Business Development

Greater Spokane Incorporated has promoted Cassidy Peterson to director of education and talent. She joined GSI in 2016 and has previously served as education and talent program coordinator and career connected learning program manager.

Daniel Stewart, professor of entrepreneurship and director of the Hogan Entrepreneurial Leadership Program in the Gonzaga University School of Business, has co-edited the book Creating Private Sector Economies in Native America: Sustainable Development through Entrepreneurship. The textbook is intended to be adopted in upper-division and graduate-level courses covering economics, business and tribal law.

The University of Washington School of Medicine and Gonzaga University jointly have hired Jake Deckert to teach medical students and Gonzaga undergraduates. He will teach first-year UW medical student courses in immunology, biochemistry and cellular physiology. In spring, Deckert will be a full-time instructor in Gonzagas human physiology program.

Life Care Center of Post Falls has rehired Stephanie Bonanzino as senior executive director. She served in the role previously, and worked most recently as senior executive director at Life Care Center of Coeur dAlene.

The Association of Washington Business recently presented its top honors to six employers during its annual Evening of Excellence event, granting Avista the leading environmental practices award. The honor recognizes businesses for putting a priority on environmental improvement, education and outreach.

The Spokane Estate Planning Council has been awarded the highest honor available to an estate planning council affiliated with the National Association of Estate Planners and Councils, the Leonard H. Neiman and Walter Lee Davis Jr. Council of Excellence Award. The award recognizes councils that provide an exceptional member experience, work to grow their programs and services and are successful in providing a multidisciplinary environment for estate planning professionals within their community.

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Business Beat - The Spokesman-Review

Putting JNK1 on the immunodeficiency map

Impaired TH17 immunity is the shared element among the group of inherited immunodeficiencies associated with chronic mucocutaneous candidiasis (CMC). Li et al. studied three patients from a single family who had CMC associated with an atypical form of connective tissue disorder with some features of Ehlers-Danlos syndrome. Whole-exome sequencing identified a previously unreported loss-of-function splice-site mutation in the MAPK8 gene encoding c-Jun N-terminal kinase 1 (JNK1) that causes JNK1 haploinsufficiency with autosomal dominant inheritance. The complex clinical phenotype in these patients results from defects in signaling downstream of both IL-17 and TGF- cytokines. These findings demonstrate that JNK1-mediated signaling plays a critical role in maintaining normal immunity to Candida as well as supporting TGF-dependent homeostasis of connective tissues.

Genetic etiologies of chronic mucocutaneous candidiasis (CMC) disrupt human IL-17A/Fdependent immunity at mucosal surfaces, whereas those of connective tissue disorders (CTDs) often impair the TGF-dependent homeostasis of connective tissues. The signaling pathways involved are incompletely understood. We report a three-generation family with an autosomal dominant (AD) combination of CMC and a previously undescribed form of CTD that clinically overlaps with Ehlers-Danlos syndrome (EDS). The patients are heterozygous for a private splice-site variant of MAPK8, the gene encoding c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway. This variant is loss-of-expression and loss-of-function in the patients fibroblasts, which display AD JNK1 deficiency by haploinsufficiency. These cells have impaired, but not abolished, responses to IL-17A and IL-17F. Moreover, the development of the patients TH17 cells was impaired ex vivo and in vitro, probably due to the involvement of JNK1 in the TGF-responsive pathway and further accounting for the patients CMC. Consistently, the patients fibroblasts displayed impaired JNK1- and c-Jun/ATF-2dependent induction of key extracellular matrix (ECM) components and regulators, but not of EDS-causing gene products, in response to TGF-. Furthermore, they displayed a transcriptional pattern in response to TGF- different from that of fibroblasts from patients with Loeys-Dietz syndrome caused by mutations of TGFBR2 or SMAD3, further accounting for the patients complex and unusual CTD phenotype. This experiment of nature indicates that the integrity of the human JNK1-dependent MAPK signaling pathway is essential for IL-17A and IL-17Fdependent mucocutaneous immunity to Candida and for the TGF-dependent homeostasis of connective tissues.

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent lesions of the skin, nails, oral, and genital mucosae caused by Candida albicans (1). Patients with profound and broad inherited T cell immunodeficiencies present with CMC as one of their many infections (2). Most patients heterozygous for dominant-negative STAT3 mutations (3) or gain-of-function STAT1 mutations (4), and most patients with autosomal recessive (AR) RORC (5) or ZNF341 deficiency (6, 7) have CMC among the infections suffered, the range of which is smaller than for patients with severe T cell deficiencies. Patients with these various forms of syndromic CMC (SCMC) share a paucity of circulating T helper 17 (TH17) cells (513). Patients with AR autoimmune regulator (AIRE) deficiency display not only autoimmunity but also CMC as their only infection due to the production of neutralizing autoantibodies against interleukin-17A (IL-17A) and/or IL-17F (14, 15). Last, isolated forms of CMC (ICMC), in which CMC is the predominant or only clinical manifestation in otherwise healthy individuals, can be due to autosomal dominant (AD) IL-17F deficiency, or inborn errors of the IL-17responsive pathway, such as AR IL-17RA, IL-17RC, and ACT1 deficiencies (1620). Fibroblasts and keratinocytes derived from these patients display impaired (AD IL-17F deficiency) (16) or abolished (AR IL-17RA, IL-17RC, or ACT1 deficiency) responses to IL-17A and IL-17F (1619).

Patients with inherited ICMC do not, however, display any overt signs of connective tissue disorders (CTDs) as their skin, joints, bones, and blood vessels are unaffected. Conversely, patients with CTDs, such as Ehlers-Danlos syndrome (EDS), Loeys-Dietz syndrome (LDS), and Marfan syndrome (MS), do not suffer from CMC (21). Whereas the genetic basis of hypermobile EDS (hEDS) is unknown (22), the other 13 subtypes of EDS are caused by various inborn errors of genes, many of which encode collagen or collagen-modifying enzymes (e.g., COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, ADAMTS2, and PLOD1) (22, 23). LDS is caused by inborn errors of the transforming growth factor (TGF-) signaling pathway (TGFBR1, TGFBR2, SMAD2, SMAD3, TGFB2, and TGFB3) (24), and MS by inborn errors of FBN1, which encodes fibrillin-1 (25). In these disorders, the homeostasis and integrity of connective tissues are impaired by dysfunctional extracellular matrix (ECM) proteins, the production of which is controlled by TGF- in fibroblasts (24, 26).

We studied three patients (P1, P2, and P3) from three generations of a French family with AD CMC and a CTD overlapping with hEDS (Fig. 1A, fig. S1A, table S1, and the Case reports section in the Supplementary Materials). We performed whole-exome sequencing (WES) and found no rare nonsynonymous coding variants in any of the known CMC-, EDS-, LDS-, and MS-causing genes, all of which were well covered by WES (table S2). Under a complete penetrance model, we found 18 heterozygous nonsynonymous variants common to the three patients and private to this family, i.e., not previously reported in the 1000 Genomes Project, the Single-Nucleotide Polymorphism Database, the National Heart, Lung, and Blood Institute (NHLBI) GO Exome Sequencing Project, the Exome Aggregation Consortium Genome Aggregation Database, the NHLBIs TOPMed program (Bravo), or our in-house database of more than 6000 exomes from patients with various infectious diseases (fig. S1B and table S3). The most plausible candidate was a splice-site mutation in the MAPK8 gene, for which the biological distance to six of eight known SCMC- and ICMC-causing genes other than AIRE (IL17F, IL17RA, ACT1, STAT1, STAT3, and RORC) was shortest in the human gene connectome, and the distance to the other two (IL17RC and ZNF341) ranked second shortest (27, 28). The familial segregation of this private mutant MAPK8 allele was consistent with a fully penetrant AD trait (Fig. 1, A and B). This nucleotide substitution (c.311+1G>A), 1 base pair (bp) downstream from exon IV (Fig. 1C), was predicted to affect splicing by altering the donor splice site (29). The c.311+1G>A mutation has a combined annotation-dependent depletion (CADD) score of 26 (30), which is above the mutation significance cutoff (MSC) threshold of 19.034 for MAPK8 (fig. S1C) (31). Moreover, three of four nonsense or frameshift mutations in MAPK8 present in public databases have a minor allele frequency (MAF) of <105, whereas the fourth, with a MAF of 0.0000114, has a CADD score below the MSC threshold (fig. S1C). Consistent with these findings, MAPK8 has a gene damage index of 0.32 (32), a neutrality index of 0.06 (33), and a SnIPRE f parameter of 0.329 (within the top 11% of genes within the genome subject to the greatest constraints) (fig. S1D) (34), indicating that this gene is highly conserved in human populations and has evolved under purifying selection. Last, MAPK8 has a probability of loss-of-function intolerance score of 0.98, which is greater than the threshold of 0.9, above which genes are considered to be extremely intolerant to loss-of-function variants (35). The MAPK8 mutation found in this kindred was therefore probably deleterious, with the potential to cause an AD disease.

(A) Pedigree and segregation of the MAPK8 mutation. The patients, indicated with filled black symbols, are heterozygous for the mutation. E? indicates individuals whose genetic status could not be evaluated. (B) Electropherograms of partial sequences of MAPK8 corresponding to the mutation in a healthy control (C) and four members of the kindred (II.1, P1, P2, and P3). (C) Schematic illustration of the genomic locus and of the protein encoded by the MAPK8 gene extracted from the Ensembl database. It has 13 exons (I to XIII), 12 of which are coding exons (II to XIII), encoding four isoforms (JNK11, JNK12, JNK11, and JNK12), with alternative usage of exon VII or VIII and alternative splicing of exon XIII. The red arrow indicates the position of the mutation.

The MAPK8 gene encodes c-Jun N-terminal kinase 1 (JNK1), one of three members of the JNK family. This protein is a component of the mitogen-activated protein kinase (MAPK) pathway that converts extracellular stimuli into cellular responses (36, 37). JNK1 is phosphorylated by upstream MAPK kinases and, in turn, phosphorylates downstream activator protein-1 (AP-1) transcription factors, including c-Jun and ATF-2 (37). There are two long (JNK12 and JNK12, 54 kDa) and two short (JNK11 and JNK11, 46 kDa) isoforms, generated by alternative usage of exon VII or VIII and alternative splicing of exon XIII (Fig. 1C) (38). We amplified a complementary DNA (cDNA) fragment extending from exons III to V from Epstein-Barr virus (EBV)transformed B cells and simian virus 40 (SV40)transformed fibroblasts from the patients. In addition to the wild-type (WT) transcript (band 4), we detected four aberrant products (bands 1, 2, 3, and 5) (Fig. 2A). TA cloning and subsequent sequencing identified two aberrantly spliced transcripts: one in which intron IV was retained (band 2) and one in which exon IV was skipped (band 5) (Fig. 2A). Bands 1 and 3 were artifacts of heteroduplex formation (39). We then inserted a genomic fragment containing the WT or mutant intron IV together with the surrounding exons (IV and V) into an exon-trapping vector (Fig. 2B). The WT minigene was normally spliced, whereas the mutant minigene generated two aberrant splicing products: one in which exon IV was skipped and another in which intron IV was retained (Fig. 2B). This assay confirmed the direct impact of the c.311+1G>A mutation on MAPK8 mRNA splicing, with no detectable leakiness. Both aberrant mRNAs were predicted to result in the creation of premature stop codons (Fig. 2C). Consistent with this prediction, the levels of WT MAPK8 mRNA and JNK1 protein in the patients cells were about half those in control cells (Fig. 2, D and E). Moreover, no truncated proteins were detected in the patients cells (Fig. 2E) or in human embryonic kidney (HEK) 293T cells transfected with the corresponding mutant constructs, with or without the N-terminal Myc tag (Fig. 2F). The three patients were, therefore, heterozygous for a private loss-of-expression MAPK8 allele.

(A) MAPK8 mRNA levels in EBV-B cells and SV40-fibroblasts from healthy controls (C1, C2, and C3) and patients (P1, P2, and P3). TA cloning and subsequent sequencing of the five bands generated by amplification from exon III to exon V identified three spliced transcripts: band 1 corresponding to the WT sequence together with intron IV retention and exon IV skipping; band 2 (376 bp) corresponding to intron IV retention; band 3 corresponding to the WT sequence together with exon IV skipping; band 4 (284 bp) corresponding to the WT sequence; band 5 (225 bp) corresponding to exon IV skipping. (B) Schematic diagram of the constructs used for exon trapping. pET01, exon-trapping vector; RSV, Rous sarcoma virus long terminal repeat promoter; pA, polyadenylation; E in black, exon of the pET01 vector; IV and V in blue, MAPK8 exons IV and V; in yellow, MAPK8 intron IV. The red arrow indicates the position of the mutation. Reverse transcription PCR and subsequent sequencing identified three spliced transcripts: band 1 corresponding to intron IV retention and exon IV skipping; band 2 (354 bp) corresponding to the WT sequence; band 3 (295 bp) corresponding to exon IV skipping. (C) Schematic illustration of the mutant proteins. JNK1ES (JNK1 exon skipping) represents exon IV skipping, whereas JNK1IR (JNK1 intron retention) denotes intron IV retention. Both transcripts are predicted to encode proteins of about 10 kDa in size. Red arrows indicate the positions of premature stop codons. (D) mRNA levels for MAPK8 isoforms in EBV-B cells (top) and SV40-fibroblasts (bottom) from healthy controls (C1, C2, and C3) and patients (P1, P2, and P3). Quantitative reverse transcription PCR was performed with primers specific for JNK11/JNK12 and JNK11/JNK12 mRNAs. /, total mRNA corresponding to JNK11, JNK12, JNK11, and JNK12; , total mRNA corresponding to JNK11 and JNK12; , total mRNA corresponding to JNK11 and JNK12. The values shown are the means SEM of three independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001, unpaired t tests. (E and F) Immunoblot of JNK1 in EBV-B cells and SV40-fibroblasts from healthy controls (C1, C2, and C3) and patients (P1, P2, and P3) (E), and in HEK293T cells transfected with plasmids encoding four WT JNK1 isoforms (1, 2, 1, and 2) and two mutants (ES and IR) inserted into the pTRIP-SFFV vector or the pCMV6-AN-Myc-DDK vector (F). Endogenous JNK1 was detected with an anti-JNK1 antibody recognizing the N terminus of JNK1. Myc-tagged JNK1 was detected with an anti-Myc antibody. EV, empty vector. The data shown are representative of three independent experiments (A, B, E, and F).

Human IL-17A, IL-17F, and IL-17A/F (referred to collectively as IL-17A/F) can activate JNK1 after binding to IL-17RA/IL-17RC, which is mostly expressed in various nonhematopoietic cells, thereby inducing the production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides (40, 41). Upon stimulation with IL-17A/F, SV40-fibroblasts from the patients produced abnormally small amounts of growth-regulated oncogene- (GRO-) and IL-6, whereas SV40-fibroblasts from an IL-17RAdeficient patient did not respond at all (Fig. 3A). Similar results were obtained with primary fibroblasts (fig. S2A). The patients cells had subnormal-to-normal responses to tumor necrosis factor (TNF-) and IL-1 (Fig. 3B and fig. S2B). Moreover, the activation of AP-1 (c-Jun/ATF-2), unlike that of extracellular signalregulated kinase 1/2 (ERK1/2), p38, and nuclear factor B (NF-B), was impaired in the patients SV40-fibroblasts after stimulation with IL-17A, as shown by Western blotting (fig. S2C). By contrast, AP-1 was normally activated by TNF- and IL-1 (fig. S2D). Fibroblasts and leukocytes from the patients also responded normally to lymphotoxin 12 (IL-8 production) and Toll-like receptor agonists (IL-6 and IL-8 production), respectively (fig. S2, E to G). Peripheral blood mononuclear cells (PBMCs) responded normally to IL-2 in combination with IL-17E (IL-5 production) (fig. S2H). Lentiviral transduction of the patients SV40-fibroblasts with cDNAs encoding WT JNK1 isoforms, JNK11 and JNK11 in particular, but not with any of the mutant isoforms, restored the response to IL-17A (Fig. 3C and fig. S2I). This finding is consistent with the predominant protein expression of JNK11 and JNK11 in control SV40-fibroblasts (Fig. 2E). Moreover, the induction of GRO- and IL-6 in control SV40-fibroblasts was not affected by the overexpression of any mutant JNK1 isoform, suggesting that the mutant allele is not dominant negative (Fig. 3C and fig. S2I). This is consistent with the purifying selection exerted on the MAPK8 locus (fig. S1D) (34). By contrast, the RNA interference (RNAi)mediated knockdown of MAPK8 impaired the response to IL-17A in control fibroblasts (Fig. 3D and fig. S2, J and K). Last, we performed RNA sequencing (RNA-Seq) to delineate the range of IL-17Aresponsive genes in primary fibroblasts. The number of up-regulated or down-regulated genes in response to IL-17A was much lower in the patients (fig. S2L). Several IL-17A/F target genes, including CXCL1, CXCL2, IL6, IL8, C3, and ICAM1, were less induced in the patients cells (fig. S2M). About 60% of IL-17RA/IL-17RCdependent genes were JNK1 dependent (fig. S2N). Collectively, these findings indicate that heterozygosity for the private MAPK8 c.311+1G>A loss-of-expression variant underlies a distinctive AD cellular phenotype, with impaired responses to IL-17A/F in fibroblasts, by haploinsufficiency. Moreover, impaired cellular responses to IL-17A/F in fibroblasts, and possibly in other cells, contribute to CMC (42, 43).

(A) Production of GRO- (top) and IL-6 (bottom) by SV40-fibroblasts from healthy controls (C1 and C2), patients (P2 and P3), and an IL-17RAdeficient (IL17RA/) patient (16) stimulated with IL-17A, IL-17F, or IL-17A/F (10, 100, or 1000 ng/ml) for 24 hours. (B) Production of GRO- (top) and IL-6 (bottom) by SV40-fibroblasts from healthy controls (C1 and C2), patients (P2 and P3), and a NEMO-deficient (NEMO/) patient (92) stimulated with TNF- (20 ng/ml) or IL-1 (10 ng/ml) for 24 hours. (C) Production of GRO- (top) and IL-6 (bottom) by SV40-fibroblasts from healthy controls (C1 and C2) and patients (P2 and P3) transfected with empty vector (EV) or plasmids encoding WT JNK11 (1), JNK12 (2), JNK11 (1), JNK12 (2), all four isoforms (1/2/1/2), JNK1ES (ES), or JNK1IR (IR) in the presence of IL-17A (100 ng/ml) for 24 hours. (D) Production of GRO- (left) and IL-6 (right) by primary fibroblasts from healthy controls (C1, C2, and C3) transfected with control siRNA (50 nM) or MAPK8 siRNA (50 nM) for 24 hours and then stimulated with IL-17A (100 ng/ml) for an additional 24 hours. The values shown are the means SEM of three independent experiments (A to D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, unpaired t tests (A to D).

Given that mouse JNK1 is important for T cell activation and differentiation (4446), and that human TGF- activates JNK1 (47) and is essential for TH17 differentiation in vitro (4850), we also investigated the development and function of T cells in the patients, testing the hypothesis that impaired TH17 development in the patients might also contribute to their CMC. The frequencies of nave and CD45RA+ effector memory CD4+ and CD8+ T cells in the patients were slightly higher, whereas those of central and effector memory CD4+ and CD8+ T cells were correspondingly slightly lower than those in healthy controls (Fig. 4A). The patients had higher proportions of TH1 cells and lower proportions of TH17 cells than controls but normal proportions of the TH2, TH1*, TFH (T follicular helper), and (Treg) (T regulatory) subsets among circulating CD4+ T cells, as shown by flow cytometry (Fig. 4B) (51). Normal amounts of IL-17A and IL-22 were secreted by whole blood stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Fig. 4C). Ex vivo memory CD4+ T cells also expressed IL-17A and IL-17F, albeit in the lower part of the control range, and interferon- (IFN-) after stimulation with T cell activation and expansion (TAE) beads (anti-CD2/CD3/CD28 monoclonal antibodyconjugated beads) and PMA plus ionomycin (Fig. 4D). The patients nave CD4+ T cells produced less IL-17A and IL-17F than control cells when cultured under TH17-polarizing conditions (Fig. 4, E and F). This difference was more pronounced when memory CD4+ T cells were tested under the same conditions (Fig. 4G). Last, the percentages of transitional, nave, and memory B cells and of class-switched memory B cells were normal in these patients (fig. S3, A and B). The abilities of nave and memory B cells to differentiate into antibody-secreting cells were also intact (fig. S3, C and D). Overall, the ability of T cells to produce IL-17A and IL-17F was 50% lower (ex vivo) and 75% lower (in vitro) in patients heterozygous for the MAPK8 mutation. The ex vivo development of Treg cells was largely unaffected, consistent with the absence of overt autoimmunity in the patients. The CMC in these patients is, thus, a combined consequence of lower proportions of TH17 cells and impaired cellular responses to IL-17A/F. Both human IL-17A/F and IL-17RA/IL-17RCdependent mucocutaneous immunity to C. albicans are, therefore, dependent on JNK1.

(A) Percentage of total, nave (CCR7+CD45RA+), central memory (CM; CCR7+CD45RA), effector memory (EM; CCR7CD45RA), or CD45RA+ effector memory (EMRA; CCR7CD45RA+) CD4+ and CD8+ T cells from healthy controls (n = 40) and patients (P2 and P3). (B) Frequency of TH1 (CXCR5CXCR3+CCR6), TH2 (CXCR5CXCR3CCR6CCR4+), TH17 (CXCR5CXCR3CCR6+CCR4+), TH1* (CXCR5CXCR3+CCR6+CCR4+), TFH (CXCR5+), and Treg (CD25+FOXP3+) subsets among CD4+ T cells from healthy controls (TH1, TH2, TH17, TH1*, and TFH, n = 34; Treg, n = 17) and patients (P2 and P3). (C) Production of IL-17A and IL-22 by whole blood from healthy controls (n = 33) and patients (P2 and P3) after stimulation with PMA plus ionomycin for 24 hours. (D) Percentage of IL-17A+, IL-17F+, and IFN-+ cells among memory CD4+ T cells from healthy controls (n = 36) and patients (P2 and P3) activated by TAE beads or PMA plus ionomycin (P/I) for 12 hours. (E) Cytokine production by nave CD4+ T cells from healthy controls (n = 8) and patients (P2 and P3) cultured under TH0-, TH17-, or TH1-polarizing conditions. (F and G) Frequency of IL-17A+ and IFN-+ cells among nave (F) and memory (G) CD4+ T cells from healthy controls (n = 10) and patients (P2 and P3) cultured under TH0-, TH17-, or TH1-polarizing conditions. C, healthy controls; P, P2 and P3. Horizontal bars represent median values (A to G). *P < 0.05 and **P < 0.01, two-tailed Mann-Whitney tests (A to G).

We subsequently investigated the pathogenesis of the complex CTD phenotype of the patients. Previous studies have proposed an in vitro fibroblast phenotype common to most patients with EDS but apparently not observed in other inherited CTDs (5255). This phenotype is characterized by generalized fibronectin-ECM (FN-ECM) disarray, low levels of expression of the canonical integrin receptor 51, and the recruitment of v3 integrin (52, 53, 56). EDS fibroblasts also seem to display little or no type III collagen deposition in the ECM (COLLIII-ECM) and a variable disorganization of type V collagen (COLLV-ECM) (52, 53, 56). A specific myofibroblast-like phenotype of hEDS has also been proposed, on the basis of the organization of -smooth muscle actin (-SMA), cadherin-11 (CAD-11) expression, and enhanced cell migration (56). Unlike cells from patients with EDS, the primary fibroblasts of P2 displayed no FN-ECM disarray, and 51 integrin was organized as in control fibroblasts (Fig. 5A). Despite the low levels of COLLIII-ECM and a barely detectable organization of COLLV-ECM, P2s fibroblasts expressed the canonical collagen receptor, 21 integrin, normally, unlike EDS cells (Fig. 5A). The myofibroblast-specific markers -SMA and CAD-11 were absent from the cells of P2, whereas they were present on hEDS fibroblasts (Fig. 5A). Consistent with this finding, the fibroblasts of P2 did not have the enhanced migratory capability reported for some hEDS fibroblasts, as shown by in vitro scratch and Transwell assays (Fig. 5, B and C). Instead, the fibroblasts of P2, like some classic EDS (cEDS) cells, migrated poorly (Fig. 5, B and C), probably accounting for the poor wound healing observed in the patients (see the Case reports section in the Supplementary Materials). Overall, these data suggest that, although the clinical presentation in these patients overlaps with EDS, and despite the 2017 EDS diagnostic criteria for hEDS being met (22), the in vitro fibroblast phenotype of these patients is apparently different from that proposed for EDS, in general, and for hEDS, in particular (52, 53, 56).

(A) Immunofluorescence of FN; type V collagen (COLLV); type III collagen (COLLIII); 21, 51, and v3 integrins; -SMA; and CAD-11 in primary fibroblasts from a healthy control (C), P2, a patient with hEDS (56), and a patient with cEDS (93). Scale bar, 10 m. (B) In vitro scratch assay with primary fibroblasts from a healthy control (C), P2, a patient with hEDS (56), and a patient with cEDS (93). Images were captured at 0 and 48 hours after scratching. Scale bar, 100 m. (C) Transwell assay with primary fibroblasts from a healthy control (C), P2, a patient with hEDS (56), and a patient with cEDS (93). (D) mRNA induction in primary fibroblasts from healthy controls (C1 and C2) and patients (P2 and P3) stimulated with TGF- (10 ng/ml) for the indicated times. (E) Top 10 up-regulated or down-regulated genes in terms of absolute fold change, in primary fibroblasts from healthy controls (C1 and C2) stimulated with TGF- (10 ng/ml) for 2, 6, and 24 hours, with a greater than 1.5-fold change relative to patients (P2 and P3) at each time point. (F and G) Expression of JNK1 protein (F) and production of FN (top) and IL-11 (bottom) (G) by primary fibroblasts from healthy controls (C1 and C2) transfected with control siRNA (50 nM) or MAPK8 siRNA (50 nM) for 48 hours and then stimulated with TGF- (10 ng/ml) for an additional 24 hours. NS, nonstimulated conditions. The values shown are the means SEM of two (C) or three (D and G) independent experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001, unpaired t tests (D and G).

We tested the hypothesis that the patients CTD resulted from dysfunctional TGF- signaling because this pathway controls the expression of key genes involved in the development and maintenance of the ECM (24). Upon TGF- stimulation, the patients SV40-fibroblasts displayed impaired AP-1 (c-Jun/ATF-2) activation, whereas ERK1/2, p38, and SMAD2/3 were normally activated, as shown by Western blotting (fig. S4A). Previous reports have suggested that TGF- induces the expression of FN in a JNK1-dependent manner (57, 58). Consistent with these findings, the induction of FN production by TGF- was impaired at both the mRNA and protein levels in the patients fibroblasts (Fig. 5D and fig. S4, B and C). The patients did not display spondylometaphyseal dysplasia, which can be caused by heterozygous FN1 mutations (59), probably because their baseline FN-ECM organization levels were normal (Fig. 5A). By contrast, various SMAD2/3-dependent TGF- target genes (58, 60), such as COL1A1, COL1A2, COL3A1, COL5A1, and COL5A2, encoding key components of the ECM and mutated in patients with cEDS and other forms of EDS (22), were normally induced by TGF- in the patients cells (Fig. 5D and fig. S4, B and C). Last, we performed a transcriptomic analysis of the cellular response to TGF- in primary fibroblasts. The genome-wide transcriptional response to TGF- was impaired in the patients cells (fig. S4D). A number of TGF-responsive genes, including ELN, EDN1, IL11, and COMP, were not induced in the patients cells (Fig. 5E and fig. S4E). Consistently, their induction in control fibroblasts stimulated with TGF- was impaired by the RNAi-mediated knockdown of MAPK8 (Fig. 5, F and G, and fig. S4F). These findings are consistent with previous reports of the presence of AP-1binding motifs in the regulatory regions of COMP and ELN (61, 62) or of the AP-1-dependent induction of EDN1 and IL11 by TGF- (63, 64). Mutations in these genes (59, 6568) or in those encoding the corresponding receptors (69, 70) have already been reported in patients with various CTDs other than EDS, LDS, and MS (table S4). The study of the patients fibroblasts thus delineated the transcriptomic impact of impaired JNK1-dependent, SMAD2/3-independent TGF- signaling. Moreover, fibroblasts from patients with LDS, heterozygous for mutations in TGFBR2 or SMAD3, also showed impaired responses to TGF- (fig. S4D), consistent with previous studies showing these mutations to be loss-of-function in vitro (7173). However, their impact differed from that of JNK1 haploinsufficiency, because about 40% of JNK1-dependent genes were TGFBR2/SMAD3 independent (fig. S4G). This is consistent with the clinical differences observed between our patients particular CTD (displaying some overlap with hEDS) and LDS. In addition, about 30% of TGFBR2-dependent genes were SMAD3 independent (fig. S4H), potentially accounting for some of the phenotypic differences between LDS patients with TGFBR2 and SMAD3 mutations. Our findings provide a molecular and cellular basis for the complex new form of CTD displayed by the patients, with an impairment of the TGF-dependent induction of key ECM components and regulators different from that of patients with another CTD, LDS, who are heterozygous for TGFBR2 or SMAD3 mutations.

We have found a heterozygous loss-of-expression and loss-of-function mutation of MAPK8 in a three-generation multiplex kindred with a rare combination of classic CMC and a previously undescribed form of CTD (Fig. 6). Human JNK1 haploinsufficiency impairs IL-17A/F immunity in two ways, by reducing the responses of fibroblasts to IL-17RA/IL-17RC ligation and by compromising the TGF-dependent development of TH17 cells, accounting for the impaired mucocutaneous immunity to C. albicans and subsequent development of CMC in these patients. These findings indicate that IL-17RA/IL-17RCdependent protective mucocutaneous immunity to C. albicans is JNK1 dependent. We previously described CMC patients with biallelic mutations of ACT1 (19). The findings reported here identify JNK1 as a key component of this antifungal pathway acting downstream from ACT1. They also indicate that JNK1 haploinsufficiency has an impact on the development of TH17 cells, probably due to the involvement of JNK1 in the TGF- pathway.

The binding of IL-17A/F to the IL-17RA/IL-17RC receptor facilitates the recruitment of ACT1 to the receptor, which mediates the activation of JNK1, ERK, p38, and NF-B (p65/p50) signaling, leading to the production of pro-inflammatory cytokines and chemokines (e.g., CXCL1 and IL6). Similarly, TGF- binds to its receptor (TGFBR1/TGFBR2), leading to the activation of JNK1, ERK, p38, and SMAD (SMAD2/3/4) signaling. This pathway ultimately results in the production of ECM proteins and regulators (e.g., FN1 and IL11). The mutation (yellow star) in MAPK8 encoding JNK1 impairs the JNK1-dependent activation of downstream AP-1 (c-Jun/ATF-2), thereby reducing the JNK1-dependent cellular responses to IL-17 and TGF-.

Our data also suggest that JNK1 haploinsufficiency impairs the c-Jun/ATF-2dependent, and SMAD2/3-independent, TGF-responsive pathway in fibroblasts, a cellular phenotype that probably accounts for the patients complex and unusual CTD phenotype. The induction of collagen genes mutated in cEDS and other forms of EDS, such as COL1A1 and COL5A1, was intact, whereas that of other ECM proteins, such as COMP and ELN, mutated in patients with other types of CTD (65, 66), was impaired. The impaired induction of genes encoding ECM regulators, such as EDN1 and IL11, may also contribute to the patients CTD phenotype. It is also relevant that the impact of heterozygous mutations of MAPK8 differed from that of the TGFBR2 or SMAD3 of patients with LDS, in terms of the transcriptional response to TGF-. Haploinsufficiency for JNK1 probably defines a previously undescribed CTD entity encompassing various clinical manifestations, some of which overlap with EDS, but not LDS.

Cellular responses to cytokines other than IL-17A/F and TGF- were apparently intact in cells from the patients. JNK1-deficient mice have defects of innate and adaptive immunity to various infections (7476), but their connective tissues have not been studied. MAPK8-heterozygous mice have rarely been studied and seem to be normal (77). In conclusion, the integrity of the human JNK1 pathway is essential for IL-17A/Fdependent mucocutaneous immunity to Candida and for the TGF-dependent homeostasis of connective tissues.

We studied three patients from a kindred suffering from CMC and CTD. We analyzed this kindred by WES and found that the patients were heterozygous for a private splice-site mutation in MAPK8, the gene encoding JNK1. We evaluated the impact of this mutation in an overexpression system and in the patients cells. We assessed the cellular responses to IL-17A/F and TGF- of the patients fibroblasts as well as the development and the differentiation properties of the patients T and B cells.

The patients (P1, P2, and P3) were followed in their country of residence, France. Another family member (II.1) also participated to the genetic study. Informed consent was obtained from each patient, in accordance with local regulations and a protocol for research on human subjects approved by the institutional review board (IRB) of Institut National de la Sant et de la Recherche Mdicale (INSERM). Experiments were performed on samples from human subjects in the United States, France, Italy, and Australia, in accordance with local regulations and with the approval of the IRB of The Rockefeller University, the IRB of INSERM, the local ethical committee of Brescia, and the Sydney South West Area Health Service, respectively.

Genomic DNA was extracted from whole blood and sheared with an S2 Focused-ultrasonicator (Covaris). An adaptor-ligated library was prepared with the TruSeq DNA Sample Prep Kit (Illumina). Exome capture was performed with the SureSelect Human All Exon V5 Kit (Agilent Technologies). Paired-end sequencing was performed on the HiSeq 2500 System (Illumina) generating 100-base reads. The sequences were aligned with the GRCh37 build of the human genome reference sequence, with the Burrows-Wheeler Aligner (78). Downstream processing and variant calling were performed with the Genome Analysis Toolkit (79), SAMtools (80), and Picard tools (http://broadinstitute.github.io/picard/). All variants were annotated with in-house annotation software.

Primary fibroblasts were obtained from skin biopsy specimens and cultured in Dulbeccos modified Eagles medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). PBMCs were isolated from whole blood by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare Life Sciences). Immortalized SV40-transformed fibroblasts (SV40-fibroblasts) and EBV-transformed B (EBV-B) cells were generated as previously described (81). HEK293T (American Type Culture Collection) and GP2-293 retroviral packaging cells (Clontech) were maintained in DMEM containing 10% FBS. HEK293T and GP2-293 cells were transiently transfected with the aid of X-tremeGENE 9 DNA Transfection Reagent (Roche). Primary fibroblasts were transfected with small interfering RNA (siRNA) in the presence of Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific), in accordance with the manufacturers instructions.

Genomic DNA was isolated from primary fibroblasts or EBV-B cells with the QIAamp DNA Mini Kit (QIAGEN). A fragment encompassing exon IV and intron IV of MAPK8 was amplified by polymerase chain reaction (PCR) with specific primers (table S5). The PCR products were analyzed by electrophoresis in 1% agarose gels and sequenced with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). Sequencing products were purified by gel filtration on Sephadex G-50 Superfine columns (GE Healthcare Life Sciences), and sequences were analyzed using the ABI 3730 DNA Analyzer (Applied Biosystems).

JNK11 and JNK12 were amplified from pCDNA3 FLAG JNK11 (Addgene) and pCDNA3 FLAG JNK12 (Addgene), respectively. JNK11 and JNK12 were amplified from the cDNA derived from SV40-fibroblasts. The full-length WT isoforms and truncated mutants were inserted into pTRIP-SFFV (82) and the pCMV6-AN-Myc-DDKtagged vector (OriGene), respectively. TA cloning and exon trapping were performed with the pCR4-TOPO vector (Thermo Fisher Scientific) and the pET01 vector (MoBiTec GmbH), respectively, according to the manufacturers instructions. Control siRNA (D-001810-10) and MAPK8 siRNA (L-003514-00) were obtained from Dharmacon.

SV40-fibroblasts and primary fibroblasts were plated on 24-well plates at a density of 6 104 cells per well in 0.5 ml of DMEM supplemented with 10% FBS. After 24 hours, cells were left unstimulated or were stimulated with recombinant human IL-17A (317-ILB; R&D Systems), recombinant human IL-17F (1335-IL; R&D Systems), recombinant human IL-17A/F (5194-IL; R&D Systems), recombinant human TNF- (210-TA; R&D Systems), recombinant human IL-1 (201-LB; R&D Systems), recombinant human lymphotoxin 1/2 (8884-LY; R&D Systems), lipoteichoic acid from Staphylococcus aureus (LTA-SA) (tlrl-slta; InvivoGen), Pam3CSK4 (tlrl-pms; InvivoGen), fibroblast-stimulating lipopeptide-1 (FSL-1) (tlrl-fsl; InvivoGen), Pam2CSK4 (tlrl-pm2s-1; InvivoGen), and lipopolysaccharide (LPS) (L9764; Sigma-Aldrich) for a further 24 hours. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of GRO- (DY275; R&D Systems), IL-6 (88-7066; Invitrogen), and IL-8 (M9318; Sanquin) in the supernatants. SV40-fibroblasts and primary fibroblasts were cultured in DMEM supplemented with 1% FBS for 24 hours and then stimulated with recombinant human TGF-1 (240-B-002; R&D Systems) for various time periods. Protein levels were determined by ELISA for FN (DY1918-05; R&D Systems), procollagen I (1) (DY6220-05; R&D Systems), and IL-11 (DY218; R&D Systems). Whole blood was stimulated with IL-1, Pam3CSK4, heat-killed Staphylococcus aureus (tlrl-hksa; InvivoGen), FSL-1, Pam2CSK4, LPS, R848 (tlrl-r848; InvivoGen), and PMA (P1585; Sigma-Aldrich) plus ionomycin (I3909; Sigma-Aldrich) for 24 hours, and IL-6 production was measured by ELISA. PBMCs were cultured in X-VIVO 15 (Lonza) containing 5% human serum AB (Lonza) and recombinant human thymic stromal lymphopoietin (100 ng/ml) (1398-TS/CF; R&D Systems) for 24 hours. Cells were washed and plated on 48-well plates, at a density of 4 106 cells per well, in 0.5 ml of X-VIVO 15 supplemented with 5% human serum AB in the presence of recombinant human IL-2 (10 ng/ml) (202-IL; R&D Systems) and recombinant human IL-17E (10 ng/ml) (1258-IL; R&D Systems). After 72 hours, the amount of IL-5 present in each well was determined with an ELISA kit (DY205; R&D Systems).

Total RNA was extracted with the RNeasy Mini Kit (QIAGEN), according to the manufacturers instructions. Reverse transcription was carried out with the SuperScript III First-Strand Synthesis System (Invitrogen). Conventional PCR was performed with the Choice-Taq Blue DNA Polymerase (Denville Scientific), and the amplicons were analyzed by electrophoresis in 2% agarose gels. Quantitative PCR was performed with Fast SYBR Green Master Mix (Applied Biosystems) in the 7500 Fast Real-Time PCR System (Applied Biosystems). The primer pairs used for conventional and quantitative PCR are listed in table S5.

Whole-cell lysates were prepared in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] supplemented with cOmplete Protease Inhibitor Cocktail (Roche). Proteins were separated by electrophoresis in either 10% Criterion XT Bis-Tris Protein Gels (Bio-Rad) or 4 to 20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad), and the resulting bands were transferred onto Immobilon-P PVDF Membrane (Millipore). All blots were incubated overnight with primary antibodies and developed with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). The antibodies used in this study included antibodies (from Cell Signaling Technology) against JNK1 (3708), pc-Jun (2361), c-Jun (9165), pATF-2 (9221), ATF-2 (9226), pIB (9246), pp65 (3033), pp38 (9211), p38 (9212), pERK1/2 (4370), ERK1/2 (4695), pSMAD2 (3101), SMAD2 (5339), pSMAD3 (9520), SMAD3 (9523), SMAD4 (38454), and Myc (2040), as well as IB (610690; BD Biosciences), p65 (sc-372; Santa Cruz Biotechnology), and -actin (AM1829B; Abgent), and the following secondary antibodies: Amersham ECL Mouse Immunoglobulin G (IgG), horseradish peroxidase (HRP)linked whole antibody (from sheep) (NA931; GE Healthcare Life Sciences), and Amersham ECL Rabbit IgG, HRP-linked whole antibody (from donkey) (NA934; GE Healthcare Life Sciences).

PBMCs were cultured in 48-well plates, at a density of 3 106 cells per milliliter, in RPMI 1640 medium (Gibco) containing 10% FBS with TAE beads (130-091-441; Miltenyi Biotec) or PMA plus ionomycin in the presence of a protein transport inhibitor (GolgiPlug; BD Biosciences). After 12 hours, the cells were collected and their expression of the indicated cytokines was assessed by flow cytometry, as previously described (17).

Nave and memory CD4+ T cells were isolated and cultured under polarizing conditions, as previously described (6, 83). Briefly, cells were cultured with TAE beads alone (TH0) or under TH1 [IL-12 (20 ng/ml; R&D Systems)] or TH17 [TGF-1 (2.5 ng/ml; PeproTech), IL-1 (20 ng/ml; PeproTech), IL-6 (50 ng/ml; PeproTech), IL-21 (50 ng/ml; PeproTech), and IL-23 (20 ng/ml; eBioscience)] polarizing conditions. After 5 days, the supernatants were harvested and the cells were restimulated with PMA/ionomycin for 6 hours. The levels of specific cytokines were determined by intracellular staining and flow cytometry. The secretion of the indicated cytokines was determined with a cytometric bead array (BD Biosciences).

Nave and memory B cells were sorted and cultured in the presence of CD40L (200 ng/ml; R&D Systems), with or without IL-21 (50 ng/ml; PeproTech) for 7 days, as previously described (83). The production of IgA, IgG, and IgM was assessed by Ig heavy chainspecific ELISA (83).

Cells were surface-labeled with CD4-APC-Vio770 anti-human CD4 (clone M-T321; Miltenyi Biotec), Brilliant Violet 421 anti-human CD197 (CCR7) (clone G043H7; BioLegend), phycoerythrin (PE)CF594 anti-human CD45RA (clone HI100; BD Biosciences), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957; Thermo Fisher Scientific). Intracellular staining was performed with the Fixation/Permeabilization Solution Kit (BD Biosciences) and antibodies including Alexa Fluor 488 antiIL-17A (clone eBio64DEC17; eBioscience), PE antiIL-17F (clone SHLR17; eBioscience), and Alexa Fluor 700 antiIFN- (clone 4S.B3; eBioscience). Samples were analyzed with a Gallios flow cytometer (Beckman Coulter) and FlowJo software.

Primary fibroblasts were fixed with ice-cold methanol and incubated with antibodies against FN (Sigma-Aldrich), type III collagen (Chemicon), and type V collagen (LifeSpan BioSciences) at a dilution of 1:100, and with anti-SMA antibody (A2547; Sigma-Aldrich) at a concentration of 2 g/ml, as previously described (52, 56, 84). For analysis of the 21, 51, and v3 integrins, cells were fixed with 3% paraformaldehyde (PFA)/60 mM sucrose and permeabilized with 0.5% Triton X-100, as previously reported (84). In particular, cells were incubated with anti-51 (MAB1969; Chemicon), anti-v3 (MAB1976; Chemicon), and anti-21 (MAB1998; Chemicon) integrin antibodies at a concentration of 4 g/ml for 1 hour. CAD-11 levels were investigated by fixing cells by incubation with 4% PFA/10 mM sucrose for 10 min, permeabilizing them by incubation with 0.1% Triton X-100 for 10 min, blocking them with by incubation with 2% bovine serum albumin in phosphate-buffered saline (PBS) for 1 hour, and then incubating them with anti-CDH11/cadherin OB antibody (Thermo Fisher Scientific) at a concentration of 2 g/ml for 3 hours, as previously described (56). The cells were washed and then stained with Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse antibodies (Thermo Fisher Scientific) or with rhodamine-conjugated anti-goat IgG antibody (Chemicon) for 1 hour. Immunofluorescence signals were acquired with a black-and-white charge-coupled device TV camera (SensiCam; PCO Computer Optics GmbH), mounted on a Zeiss Axiovert fluorescence microscope, and digitized with Image-Pro Plus software (Media Cybernetics).

Primary fibroblasts were plated on 35-mm petri dishes at a density of 3 104 cells per dish and grown to confluence. The cell monolayers were wounded with a rubber policeman to generate an acellular area, and dishes were marked to ensure the recording of the correct area. The monolayers were washed with PBS, rinsed in DMEM and 10% FBS, and photographed with an inverted microscope at 0 and 48 hours after scratching.

Cell migration was evaluated in a Transwell assay with an 8-m-pore filter (Corning Costar). Primary fibroblasts (5 104 cells) were resuspended in DMEM without FBS, placed in the upper chamber, and allowed to migrate for 6 hours through the polycarbonate membrane into the bottom well, which was filled with DMEM containing 10% FBS. The cells that did not migrate were removed from the upper surface with a cotton swab. The cells that had migrated were collected in the bottom chamber. They were fixed in methanol, stained with the Diff-Quik Staining Kit (Medion Diagnostic GmbH), and quantified in 10 nonoverlapping fields of 1 mm2 with a light microscope.

Total RNA was extracted with the RNeasy Plus Micro Kit (QIAGEN), according to the manufacturers instructions. Microarray analysis was performed with the GeneChip Human Gene 2.0 ST Array (Thermo Fisher Scientific). The raw expression data were normalized in R with the robust multi-array average method (85) and the affy R package (86), and processed as previously described (87). RNA-Seq analysis was performed with TruSeq Stranded mRNA (Illumina) and standard poly(A)-based methods for library preparation. Paired-end sequencing with a read length of 150 bp and ~19 million reads per sample was carried out with a HiSeq 4000 system (Illumina). Raw reads were aligned to the human genome assembly (hg38) with STAR aligner (88). The number of reads mapping to each gene feature was determined with HTSeq (89). Differential expression was analyzed with an in-house script in R with DESeq2 (90) and ComplexHeatmap (91). In brief, fold changes in expression between nonstimulated and stimulated conditions were calculated for each individual and time point separately, and genes were further filtered based on a minimal 1.5-fold change in expression (up-regulation or down-regulation). The residual responses of the patients were calculated on the basis of the number of responsive genes passing the above filter in both healthy controls [number of responsive genes in a subject/total number of responsive genes in healthy controls) 100].

Unpaired t tests and two-tailed Mann-Whitney tests were used for comparisons of two groups. P < 0.05 was considered statistically significant in all tests performed with Prism software (GraphPad).

immunology.sciencemag.org/cgi/content/full/4/41/eaax7965/DC1

Case reports

Fig. S1. Identification of a private MAPK8 variant in the patients.

Fig. S2. Impaired IL-17A/F signaling in the patients fibroblasts.

Fig. S3. Normal B cell differentiation in the patients.

Fig. S4. Impaired TGF- signaling in the patients fibroblasts.

Table S1. Immunological parameters of P1, P2, and P3.

Table S2. Careful WES analysis of rare (MAF < 1%) nonsynonymous coding variants in the known CMC-, EDS-, LDS-, and MS-causing genes.

Table S3. Heterozygous nonsynonymous variants common to P1, P2, and P3.

Table S4. Clinical presentations of disorders caused by mutations in JNK1-dependent TGF- target genes or in genes encoding the corresponding receptors.

Table S5. Primers used for Sanger sequencing, reverse transcription PCR, and exon trapping.

Table S6. Raw data file (Excel spreadsheet).

References (94, 95)

Acknowledgments: We warmly thank the patients and their family for participating in the study. We also thank all the members of the Laboratory of Human Genetics of Infectious Diseases for fruitful discussions and the members of the genomics core facility at Sidra Medicine for their contributions to Illumina library preparation and RNA-Seq. Funding: This work was funded by the French National Research Agency (ANR) under the Investments for the future program (ANR-10-IAHU-01), the HGDIFD project (ANR-14-CE15-0006-01), the EURO-CMC project (ANR-14-RARE-0005-02), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), the INSERM, Paris Descartes University, the Cross-lab project of Imagine Institute, The Rockefeller University, Sidra Medicine, the Jeffrey Modell Foundation Translational Research Program, the Jeffrey Modell Centers Network, the St. Giles Foundation, the National Center for Research Resources of the NIH, the National Center for Advancing Translational Sciences of the NIH (UL1TR001866), and the National Institute of Allergy and Infectious Diseases of the NIH (R01AI127564). C.S.M. was supported by an Early-Mid Career Research Fellowship from the Office of Health and Medical Research of the New South Wales State Government. T.F. and L.G. were supported by the Ministry of Health of the Czech Republic (16-34414A). D.S. and F.M. were supported by the Research Foundation Flanders (FWO) of Belgium. S.G.T. was supported by the National Health and Medical Research Council of Australia. A.P. was supported by an AP-HP transversal research contract. Author contributions: J.L., M.R., C.S.M., G.R., S.C., L.G., R.L., V.B., K.P., D.T.A., M.M., and M. Chrabieh performed the experiments and analyzed the data. T.H., S. Bougarn, L.S., S. Boughorbel, A.G., F.R., and B. Bigio conducted WES, microarray, RNA-Seq, and computational analyses. E.C., M.M., and S. Boucherit provided clinical samples and analyzed clinical data. Y.I., B. Boisson, V.C.-D., D.S., F.M., N.Z., L.A., T.F., H.C.D., N.M., S.G.T., and M. Colombi provided expertise and feedback. J.L., J.-L.C., and A.P. designed the study and wrote the manuscript with the assistance of all coauthors. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The WES data are available from the NCBI Sequence Read Archive via BioProject accession number PRJNA563623. The microarray and RNA-Seq data have been deposited to the Gene Expression Omnibus and are accessible under accession number GSE137110. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.

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Chronic mucocutaneous candidiasis and connective tissue disorder in humans with impaired JNK1-dependent responses to IL-17A/F and TGF- - Science

The ability to fight disease is a driving force in human survival. Inflammation has emerged as a key weapon in this process. As pathogens change and evolve, the immune system adapts to keep up.

However, to what extent might such evolutionary adaptations also give rise to autoimmune conditions such as lupus and Crohn's disease?

This was a central question in a recent Trends in Immunology review by two scientists from Radboud University, in Nijmegen, Netherlands.

To address the issue, first author Jorge Domnguez-Andrs, a postdoctoral researcher in molecular life science, and senior author Prof. Mihai G. Netea, chair of experimental internal medicine, examined studies in the fields of virology, genetics, microbiology, and immunology.

They focused on people of African or Eurasian descent and how their ancestral origins may have influenced their risk of autoimmune diseases.

Of particular interest was how common pathogens in different communities related to changes in people's DNA, particularly when this involved inflammation.

The team found that the genetic changes made it harder for pathogen infections to take hold.

Over time, however, it seems that inflammation-related diseases, such as inflammatory bowel disease, Crohn's disease, and lupus, have emerged alongside improvements in immune defenses.

The findings also suggest that the human immune system continues to evolve and adapt to changes in environment and lifestyle.

"There seems to be a balance," says Domnguez-Andrs.

"Humans evolve to build defenses against diseases," he continues, "but we are not able to stop disease from happening, so the benefit we obtain on one hand also makes us more sensitive to new diseases on the other hand."

He observes that autoimmune diseases in today's humans tend to emerge later in life. These would not have caused health problems for our ancestors because their lives were much shorter.

"Now that we live so much longer," he explains, "we can see the consequences of infections that happened to our ancestors."

One of the examples that Domnguez-Andrs and Netea cover in detail in their review is malaria.

"Among various infectious diseases," they write, "malaria has exerted the highest evolutionary pressure on the communities across the African continent."

Malaria is a mosquito-borne disease that makes people very ill with flu-like symptoms, such as chills and a high fever.

While there has been much progress in the fight to control and eliminate the potentially fatal disease, it continues to threaten nearly half of the world's population, according to the World Health Organization (WHO).

The cause of malaria is parasites belonging to the species Plasmodium. These parasites spread to humans through the bites of infected female Anopheles mosquitoes.

Domnguez-Andrs and Netea note that Plasmodium has been infecting people in Africa for millions of years. During that period, the immune systems of those human populations have evolved stronger resistance to infection by increasing inflammation.

However, the downside of increasing inflammation to withstand infectious disease is that it favors health problems that tend to occur later in life.

Modern humans of African descent are more prone to developing such conditions, which include atherosclerosis and other cardiovascular diseases.

Another example of how ancestral changes in DNA leave imprints in the immune systems of modern humans is the interbreeding of early Eurasians with Neanderthals.

Modern humans whose genomes harbor remnants of Neanderthal DNA have immune systems that are better able to withstand staph infections and HIV-1. However, they are also more prone to asthma, hay fever, and other allergies.

Improvements in technology are making it more possible to find the downsides that can accompany disease-fighting adaptations.

Next generation sequencing, for example, is allowing scientists to delve more deeply into what happens at the DNA level between pathogens and the organisms that they infect.

Not only is new technology getting better at revealing genetic changes that occurred in our ancestors, but it is also showing that the human immune system continues to evolve and adapt.

In Africa, there are still tribes that hunt for food as their ancestors did. Thanks to new tools, scientists can see how the gut bacteria of these tribes are more diverse than those of, for example, contemporary African American people, who buy food in stores.

Other changes that have had an effect on DNA are the improvements in hygiene that have occurred in recent centuries. These have reduced exposure to pathogens and the diversity of gut bacteria.

"This reduced microbiota diversity in Western societies," the authors observe, "has been associated with a higher incidence of the so-called 'diseases of civilization,' such as cardiovascular diseases, diabetes, obesity, and autoimmune disorders, which are very unusual in hunter-gatherer societies, compared with communities living a Western-type lifestyle."

Domnguez-Andrs and Netea are extending their research to populations whose ancestry is other than African or Eurasian.

"Today, we are suffering or benefiting from defenses built into our DNA by our ancestors' immune systems fighting off infections or growing accustomed to new lifestyles."

Jorge Domnguez-Andrs, Ph.D.

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Humans and autoimmune diseases continue to evolve together - Medical News Today

SAN DIEGO--(BUSINESS WIRE)--Gossamer Bio, Inc. (Nasdaq: GOSS), a clinical-stage biopharmaceutical company focused on discovering, acquiring, developing and commercializing therapeutics in the disease areas of immunology, inflammation and oncology, today announced that members of the management team will participate in the following investor conferences:

A live webcast of the presentations will be available on the Events and Presentations page in the Investors section of the companys website at https://ir.gossamerbio.com. A replay of the webcast will be archived on the companys website for 90 days following the presentation.

About Gossamer Bio

Gossamer Bio is a clinical-stage biopharmaceutical company focused on discovering, acquiring, developing and commercializing therapeutics in the disease areas of immunology, inflammation and oncology. Its goal is to be an industry leader in each of these therapeutic areas and to enhance and extend the lives of patients suffering from such diseases.

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Gossamer Bio Announces Participation in Upcoming Investor Conferences - Business Wire

Food allergies are complex things, and people dont react the same way to their allergy trigger every time they encounter it, experts say.

Co-factors like hard exercise or certain medications can alter how someone responds to an allergen, lowering the threshold at which they react, said Dr. Anne Ellis, professor and chair of the Division of Allergy and Immunology at Queens University.

Sometimes this can be full-on anaphylaxis, such as a case reported by the Daily Mail, where a woman said she had a severe reaction when she combined fish and wine in a meal despite having consumed both independently without incident in the past.

A case involving two foods would be extremely unusual, said Dr. Harold Kim, president of the Canadian Society of Allergy and Clinical Immunology, and an associate professor at Western University.

It would be very unlikely that its an actual allergic reaction to the wine, Kim said. Its just that the wine is potentiating the reaction to fish.

There are well-known factors that can make people more sensitive to allergy triggers, and alcohol is among them.

One of the most classic examples that we see quite frequently is what we call food-dependent exercise-induced anaphylaxis, Ellis said.

You can eat the food and be fine. You can exercise and be fine. But if you exercise within two hours of eating your trigger food, youll have anaphylaxis.

Alcohol and anti-inflammatory medications, like ibuprofen, are frequent triggers, Ellis said. Illness and fever can also have a similar effect.

The most common food allergies that are triggered by a co-factor are wheat, celery and seafood, Kim said. The reaction can range from mild, like hives, to something that requires an emergency room visit.

And co-factors usually affect younger people.

It often affects young, healthy patients, often young athletic females that run and eat a salad or some carbohydrate-based food before they run, he said. Thats kind of the classic situation.

It would be unusual for this reaction to come as a complete surprise, Ellis said. Usually it would appear in people who have a known food allergy, who just became more sensitive.

For the most part, food allergies arent hidden. They arent subtle. And usually the story is quite consistent.

Doctors arent quite sure what happens to change someones reaction to a food, but suspect it has to do with increased bloodflow and changes to how allergens are absorbed.

People have thresholds for their allergies, Ellis said, with some people not reacting unless theyve had nine peanuts, but are fine with a fraction of a peanut.

Exercise and alcohol can dilate the blood vessels, she said, making people more likely to absorb the antigen, and develop symptoms at a lower dose.

Most people with food allergies react the same way to their trigger every time. Only a few have co-factors to their allergies Kim estimates that he sees approximately one such patient per month. Its a strange phenomenon, he said.

If someone reacts once to the combination of exercise and an allergy trigger, its likely they will react the same way again, Kim said.

Ellis recommends that people with food allergies be aware of the possibility of a co-factor, and avoid situations that combine their risks.

Its just perhaps something to have in the back of your mind, if you do have a food allergy, Ellis said, to be extra-cautious if youre going to be consuming alcohol, or at times when youre sick, or if youre taking certain anti-inflammatories, that these are all things that could potentially lower your threshold.

Allergists are trained to ask about things like alcohol when they assess an allergy, she said, and should be able to tell you if this might be a factor. And severe allergic reactions should always be checked out by a medical professional.

If you do have a significant reaction, where youre having more than just hives and flushing, make sure that youre presenting yourself to emergency care and not just taking your own antihistamines.

- Global News

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CANADA: How 'co-factors' can increase the likelihood of a food allergy reaction - BarrieToday

Researchers at Karolinska Institutet have developed a new method to separate between two different types of a common herpes virus (HHV-6) that has been linked to multiple sclerosis. By analyzing antibodies in the blood against the most divergent proteins of herpesvirus 6A and 6B, the researchers were able to show that MS-patients carry the herpesvirus 6A to a greater extent than healthy individuals. The findings, published in Frontiers in Immunology, point to a role for HHV-6A in the development of MS.

Multiple sclerosis, MS, is an autoimmune disease that affects the central nervous system. The cause of the disease is unclear, but one plausible explanation is a virus tricks the immune system to attack the body's own tissue. Human Herpesvirus 6 (HHV-6) has previously been associated with MS, but in those studies it wasn't possible to distinguish between 6A and 6B. Through virus isolation from ill individuals, researchers have been able to show that HHV-6B can cause mild conditions such as roseola in children, but it has been unclear if HHV-6A is the cause of any disease.

According to estimates, as many as 80 percent of all children are infected with the HHV-6 virus before 2 years of age, and many also carry protection in the form of antibodies against this particular virus for the rest of their lives. But since it hasn't been possible to tell the two variants apart post-infection, it has been difficult to say whether HHV-6A or B is a risk factor for MS.

In this study, however, the researchers were able to distinguish between the A and B virus by analyzing antibodies in the blood against the proteins--immediate early protein 1A and 1B (IE1A and IE1B)--that diverge the most between the two viruses.

"This is a big breakthrough for both the MS and herpes virus research," says Anna Fogdell-Hahn, associate professor at the Department of Clinical Neuroscience at Karolinska Institutet and one of the study's senior authors. "For one, it supports the theory that HHV-6A could be a contributing factor to the development of MS. On top of that, we are now able, with this new method, to find out how common these two different types of HHV-6 are, something we haven't been able to do previously."

The researchers compared antibody levels in blood samples of some 8,700 MS-patients against more than 7,200 healthy people whose gender, date of birth, date of blood sample and other factors matched those with MS. They concluded that people with MS had a 55 percent higher risk of carrying antibodies against the HHV-6A protein than the control group. In a sub-group of almost 500 people, whose blood samples were drawn before the onset of the disease, the risk of developing MS in the future was more than doubled if they had a 6A viral infection. The younger the people were when the virus was first discovered in the blood, the higher the risk was of developing MS in the future. HHV-6B, on the other hand, was not positively associated with MS. Instead MS-patients had lower levels of antibodies toward IE1B than those without MS.

Antibodies toward Epstein-Barr virus (EBV), another herpes virus that is also associated with MS, were analyzed with the same method and the researchers were able to show that individuals affected with both viruses had an even greater risk of MS. This indicates that several virus infections could be acting jointly to increase the risk of MS.

"Both HHV-6A and 6B can infect our braincells, but they do it in slightly different ways. Therefore, it is now interesting to go forward and attempt to map out exactly how the viruses could affect the onset of MS," says Anna Fogdell-Hahn.

Reference: Engdahl, E., Gustafsson, R., Huang, J., Bistrm, M., Lima Bomfim, I., Stridh, P., Fogdell-Hahn, A. (2019). Increased Serological Response Against Human Herpesvirus 6A Is Associated With Risk for Multiple Sclerosis. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.02715

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Herpes Virus Variant Linked to MS Onset - Technology Networks

In 2013, cancer immunotherapy was being heralded as a breakthrough in the scientific community.

This exciting development led Samantha Burugu, a recent graduate from the department of pathology and laboratory medicine, to follow her dreams of advancing cancer research.

Here she describes her PhD thesis on biomarkers in breast tumors, and shares how she is using her training to inform work with biopharmaceuticals.

Samantha Burugu is a graduate of the department of pathology and laboratory medicine.

While I was finishing up my masters degree in immunology doing foundational research, I wanted to do research that involved clinical tests conducted in hospitals. Clinical tests can be used for disease diagnoses and/or for treatment decisions. After interviewing with my doctoral thesis advisor, Dr. Torsten Nielsen, the pathology and laboratory medicine program was a clear choice. It aligned with my thesis research in biomarker development and my career aspirations.

We hear every day about increasing global incidence of cancers and that everyone in their lifetime has had, or will have, someone within their immediate circle affected by cancer. I wanted to get involved in advancing cancer research. In addition, I had trained in immunology and at the start of my program Science Magazine had just named cancer immunotherapy the 2013 Breakthrough of the Year. Cancer immunotherapy works by unleashing a patients own immune system to fight cancer and this strategy led to incredible results in some cancer patients receiving immunotherapy drugs in clinical trials.

This fall, students graduate from a range of Faculty of Medicine programs.

Read more

Although cancer immunotherapy has led to remarkable clinical results in otherwise incurable cancers, the majority of cancers do not respond to these treatments and we still have not fully elucidated the mechanisms. My doctoral thesis focused on identifying potential biomarkers in breast tumors excised from patients that would indicate a predictive ability to respond to novel cancer immunotherapy drugs.

Using conventional and novel techniques to analyze breast tumors excised from patients, I found the presence of immune cells that can be reactivated by novel immunotherapy drugs to eliminate cancer cells. This work can inform the prioritization and design of immunotherapy clinical trials for breast cancer patients.

My advice is to not be afraid to get involved in the department and try to get the most of graduate studies. By participating in different departmental committees, graduate student associations and attending professional advancement learning series (just to name a few), not only will you build a professional network but also you will develop transferable skillsets.

A healthy community for me is where you feel a sense of belonging. In that sense, you feel respected, engaged, and encouraged by the community. Everyone in a healthy community participates in their own way to make the community better.

I have recently started working at Grifols, a plasma-derived biopharmaceutical company, as a Scientific Development Manager. As part of the Medical Affairs team, I provide medical and scientific understanding of our plasma-derived therapeutic products to health care professionals and researchers.

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Advancing cancer research - UBC Faculty of Medicine

A Wayne State University School of Medicine student and two recent graduates working on a collaborative project in the laboratories of Associate Professors of Ophthalmology, Visual and Anatomical Sciences Ashok Kumar, Ph.D., and Ryan Thummel, Ph.D., have discovered that zebrafish dont contract endophthalmitis.

The eye infection can cause blindness within hours if not diagnosed and treated quickly.

Matthew Rolain, Frank Mei, M.D. 19 and Xiao Yi Zhou, M.D. 17, contributed to the study, Zebrafish are Resistant to Staphylococcus aureus Endophthalmitis, published in Pathogens, a peer-reviewed journal in the field of microbiology and immunology.

The study showed that while humans require only 10 to 100 bugs to cause endophthalmitis, and mice require 5,000 before infection, in the freshwater fish even 250,000 bacteria wont cause the eye infection. The finding indicates that zebrafish eyes are incredibly resistant to such eye infections and possess strong host defense mechanisms.

Dr. Thummel and others in the field have shown that humans and fish share similarities in eye structure and immune responses. Studying why fish, but not human eyes, are resistant, may help identify protective pathways and molecules that could be translated to humans.

Traditionally, we have used a mouse model to study the pathobiology of these infections. In recent years, zebrafish have emerged as an important model organism in biomedical research, providing insight into the pathogenic mechanisms of infectious diseases. We sought to determine their susceptibility with the ocular bacterial infection, Dr. Kumar said. I contacted my colleague Dr. Thummel and discussed the idea, and the project took off with participation of three medical students who completed the task collectively.

Dr. Kumars laboratory focuses on understanding the pathobiology of ocular infections, especially those affecting the retina, such as endophthalmitis. The infection most often occurs due to surgical complications or eye trauma.

Apart from conducting research, I truly enjoyed mentoring these medical students, Dr. Kumar said. I hope they continue develop scientific acumen as they transition to their respective residency programs.

Matthew Rolain will graduate from the School of Medicine in 2020.

Working with Dr. Kumar and Dr. Thummel was an awesome experience, he said. They gave me great guidance and were always very supportive, regardless of the outcome of our experiments. It was nice being able to learn about the research process while working on such an interesting and potentially impactful project. Hopefully the scientific community will be able to build on our results to better help future patients.

Dr. Mei is now a resident in his transitional year in Chicago before starting a two-year Ophthalmology program at the University of Texas Southwestern Medical School in Dallas.

Individually, Drs. Kumar and Thummel were well respected in their separate expertise. However, the unification of their talents into a singular project created a collaborative environment where the strengths of both labs meshed, launching and dramatically expeditingthis project to completionin a very short timeframe. Bridging the gap between Scott Hall and the KresgeEye Institute, Drs. Kumar and Thummel created a warm atmosphere to foster my growth as a researcher. This experience was invaluableand an encouragement for me to seek further collaborations in my career in academic ophthalmology, Dr. Mei said.Lastly,I would like to thank the Medical Summer Research Project through Wayne State and the Kresge Summer Internship for supporting me through this project.

Their colleague, Dr. Zhou is a resident in her transitional year at NorthShore University Health System in Illinois. She completed a one-year fellowship at Bascom Palmer Eye Institute in Miami after graduation.

Moving forward, they plan to test zebrafish susceptibility to other bacterial and fungal pathogens.

The work was supported by grants from the National Institutes of Health (R01EY027381 and R01EY026964 to Dr. Kumar, and R01EY026551 to Dr. Thummel. Histology and imaging core resources were supported by a vision core grant (P30EY04068) and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, Visual and Anatomical Sciences.

Continued here:
Medical student and alumni discover zebrafish are resistant to eye infection - The South End

The globalcancer immunotherapy marketshould reach $96.5 billion by 2021 from $73.0 billion in 2016 at a compound annual growth rate (CAGR) of 5.7%, from 2016 to 2021.

Report Scope:

The scope of this report covers current cancer immunotherapy markets for most common cancers. The market segments included in this report are therapeutic monoclonal antibodies (with special focus on checkpoint inhibitors), synthetic interleukins, interferons, and colony-stimulating factors; small kinase inhibitors of cancer-related targets; protective and therapeutic cancer vaccines; and adoptive cell therapies. This report also covers treatments that are in development for late-stage and early-stage oncolytic viruses. Detailed epidemiological information, discussion of incidence and mortality trends, overview of regulatory landscapes, and analysis of market shares for leading products and companies are also included in this report.

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Report Includes:

An overview of the global markets for cancer immunotherapies and oncolytic virology. Analyses of global market trends, with data from 2015, 2016, and projections of compound annual growth rates (CAGRs) through 2021. Analyses of factors influencing market demand, such as clinical guidelines, demographic changes, and market saturation. Information covering the latest trends, market structure, market size, key drug segments, and trends in technology. Coverage of colony stimulating factors (CSFs), interferon alfa and gamma products, interleukin products and therapeutic monoclonal antibodies, including antibody conjugates, cancer vaccines, and other cancer treatment immunology products. Technological discussions, including the current state, newly issued patents, and pending applications. Profiles of leading companies in the industry.

Report Summary

Cancer is a disease with global implications. There are many different types of cancer, of which the most common types include lung, breast, colon and rectal, stomach, head and neck, prostate, cervical, melanoma, and ovarian cancer, as well as leukemia. Cancer is a genetic disease that is conventionally treated by surgery, radiation therapy, chemotherapy, hormonal therapy, and immunotherapy. Surgery is the mainstay treatment for all cancers. Usually surgery is complimented with radiation or chemotherapy to ensure the clearance of all residual cancer. Despite the advances in treatment, cancer has great plasticity; therefore, after a certain time the effects of treatment fade and cancer returns with acquired resistance. Combination therapy, using multiple modalities including surgery and pharmaceutical or radiation therapy, improves response to treatment.

Radiation and chemotherapy have many side effects. Biological treatment options provide less impactful treatment of cancer. Immunotherapy is a type of biological therapy and it incorporates elements of the immune system in cancer treatment. The immune system has various types of cells and proteins that detect and act upon signs of a disease or infection by harmful and foreign substances such as microbes, bacteria and viruses. The immune system differentiates the bodys own cells and tissues through an evolutionary bar-coding system. This system helps the immune system understand encountered foreign substances as nonself. Cancer cells are recognized as nonself as well. The immune system monitors the body for cancer and destroys when it detects a malignancy. Cancer cells can avoid being recognized by the immune system and develop resistance through numerous methods.

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Since the early 1900s, the connection between cancer and the immune system has caught the attention of various scientists and medical practitioners. Although the early studies were bluntly done without current technological and scientific tools, they nonetheless shed insights leading to the development of the first monoclonal antibodies and to the use of biologically derived synthetic interleukins and interferons. After many decades of research, immunotherapy finally emerged as a fully functionalclinical area in the 1990s. Since then, the cancer therapeutics landscape has changed dramatically.

With the stream of product approvals in recent years, the global immunotherapy market has reached its current value. In 2015, the global cancer immunotherapy market hit $65 billion. The current immunotherapy market contains several blockbuster products reaching their end-of-market exclusivities; however, the market is mostly comprised of newly introduced and expensive therapies. In 2016, the market expanded by more than 10% over the previous year, reaching $73 billion. During the period of 2016 through 2021, the global cancer immunotherapy market is forecast to grow by a 5.7% compound annual growth rate (CAGR), reaching $96.5 billion in 2021.

The strongest growth is expected to occur in checkpoint-inhibitor drugs with a 19.4% CAGR during the forecast period. Immunomodulators are anticipated to show the second-highest growth rates among immunotherapy products, with an 8.4% CAGR during the same period. The combined sales from both segments are expected to make up for nearly one-third of the market, with a combined sales value of $28 billion in 2021. Checkpoint inhibitors are virtually comprised of monoclonal antibodies; however,they are assessed separately due to their immense commercial and clinical significance. Sales from other therapeutic antibodies accrued to $28 billion in 2016, and this value is expected to remain relatively constant through 2021, due to several patent expiries, pressure from anticipated generic entries, and newly introduced classes of drugs expected by 2021.

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Cancer Immunology And Oncolytic Virology Market Opportunities and Forecast Assessment, 2021 - Downey Magazine

A Wayne State University School of Medicine student and two recent graduates working on a collaborative project in the laboratories of Associate Professors of Ophthalmology, Visual and Anatomical Sciences Ashok Kumar, Ph.D., and Ryan Thummel, Ph.D., have discovered that zebrafish dont contract endophthalmitis.

The eye infection can cause blindness within hours if not diagnosed and treated quickly.

Matthew Rolain, Frank Mei, M.D. 19 and Xiao Yi Zhou, M.D. 17, contributed to the study, Zebrafish are Resistant to Staphylococcus aureus Endophthalmitis, published in Pathogens, a peer-reviewed journal in the field of microbiology and immunology.

The study showed that while humans require only 10 to 100 bugs to cause endophthalmitis, and mice require 5,000 before infection, in the freshwater fish even 250,000 bacteria wont cause the eye infection. The finding indicates that zebrafish eyes are incredibly resistant to such eye infections and possess strong host defense mechanisms.

Dr. Thummel and others in the field have shown that humans and fish share similarities in eye structure and immune responses. Studying why fish, but not human eyes, are resistant, may help identify protective pathways and molecules that could be translated to humans.

Traditionally, we have used a mouse model to study the pathobiology of these infections. In recent years, zebrafish have emerged as an important model organism in biomedical research, providing insight into the pathogenic mechanisms of infectious diseases. We sought to determine their susceptibility with the ocular bacterial infection, Dr. Kumar said. I contacted my colleague Dr. Thummel and discussed the idea, and the project took off with participation of three medical students who completed the task collectively.

Dr. Kumars laboratory focuses on understanding the pathobiology of ocular infections, especially those affecting the retina, such as endophthalmitis. The infection most often occurs due to surgical complications or eye trauma.

Apart from conducting research, I truly enjoyed mentoring these medical students, Dr. Kumar said. I hope they continue develop scientific acumen as they transition to their respective residency programs.

Matthew Rolain will graduate from the School of Medicine in 2020.

Working with Dr. Kumar and Dr. Thummel was an awesome experience, he said. They gave me great guidance and were always very supportive, regardless of the outcome of our experiments. It was nice being able to learn about the research process while working on such an interesting and potentially impactful project. Hopefully the scientific community will be able to build on our results to better help future patients.

Dr. Mei is now a resident in his transitional year in Chicago before starting a two-year Ophthalmology program at the University of Texas Southwestern Medical School in Dallas.

Individually, Drs. Kumar and Thummel were well respected in their separate expertise. However, the unification of their talents into a singular project created a collaborative environment where the strengths of both labs meshed, launching and dramatically expeditingthis project to completionin a very short timeframe. Bridging the gap between Scott Hall and the KresgeEye Institute, Drs. Kumar and Thummel created a warm atmosphere to foster my growth as a researcher. This experience was invaluableand an encouragement for me to seek further collaborations in my career in academic ophthalmology, Dr. Mei said.Lastly,I would like to thank the Medical Summer Research Project through Wayne State and the Kresge Summer Internship for supporting me through this project.

Their colleague, Dr. Zhou is a resident in her transitional year at NorthShore University Health System in Illinois. She completed a one-year fellowship at Bascom Palmer Eye Institute in Miami after graduation.

Moving forward, they plan to test zebrafish susceptibility to other bacterial and fungal pathogens.

The work was supported by grants from the National Institutes of Health (R01EY027381 and R01EY026964 to Dr. Kumar, and R01EY026551 to Dr. Thummel. Histology and imaging core resources were supported by a vision core grant (P30EY04068) and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology, Visual and Anatomical Sciences.

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Medical student and alumni discover zebrafish are resistent to eye infection - The South End