's T). (C) IFNγ expression following stimulation with PMA and ionomycin using T cells harvested from WT or C3-/- mice at day eight post infection (n = 7 unstimulated and 10 activated replicates from three independent experiments; one-way ANOVA, Bonferroni). (D) Adoptive transfer schematic and corneal sensation measurements in TCRα-/- mice following reconstitution with purified splenic T cells from HSV-infected WT and C3-/- mice (n = 5–9 TCRα-/- mice/group; three independent experiments; two-way ANOVA, Bonferroni). https://doi.org/10.7554/eLife.48378.005 In support of our hypothesis that C3 and T cells jointly coordinate corneal sensation loss, frank corneal sensation loss was not observed following ocular HSV-1 infection in C3-sufficient, alpha-beta T cell receptor-deficient mice (TCRα-/-) which congenitally lack classical CD4+ and CD8+ T cells (Figure 2D). To investigate potential functional defects of C3-/- T cells in an in vivo context, CD3+ T cells were harvested from HSV-infected WT or C3-/- mice at day 8 p.i. and adoptively transferred into TCRα-/- mice. Six days following cell transfer, TCRα-/- recipients were infected with HSV-1 and corneal sensation monitored longitudinally. Adoptive transfer of CD3+ T cells from either WT or C3-/- donors evoked progressive corneal sensation loss in TCRα-/- recipients by day 8 p.i. (Figure 2D). These findings further corroborate that C3-/- T cells remain functional in vivo. Analysis of corneal sensation was not feasible beyond 8 days p.i., as the TCRα-/- mice succumbed to herpetic encephalitis. However, engraftment of donor cells was confirmed by analysis of T cells in the eye-draining MLN of recipient mice by flow cytometry at day 8 p.i. The baseline lymphocyte counts in MLN from TCRα-/- controls likely reflect expansion of non-classical T cell populations (Viney et al., 1994). An increase in total cell number was observed for CD4+ but not CD8+ T cells upon adoptive transfer of purified CD3+ T cells into TCRα-/- recipient mice (Figure 2—figure supplement 1A). Consistent with this observation, our data show that transfer of CD3+ T cells from HSV-infected mice have no appreciable impact on HSV-1 titers in the TG of TCRα-/- recipients by day 8 p.i. (Figure 2—figure supplement 1B). Notably, bulk transfer of HSV-specific CD8+ T cells has been shown to reduce viral burden in the peripheral nervous system of C57BL/6 mice during acute HSV-1 infection (Conrady et al., 2009; Royer et al., 2016). Our collective findings reveal that C3 and T cells are individually necessary and interdependent in the pathogenesis of HSV-1-associated corneal sensation loss. Whether by direct or indirect mechanisms, these data support a paradigm in which C3 activation and T cell engagement coordinate corneal nerve damage in herpetic keratitis. Antigen-specific CD4+ T cells facilitate corneal sensation loss in HSV-1 keratitis Our data show that T cells contribute to corneal sensation loss during acute HSV-1 infection, yet whether this pathology is dependent upon CD4+ or CD8+ T cells remained unclear. To better discern the contributions of each subset, CD4+ and CD8+ T cells were harvested from infected WT mice, transferred into separate groups of TCRα-/- mice, and recipient mice subsequently infected with HSV-1. Corneal sensation loss during acute HSV-1 infection was only observed upon reconstitution with CD4+ T cells (Figure 3A). Donor cell engraftment was confirmed by flow cytometry, although a statistically significant increase in cell number within the eye-draining MLN was only observed upon transfer of CD4+ T cells (Figure 3—figure supplement 1). The requirement for antigen specificity was subsequently evaluated during acute HSV-1 infection by monitoring corneal sensation in WT and OT-II transgenic mice that generate ovalbumin (OVA)-specific CD4 T cells. Transgenic OT-II mice did not exhibit corneal sensation loss following infection despite evidence of bystander CD4+ T cell activation and recruitment into the cornea (Figure 3B–D). Adoptive transfer of CD4+ T cells from HSV-infected WT and OT-II mice into TCRα-/- recipients corroborated these findings (Figure 3—figure supplement 2). Abrasion of the corneal epithelium, which is required to mediate infection in this model, induces local chemokine expression capable of recruiting T cells to the cornea even in the absence of viral infection (Liu et al., 2012). Nonetheless, active corneal HSV-1 infection was necessary to provoke corneal sensation loss, as TCRα-/- mice reconstituted with CD4+ T cells harvested from HSV-infected WT mice did not elicit auto/allo-antigen-associated sensation loss within two weeks of corneal scratch injury (i.e. mock infection) (Figure 3—figure supplement 3). Collectively, our data show that complement C3 and antigen-specific CD4+ T cells are simultaneously necessary to drive corneal sensation loss during HSV-1 infection. These results strongly portend that a coordinated inflammatory axis exists involving C3 and antigen-specific CD4+ T cells, and that it is responsible for sensory neuropathy in herpetic keratitis. Figure 3 with 3 supplements see all Download asset Open asset Antigen-specific CD4 T cells drive corneal sensation loss. (A) Adoptive transfer schematic and corneal sensation measurements in TCRα-/- mice following reconstitution with purified splenic CD4 or CD8 T cells from HSV-infected WT mice (n = 6–7 TCRα-/- mice/group; two independent experiments; two-way ANOVA, Bonferroni). (B) Corneal sensation measurements at baseline and day seven post infection (p.i.) in WT and OT-II mice following ocular HSV-1 infection (n = 4–6 mice/group; three independent experiments). (C) Percentage of CXCR3-expressing CD4 T cells in peripheral blood from WT and OT-II mice at day 7 p.i.; (n = 5 mice/group; two independent experiments). (D) Verification of CD4 T cell infiltration into corneas of WT and OT-II mice at day 7 p.i. (n = 3 mice/group; two independent experiments). Data in panels B-D were analyzed using Student's T tests. https://doi.org/10.7554/eLife.48378.007 Nonhematopoietic cornea-resident cells and CSF1R+ leukocytes augment local C3 synthesis during herpetic keratitis Complement-mediated tissue pathology can arise from complement activators synthesized by the liver (systemic complement) or in tissue microenvironments (local complement). While local complement activation and regulation have been reported in the cornea in health and disease (Bora et al., 1993; Clark and Bishop, 2018; Mondino and Brady, 1981; Verhagen et al., 1992), the expression profile and cellular sources of various complement components have not been investigated in the context of ocular HSV-1 infection. To this end, a modest array of complement component transcripts was evaluated by semiquantitative real-time PCR on corneal buttons from healthy and HSV-infected mice at day 2 and 7 p.i. Upregulation of complement effectors and anaphylatoxin receptors were noted including C3, C5, C3ar1, and C5ar1 (Figure 4A,B). Constitutive expression of complement receptor 2 (CD21) has been reported in the corneal epithelium (Levine et al., 1990), but variances in its expression during HSV-1 infection were not statistically significant across the time points evaluated (Figure 4B). Likewise, no differences in the local expression of various complement regulators were observed in HSV-infected corneas aside from the C1-inhibitor SerpinG1 (Table 1, Figure 4C). Collectively, this expression profile favors local complement activation, as effectors are upregulated without proportional enhancement of pathway regulatory components. Figure 4 Download asset Open asset Corneal HSV-1 infection enhances local complement synthesis. Gene expression of complement effectors (A), receptors (B), and regulators (C) upregulated in the corneas of B6 mice during acute HSV-1 infection (n = 7 WT mice/group; two independent experiments; Kruskal-Wallis, Dunn's multiple comparisons test). (D) Relative C3 expression among selected cornea-resident and infiltrating cell subsets at day three post-infection (n = 3–4 pooled samples from two mice each for cell subsets or three independent cornea pairs; two independent experiments; one-way ANOVA, Bonferroni; ND, not detected/amplification cycle >35). Final PCR products were resolved on an agarose gel to verify amplification. Data are relative to GAPDH expression and normalized to uninfected control samples for panels A-C or to purified CSF1R-expressing peripheral blood monocytes/macrophages in panel D. https://doi.org/10.7554/eLife.48378.011 Table 1 Complement regulatory factor expression in the cornea during HSV-1 infection https://doi.org/10.7554/eLife.48378.012 GeneUninfectedDay 2 p.i.Day 7 p.i.C4bpNDNDNDCrry (CD46 homolog)1.193 ± 0.3380.817 ± 0.1721.137 ± 0.331CD55 (DAF)1.087 ± 0.2011.035 ± 0.1990.791 ± 0.251CD59a (MIRL)1.078 ± 0.1920.611 ± 0.1230.728 ± 0.191CfiNDNDNDCr1l1.026 ± 0.0960.7442 ± 0.2220.889 ± 0.133 Data are expressed as mean ± SEM; n = 7 samples/group. Gene expression is standardized to internal GAPDH expression and relative to uninfected control tissue. Abbreviations: C4bp, C4 binding protein; CD, cluster of differentiation; Cfi, complement factor I, Cr1l, complement C3b/C4b receptor-1-like; DAF, decay accelerating factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MIRL, membrane inhibitor of reactive lysis; ND, not detected (amplification cycle >35); p.i., post-infection. As C3 is the central component of the complement activation pathway, the cellular sources of complement C3 were evaluated in corneas infected with HSV-1. Monocytes and tissue macrophages are known sources of C3 (Einstein et al., 1977; Lubbers et al., 2017; Morgan and Gasque, 1997; Verschoor et al., 2001), thus CSF1R+ cells from the peripheral blood of transgenic CSF1R-GFP mice (MAFIA) were utilized as the relative standard for C3 transcript expression. Expression of C3 was noted in CD45+ leukocytes isolated from infected corneas, yet detection of C3 expression was lost when CSF1R+ cells were removed from the total CD45+ pool. In addition, isolated EpCAM+ corneal epithelial cells expressed C3 at levels comparable to blood monocytes. However, the C3 expression level was greater in whole-cornea preparations than among individual cell fractions (Figure 4D). Taken together, our data show that both tissue-resident non-hematopoietic cells and resident/infiltrating CSF1R+ leukocytes contribute to local C3 expression in the cornea during acute HSV-1 infection. Localized pharmacologic C3 depletion preserves corneal sensation in HSV-1 keratitis Ocular HSV-1 infection amplifies local complement gene expression, yet whether local control of complement activation can be harnessed to prevent corneal nerve damage has not been explored. From a clinical perspective, modulating complement activation at the ocular surface may be a viable therapeutic option. As a proof of concept, daily ocular cobra venom factor (CVF) treatment was explored as a putative method to deplete C3 and preserve corneal sensation during acute HSV-1 infection. Vehicle (PBS)-treated mice exhibited corneal sensation loss following HSV-1 infection, but CVF treatment preserved corneal sensation (Figure 5A). Protein levels of C3 remained near baseline in the cornea following CVF treatment during HSV-1 infection, yet vehicle-treated animals exhibited a 300–600% increase in C3 protein levels in the cornea at days 3 and 7 p.i. (Figure 5B). Ocular CVF treatment did not significantly impact systemic serum C3 concentrations throughout the study (Figure 5C). Figure 5 with 2 supplements see all Download asset Open asset Local C3 depletion prevents HSV-associated corneal sensation loss. B6 mice were given PBS (vehicle) or 5.0 μg cobra venom factor (CVF) via subconjunctival injection to degrade C3, and ocularly infected with HSV-1 18 hr later. C3 depletion was maintained by daily topical treatment (eyedrop) containing 0.5 μg CVF. (A) Corneal sensation following HSV-1 infection in animals treated with CVF or PBS (n = 5–11 mice/group; three independent experiments; two-way ANOVA, Bonferroni). Impact of CVF treatment on C3 protein concentrations in the cornea (B) and serum (C) (n= 3 cornea pairs, 5–9 serum samples/timepoint; 2–3 independent experiments; one-way ANOVA, Tukey). (D) Corneal edema measurements (central corneal thickness) determined via spectral domain optical coherence tomography (SD-OCT) on uninfected (UI) or HSV-1 infected mice treated with CVF or PBS at day 5 p.i. (n = 4–5 mice/group; two experiments; one-way ANOVA, Newman-Keuls). (E) Impact of CVF treatment on total leukocyte (CD45+) and monocyte/macrophage (CSF1R+) infiltration into the corneas of CVF and PBS-treated MaFIA (CSF1R-GFP) mice at day 3 p.i. (n = 5–6 mice/group; two independent experiments; Student's T). (F) Representative flow plots showing cell
The electromagnetic interference of the motor drive system has a great influence on the performance. For the motor drive systems, the interference of CM current is important cause of conducted emission. AC side of motor drive system is high voltage and load is large inductive motor, so the AC side has a big impact on the DC side in the process of rapid on-off devices. The CM EMI current paths at frequency 30 MHz are analyzed based on the distribution parameters of elements in current flowing circuit. The change of the CM EMI current will be analyzed through circuit and equation of the CM EMI current when the AC cable is added ferrite chokes or ferrite. The DC conducted emission electromagnetic interference suppression effect is obvious to add ferrite chokes or ferrite the AC cable in high frequency in the experiment, which is consistent with theoretical analysis. The results show that adding ferrite is more effective measure than adding ferrite choke to suppress the DC side conduction emission electromagnetic interference both in decreasing amplitude and larger frequency band.
Despite advances in management of immunosuppression, graft rejection remains a significant clinical problem in solid organ transplantation. Non-invasive biomarkers of graft rejection can facilitate earlier diagnosis and treatment of acute rejection. The purpose of this study was to investigate the potential role of heparan sulfate as a novel biomarker for acute cellular rejection. Heparan sulfate is released from the extracellular matrix during T-cell infiltration of graft tissue via the action of the enzyme heparanase. In a murine heart transplant model, serum heparan sulfate is significantly elevated during rejection of cardiac allografts. Moreover, expression of the enzyme heparanase is significantly increased in activated T-cells. In human studies, plasma heparan sulfate is significantly elevated in kidney transplant recipients with biopsy-proven acute cellular rejection compared to healthy controls, recipients with stable graft function, and recipients without acute cellular rejection on biopsy. Taken together, these findings support further investigation of heparan sulfate as a novel biomarker of acute cellular rejection in solid organ transplantation.
This study evaluated the short-term effects of tofacitinib treatment on peripheral blood leukocyte phenotype and function, and the reversibility of any such effects following treatment withdrawal in healthy volunteers. Cytomegalovirus (CMV)-seropositive subjects received oral tofacitinib 10 mg twice daily for 4 weeks and were followed for 4 weeks after drug withdrawal. There were slight increases in total lymphocyte and total T-cell counts during tofacitinib treatment, and B-cell counts increased by up to 26%. There were no significant changes in granulocyte or monocyte counts, or granulocyte function. Naïve and central memory T-cell counts increased during treatment, while all subsets of activated T cells were decreased by up to 69%. T-cell subsets other than effector memory cluster of differentiation (CD)4+, activated naïve CD4+ and effector CD8+ T-cell counts and B-cell counts, normalized 4 weeks after withdrawal. Following ex vivo activation, measures of CMV-specific T-cell responses, and antigen non-specific T-cell-mediated cytotoxicity and interferon (IFN)-γ production, decreased slightly. These T-cell functional changes were most pronounced at Day 15, partially normalized while still on tofacitinib and returned to baseline after drug withdrawal. Total natural killer (NK)-cell counts decreased by 33%, returning towards baseline after drug withdrawal. NK-cell function decreased during tofacitinib treatment, but without a consistent time course across measured parameters. However, markers of NK-cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity and IFN-γ production were decreased up to 42% 1 month after drug withdrawal. CMV DNA was not detectable in whole blood, and there were no cases of herpes zoster reactivation. No new safety concerns arose. In conclusion, the effect of short-term tofacitinib treatment on leukocyte composition and function in healthy CMV+ volunteers is modest and largely reversible 4 weeks after withdrawal.
BackgroundWe aim to investigate genes associated with myasthenia gravis (MG), specifically those potentially implicated in the pathogenesis of dilated cardiomyopathy (DCM). Additionally, we seek to identify potential biomarkers for diagnosing myasthenia gravis co-occurring with DCM.MethodsWe obtained two expression profiling datasets related to DCM and MG from the Gene Expression Omnibus (GEO). Subsequently, we conducted differential gene expression analysis and weighted gene co-expression network analysis (WGCNA) on these datasets. The genes exhibiting differential expression common to both DCM and MG were employed for protein-protein interaction (PPI), Gene Ontology (GO) enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Additionally, machine learning techniques were employed to identify potential biomarkers and develop a diagnostic nomogram for predicting MG-associated DCM. Subsequently, the machine learning results underwent validation using an external dataset. Finally, gene set enrichment analysis (GSEA) and machine algorithm analysis were conducted on pivotal model genes to further elucidate their potential mechanisms in MG-associated DCM.ResultsIn our analysis of both DCM and MG datasets, we identified 2641 critical module genes and 11 differentially expressed genes shared between the two conditions. Enrichment analysis disclosed that these 11 genes primarily pertain to inflammation and immune regulation. Connectivity map (CMAP) analysis pinpointed SB-216763 as a potential drug for DCM treatment. The results from machine learning indicated the substantial diagnostic value of midline 1 interacting protein1 (MID1IP1) and PI3K-interacting protein 1 (PIK3IP1) in MG-associated DCM. These two hub genes were chosen as candidate biomarkers and employed to formulate a diagnostic nomogram with optimal diagnostic performance through machine learning. Simultaneously, single-gene GSEA results and immune cell infiltration analysis unveiled immune dysregulation in both DCM and MG, with MID1IP1 and PIK3IP1 showing significant associations with invasive immune cells.ConclusionWe have elucidated the inflammatory and immune pathways associated with MG-related DCM and formulated a diagnostic nomogram for DCM utilizing MID1IP1/PIK3IP1. This contribution offers novel insights for prospective diagnostic approaches and therapeutic interventions in the context of MG coexisting with DCM.
3083 Background: PG545 (pixatimod, pINN) is a novel immunomodulatory agent which stimulates dendritic cells (DC) via TLR9/IL-12 pathway to activate natural killer (NK) cells. It also inhibits tumour-associated macrophages in cancer models. We report on safety, PK, PD, and antitumor activity of PG545 monotherapy. Methods: In this dose escalation (3+3 design) study, eligible pts (ECOG≤1) with advanced solid malignancies who failed standard therapies received PG545 once weekly as a 1-hour i.v. infusion until disease progression or discontinuation due to intolerability. The primary objective was determination of the maximum tolerated dose (MTD). Secondary objectives evaluated safety, antitumor activity based on RECIST (1.1) criteria, PK and PD (plasmacytoid DC & NKp46 + NK cells from PBMC, and plasma cytokines/chemokines). Results: The study recruited 23 subjects across four cohorts (25, 50, 100 & 150 mg). Three dose limiting toxicities (DLTs) - hypertension (2), epistaxis (1) - occurred in the 150 mg cohort, which was identified as a non-tolerated dose level. No DLTs occurred in the 100 mg cohort, which was identified as the MTD. Six SAEs were reported to be possibly or likely related to PG545 treatment. No RECIST 1.1 objective responses were reported; best response was prolonged stable disease up to 24 weeks (mCRC), with disease control rate in evaluable subjects of 38% (6/16) at eight weeks. Exposure (AUC 0-last ) was proportional up to 100mg and mean half-life was 144 hours. At 50 and 100mg dose levels, two subjects in each cohort exhibited up to 4-fold increased numbers of NKp46 + NK cells, IFN-α-producing pDCs, and increases (up to 25-fold) in plasma IFN-γ, TNF-α, IP-10 and MCP-1. Conclusions: PG545 is well tolerated up to 100 mg once-weekly via i.v. infusion. Human exposure data at 50mg and 100mg reach exposures consistent with those required for preclinical efficacy. Preliminary PD data support the proposed mechanism of action, which represents a promising approach to improve the efficacy of existing therapies. These data, and the absence of toxicities associated with chemo- or immunotherapies, support the development of PG545 in combination clinical trials. Clinical trial information: NCT02042781.
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