Human gut microbiota stimulate defined innate immune responses that vary from phylum to strain.

The potential of commensal bacteria to modulate host immunity remains largely uncharacterized, largely due to the vast number of strains that comprise the human gut microbiota. We have developed a screening platform to measure the innate immune responses of myeloid cells to 277 bacterial strains isolated from the gut microbiota of healthy individuals and those with inflammatory bowel diseases. The innate immune responses to gut-derived bacteria are as strong as those toward pathogenic bacteria, and they vary from phylum to strain. Myeloid cells differentially rely upon innate receptors TLR2 or TLR4 to sense taxa, with differential sensing of Bacteroidetes and Proteobacteria that predict in vivo functions. These innate immune responses can be modeled using combinations of up to 8 Toll-like receptor (TLR) agonists. Furthermore, the immunogenicity of strains is stable over time and following fecal microbiota transplantation into new human recipients. Collectively, this high-throughput approach provides an insight into how commensal microorganisms shape innate immune phenotypes.

Limited intestinal inflammation despite diarrhea, fecal viral RNA and SARS-CoV-2-specific IgA in patients with acute COVID-19.

Gastrointestinal symptoms are common in COVID-19 patients but the nature of the gut immune response to SARS-CoV-2 remains poorly characterized, partly due to the difficulty of obtaining biopsy specimens from infected individuals. In lieu of tissue samples, we measured cytokines, inflammatory markers, viral RNA, microbiome composition, and antibody responses in stool samples from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.

Limited intestinal inflammation despite diarrhea, fecal viral RNA and SARS-CoV-2-specific IgA in patients with acute COVID-19.

We sought to characterize the role of the gastrointestinal immune system in the pathogenesis of the inflammatory response associated with COVID-19. We measured cytokines, inflammatory markers, viral RNA, microbiome composition and antibody responses in stool from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.

Fecal IgA Levels Are Determined by Strain-Level Differences in Bacteroides ovatus and Are Modifiable by Gut Microbiota Manipulation.

Fecal IgA production depends on colonization by a gut microbiota. However, the bacterial strains that drive gut IgA production remain largely unknown. Here, we assessed the IgA-inducing capacity of a diverse set of human gut microbial strains by monocolonizing mice with each strain. We identified Bacteroides ovatus as the species that best induced gut IgA production. However, this induction varied bimodally across different B. ovatus strains. The high IgA-inducing B. ovatus strains preferentially elicited more IgA production in the large intestine through the T cell-dependent B cell-activation pathway. Remarkably, a low-IgA phenotype in mice could be robustly and consistently converted into a high-IgA phenotype by transplanting a multiplex cocktail of high IgA-inducing B. ovatus strains but not individual ones. Our results highlight the critical importance of microbial strains in driving phenotype variation in the mucosal immune system and provide a strategy to robustly modify a gut immune phenotype, including IgA production.

Acanthamoeba Keratitis: Are Recent Cases More Severe?

PURPOSE: To identify a recent change in disease severity and visual outcomes of patients with Acanthamoeba keratitis (AK) at Wills Eye Hospital (WEH). METHODS: A retrospective chart review was performed of all cases diagnosed with AK at WEH between January 1, 2009, and December 31, 2015. Failure of treatment was defined as having a final best-corrected visual acuity worse than 20/100 and/or requiring keratoplasty. The patients were grouped into 2 outcome categories: successfully treated and those who failed treatment; and prognostic factors associated with failure status were evaluated. Additionally, patients who presented from 2009 to 2012 (labeled "first interval") were compared with those who presented more recently from 2013 to 2015 (labeled "second interval") to determine whether the treatment outcomes and patients' characteristics from the 2 intervals were different. RESULTS: Fifty-nine patients were diagnosed. The rate of treatment failure in the second interval was significantly higher than in the first interval (68% vs. 28%, P = 0.004). Compared with the patients in the first interval, those who presented in the second interval were significantly older, were treated with more corticosteroids before diagnosis, had more days from the symptom onset to initiation of AK treatment, and presented to WEH with poorer vision. CONCLUSIONS: In recent years, the general profile of patients with AK has changed at WEH. Currently, we are treating older patients with more severe keratitis who are presenting later and with worse vision compared with our previous patients. At the same time, treatment outcomes have been poorer.

Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability.

Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.

Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism.

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm.

Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.

AMG 595, an Anti-EGFRvIII Antibody-Drug Conjugate, Induces Potent Antitumor Activity against EGFRvIII-Expressing Glioblastoma.

Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 µg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients.

Recombinant human lecithin-cholesterol acyltransferase Fc fusion: analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region.

Recombinant human lecithin-cholesterol acyltransferase Fc fusion (huLCAT-Fc) is a chimeric protein produced by fusing human Fc to the C-terminus of the human enzyme via a linker sequence. The huLCAT-Fc homodimer contains five N-linked glycosylation sites per monomer. The heterogeneity and site-specific distribution of the various glycans were examined using enzymatic digestion and LC-MS/MS, followed by automatic processing. Almost all of the N-linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N-linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N-linked site exclusively contain typical asialobiantennary structures. HuLCAT-Fc was also confirmed to have mucin-type glycans attached at T407 and S409 . When LCAT-Fc fusions were constructed using a G-S-G-G-G-G linker, an unexpected +632 Da xylose-based glycosaminoglycan (GAG) tetrasaccharide core of Xyl-Gal-Gal-GlcA was attached to S418 . Several minor intermediate species including Xyl, Xyl-Gal, Xyl-Gal-Gal, and a phosphorylated GAG core were also present. The mucin-type O-linked glycans can be effectively released by sialidase and O-glycanase; however, the GAG could only be removed and localized using chemical alkaline ß-elimination and targeted LC-MS/MS. E416 (the C-terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O-xylosyltransferase. HuLCAT-Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPPE416 GS418 GGGGDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.

Retinal pigment epithelial trough: a sign of chronicity of choroidal nevi.

PURPOSE: To describe the clinical features and natural course of choroidal nevi with retinal pigment epithelial (RPE) trough. METHODS: Retrospective observational case series. RESULTS: Of the 90 patients (91 choroidal nevi), 88 (98%) were Caucasian and 59 (66%) were women, with a mean age of 56 years (range 18-79 years) at presentation. The mean largest nevus basal diameter was 7 mm (range 3-12 mm) and the mean thickness was 2.2 mm (range 0.8-3.4 mm). Orange pigment and subretinal fluid were associated with 9 (10%) and 16 (18%) of the nevi, respectively. Overlying RPE alterations included RPE atrophy (93%), RPE hyperplasia (74%), drusen (54%), and RPE fibrous metaplasia (36%). Retinal pigment epithelial trough was present at initial evaluation in 88 eyes and developed during follow-up from a long-standing pocket of shallow subretinal fluid in 3 other eyes. The mean largest dimension of the RPE trough was 4 mm (range 1-16 mm) and most (76%) were located at the inferior margin of the nevus. Of 91 nevi, 82 had subsequent follow-up with a mean follow-up duration of 79 months (range 3-464 mo). Of these, 19 (23%) showed slight growth and only 3 (4%) had presumed transformation into melanoma. None of the patients showed evidence of distant metastasis. CONCLUSIONS: Retinal pigment epithelial trough results from resorption of long-standing serous subretinal fluid and should therefore be considered a sign of chronicity of choroidal nevi. Choroidal nevi associated with RPE trough, despite their greater thickness and larger size, are relatively stable with minimal risk of progression into melanoma.

Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31.

Interleukin-31 (IL-31) is a member of the four helical-bundle gp130/IL-6 cytokine family. Despite its implicated roles in inflammatory diseases, the biosynthetic processes of IL-31 have been poorly investigated. A detailed understanding of IL-31 biosynthesis and the nature of ligand-receptor interactions can provide insights into effective strategies for the design of therapeutic approaches. By using various heterologous protein expression systems, we demonstrated that murine IL-31 was secreted as inter-molecularly disulfide-bonded covalent aggregates. Covalently aggregated IL-31 appeared while trafficking in the secretory pathway, but was not actively retained in the ER. The aggregate formation was not caused by a dysfunctional ER quality control mechanism or an intrinsic limitation in protein folding capacity. Furthermore, secreted IL-31 aggregates were part of a large complex composed of various pleiotropic secretory factors and immune-stimulators. The extent and the heterogeneous nature of aggregates may imply that IL-31 was erroneously folded, but it was capable of signaling through cognate receptors. Mutagenesis revealed the promiscuity of all five cysteines in inter-molecular disulfide formation with components of the hetero-aggregates, but no cysteine was required for IL-31 secretion itself. Our present study not only illustrated various functions that cysteines perform during IL-31 biosynthesis and secretion, but also highlighted their potential roles in cytokine effector functions.

Slow enlargement of choroidal nevi: a long-term follow-up study.

PURPOSE: Choroidal nevi are generally considered to be stable lesions, and growth of a choroidal nevus is usually believed to be a sign of malignant transformation. We performed this study to determine whether choroidal nevi enlarge over a long period of follow-up without undergoing malignant transformation. DESIGN: Retrospective observational case series. PARTICIPANTS: A total of 278 patients with 284 nevi who had at least 7 years of photographic follow-up without clinical signs of transformation into melanoma were included in the study. METHODS: Data on demographic and clinical information were extracted from patients' charts. Detailed fundus drawings and color fundus photographs were reviewed and compared for evidence of enlargement. MAIN OUTCOME MEASURES: Nevus enlargement without clinical evidence of transformation into melanoma. RESULTS: Of the 278 patients, 69% were female and more than 99% were White with a median age at presentation of 57 years (range, 4-87 years). The largest nevus basal diameter was a median of 5 mm (range, 0.5-14 mm), and the median thickness was 1.5 mm (range, 0.1-3.6 mm). Only 14 nevi (5%) had subretinal fluid outside the nevus, and 6% showed overlying orange pigment. Overlying retinal pigment epithelial alterations included drusen (61%), atrophy (6%), hyperplasia (10%), and fibrous metaplasia (6%). Of 284 nevi, 31% showed slight enlargement over a mean follow-up of 15 years. The median increase in diameter was 1 mm (mean, 0.9 mm; range, 0.2-3.0 mm), and the median rate of enlargement was 0.06 mm/yr (mean, 0.06 mm/yr; range, 0.01-0.36 mm/yr). None of the lesions that enlarged developed new risk factors that are generally associated with malignant transformation. Frequency of enlargement was 54% in patients aged less than 40 years and 19% in patients aged more than 60 years. On multivariate analysis, younger patient age was the only factor predictive of nevus enlargement (P<0.001). CONCLUSIONS: With long-term follow up, 31% of choroidal nevi showed slight enlargement without clinical evidence of transformation into melanoma. The frequency of enlargement was inversely related to patient age.

Fusion partners as a tool for the expression of difficult proteins in mammalian cells.

The expression of proteins which do not express well on their own can be enhanced by linking them to human serum albumin (HSA) or antibody crystallizable fragment (Fc). The constructs shown here are designed to secrete the proteins after transient transfection of mammalian cell lines. The fusion partners are appended to the N-terminus of the proteins and contain a linker designed to be proteolytically cleaved. Transient transfection and purification protocols are provided as well as experimental results with five interleukins.

In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells.

The expression levels of five secreted target interleukins (IL-11, 15, 17B, 32, and IL23 p19 subunit) were tested with three different fusion partners in 2936E cells. When fused to the N-terminus, human serum albumin (HSA) was found to enhance the expression of both IL-17B and IL-15, cytokines which did not express at measurable levels on their own. Although the crystallizable fragment of an antibody (Fc) was also an effective fusion partner for IL-17B, Fc did not increase expression of IL-15. Fc was superior to HSA for the expression of the p19 subunit of IL-23, but no partner led to measurable levels of IL-32gamma secretion. Glutathione S-transferase (GST) did not enhance the expression of any target and suppressed the production of IL-11, a cytokine which expressed robustly both on its own and when fused to HSA or Fc. Cleavage of the fusion partner was not always possible. The use of HSA or Fc as N-terminal fusions can be an effective technique to express difficult proteins, especially for applications in which the fusion partner need not be removed.

The IL-1 receptor accessory protein (AcP) is required for IL-33 signaling and soluble AcP enhances the ability of soluble ST2 to inhibit IL-33.

Interleukin (IL)-33 (or IL-1F11) was recently identified as a ligand for the orphan IL-1 receptor family member T1/ST2 (ST2). IL-33 belongs to the IL-1 cytokine family and, upon binding to ST2, induces intracellular signals similar to those utilized by IL-1. The effects of other IL-1 family cytokines are mediated by their binding to a specific receptor and the recruitment of a co-receptor required for elicitation of signaling. The aim of this study was to characterize the co-receptor involved in IL-33 signaling. Immunoprecipitation confirmed that IL-33 specifically binds ST2 and revealed that cellular IL-1 receptor accessory protein (AcP) associates with ST2 in a ligand-dependent manner. Receptor binding measurements demonstrated that the affinity of mouse (m)IL-33 for ST2 is increased by 4-fold in presence of AcP. IL-33 dose-dependently stimulated IL-6 secretion from wild-type (WT) mast cells, while no effect of IL-33 was observed with mast cells derived from AcP-deficient mice. Finally, soluble (s)ST2-Fc and sAcP-Fc acted synergistically to inhibit IL-33 activity. These observations identify AcP as a shared co-receptor within the IL-1 family that is essential for IL-33 signaling and suggest a novel role for sAcP in modulating the activity of IL-33.