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Vitamin A (VA) deficiency (VAD) is observed in both humans and mice with lupus nephritis. However, whether VAD is a driving factor for accelerated progression of lupus nephritis is unclear. In this study, we investigated the effect of VAD on the progression of lupus nephritis in a lupus-prone mouse model, MRL/lpr. We initiated VAD either during gestation or after weaning to reveal a potential time-dependent effect. We found exacerbated lupus nephritis at ∼15 wk of age with both types of VAD that provoked tubulointerstitial nephritis leading to renal failure. This was concomitant with significantly higher mortality in all VAD mice. Importantly, restoration of VA levels after weaning reversed VAD-induced mortality. These results suggest VAD-driven acceleration of tubulointerstitial lupus nephritis. Mechanistically, at the earlier time point of 7 wk of age and before the onset of clinical lupus nephritis, continued VAD (from gestation until postweaning) enhanced plasma cell activation and augmented their autoantibody production, while also increasing the expansion of T lymphocytes that could promote plasma cell autoreactivity. Moreover, continued VAD increased the renal infiltration of plasmacytoid dendritic cells. VAD initiated after weaning, in contrast, showed modest effects on autoantibodies and renal plasmacytoid dendritic cells that were not statistically significant. Remarkably, analysis of gene expression in human kidney revealed that the retinoic acid pathway was decreased in the tubulointerstitial region of lupus nephritis, supporting our findings in MRL/lpr mice. Future studies will elucidate the underlying mechanisms of how VAD modulates cellular functions to exacerbate tubulointerstitial lupus nephritis.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease caused by environmental factors and loss of key proteins. One such protein is a serum endonuclease secreted by macrophages and dendritic cells, Dnase1L3. Loss of Dnase1L3 causes pediatric-onset lupus in humans is Dnase1L3. Reduction in Dnase1L3 activity occurs in adult-onset human SLE. However, the amount of Dnase1L3 necessary to prevent lupus onset, if the impact is continuous or requires a threshold, and which phenotypes are most impacted by Dnase1L3 remain unknown. To reduce Dnase1L3 protein levels, we developed a genetic mouse model with reduced Dnase1L3 activity by deletingDnase1L3from macrophages (cKO). Serum Dnase1L3 levels were reduced 67%, though Dnase1 activity remained constant. Sera were collected weekly from cKO and littermate controls until 50 weeks of age. Homogeneous and peripheral anti-nuclear antibodies were detected by immunofluorescence, consistent with anti-dsDNA antibodies. Total IgM, total IgG, and anti-dsDNA antibody levels increased in cKO mice with increasing age. In contrast to global Dnase1L3−/−mice, anti-dsDNA antibodies were not elevated until 30 weeks of age. The cKO mice had minimal kidney pathology, except for deposition of immune complexes and C3. Based on these findings, we conclude that an intermediate reduction in serum Dnase1L3 causes mild lupus phenotypes. This suggest that macrophage-derived DnaselL3 is critical to limiting lupus.

Re-epithelialization of wounds is a critical element of wound closure. Growth factors have been used in combination with conventional wound management to promote closure, but the method of delivery has been limited to the topical application of ointment formulations. Cytoactive factors delivered in this way have short resident times in wounds and have met with limited success. Here, we demonstrate that methods used to covalently immobilize proteins on synthetic materials can be extended to immobilize cytoactive factors such as the epidermal growth factor (EGF) onto the wound beds of genetically diabetic mice that exhibit impaired healing. Full-thickness splinted excisional wounds were created in diabetic (db/db) mice with a well-defined silicone splint to limit wound contracture. Wound surfaces were treated with a reducing agent to expose sulfhydryl groups and subsequently treated with EGF modified with a heterobifunctional crosslinker. This allowed for the covalent immobilization of the EGF to the wound surface. The conjugation chemistry was validated in vitro and in vivo. In a separate group of mice, wounds were topically treated twice daily with soluble EGF. The mice were evaluated over 11 days for wound closure. This covalent immobilization strategy resulted in EGF being retained on the wound surface for 2 days and significantly increased epithelial wound closure by 20% compared to wounds treated with topical EGF or topical vehicle. Covalent immobilization was not only therapeutically effective but also delivered a markedly reduced load of growth factor to the wound surface compared to topical application (when only 180 ng of EGF was immobilized onto the wound surface in comparison with 7200 ng of topically applied EGF over a period of 11 days). No adverse effects were observed in treated wounds. Results obtained provide proof of concept for the effectiveness of covalent immobilization in the treatment of dysregulated wounds. The covalent immobilization of cytoactive factors represents a potentially transformative approach to the management of difficult chronic wounds.

Commensal bacteria and the immune system have a close and strong relationship that maintains a balance to control inflammation. Alterations of the microbiota, known as dysbiosis, can direct reactivity to self-antigens not only in the intestinal mucosa but also at the systemic level. Our laboratory previously reported gut dysbiosis, particularly lower abundance of bacteria in the familyLactobacillaceae, in lupus-prone MRL/lprmice, a model of systemic autoimmunity. Restoring the microbiota with a mix of 5 differentLactobacillusspecies (spp.),L. reuteri, L. oris, L. johnsonii, L. gasseriandL. rhamnosus, attenuated lupus-liked clinical signs, including splenomegaly and lymphadenopathy. However, our understanding of the mechanism was limited. In this study, we first investigated the effects of individual species. Surprisingly, none of the species individually recapitulated the benefits of the mix. Instead,Lactobacillusspp. acted synergistically to attenuate splenomegaly and renal lymphadenopathy through secreted factors and a CX3CR1-dependent mechanism. Interestingly, oral administration of MRS broth exerted the same benefits likely through increasing the relative abundance of endogenousLactobacillusspp. Mechanistically, we found increased percentages of FOXP3-negative type 1 regulatory T cells with administration of the mix in both spleen and mesenteric lymph nodes. In addition, oral gavage ofLactobacillusspp. decreased the percentage of central memory T cells while increasing that of effector memory T cells in the lymphoid organs. Furthermore, a decreased percentage of double negative T cells was observed in the spleen with the mix. These results suggest thatLactobacillusspp. might act on T cells to attenuate splenomegaly and lymphadenopathy. Together, this study advances our understanding of howLactobacillusspp. attenuate lupus in MRL/lprmice. The synergistic action of these bacteria suggests that multiple probiotic bacteria in combination may dampen systemic autoimmunity and benefit lupus patients.

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

Brain–machine interfaces (BMIs), which enable a two-way flow of signals, information, and directions between human neurons and computerized machines, offer spectacular opportunities for therapeutic and consumer applications, but they also present unique dangers to the safety, privacy, psychological health, and spiritual well-being of their users. The sale of these devices as commodities for profit exacerbates such issues and may subject the user to an unequal exchange with corporations. Catholic healthcare professionals and bioethicists should be especially concerned about the implications for the essential dignity of the persons using the new BMIs. Summary: The commercial sale of brain-machine interfaces (BMIs) generates and exacerbates problems for end-users' safety, psychological health, and spiritual well-being.

Background Recent studies have shown that early growth response 2 (EGR2) is highly induced in activated T cells and regulates T cell functions. In normal C57BL/6 (B6) mice, deletion of EGR2 in lymphocytes results in the development of lupus-like systemic autoimmune disease, which implies indirectly an autoimmune protective role of EGR2. Conversely, increased EGR2 gene expression is suggested to link with high risk of human lupus. In the present studies we sought to clarify the expression and inflammation regulatory role of EGR2 in murine lupus T cells directly. Results We performed RT-qPCR analysis and found a significant increase of EGR2 mRNA expression in human lupus PBMCs and in CD4+ T cells from three different murine lupus models including MRL-lpr, B6-lpr, and B6.sle123 mice at diseased stage when compared to age-matched control MRL or B6 mice. By performing intracellular flow cytometry analysis, we found that EGR2 protein expression was significantly increased in resting lupus (either MRL-lpr or B6.sle123) CD4+ T cells when compared to CD4+ T cells from their respective non-autoimmune controls. However, there was no difference of EGR2 protein expression in anti-CD3 and anti-CD28 stimulated control and lupus CD4+ T cells since there was a stronger induction of EGR2 in activated control CD4+ T cells. EGR2 expression was significantly increased in MRL-lpr mice at an age when lupus is manifested. To understand further the function of elevated EGR2 in lupus CD4+ T cells, we inhibited EGR2 with a specific siRNA in vitro in splenocytes from MRL-lpr and control MRL mice at 15 weeks-of-age. We found that EGR2 inhibition significantly reduced IFNγ production in PMA and ionomycin activated MRL-lpr lupus CD4+ T cells, but not control MRL CD4+ T cells. We also found that inhibition of EGR2 in vitro suppressed the Th1 differentiation in both MRL and MRL-lpr naïve CD4+ T cells. Conclusions EGR2 is highly upregulated in human and murine lupus cells. Our in vitro data suggest a positive role of EGR2 in the regulation of Th1 differentiation and IFNγ production in lupus effector CD4+ T cells.

Objective To report the surgical excision of an iridociliary adenoma and iridal melanocytoma using a postero-anterior cyclo-iridectomy in two dogs. Procedure A 7 year old neutered male English springer spaniel (case 1) and a 7 year old neutered male Labrador mix (case 2) were presented for evaluation of an intrairidal mass OS. Results Complete ophthalmic examination revealed a large, dorsonasal, well-demarcated, intrairidal mass OS. A tan to pink intrairidal mass extending into the iridocorneal angle (case 1) and a pigmented intrairidal mass (case 2) were present. B-mode ultrasonography showed a focal, soft tissue, homogenous mass within the uvea adjacent to and contacting the lens. Neither pars plana involvement nor vitreal extension was present. A postero-anterior cyclo-iridectomy was performed through a polyhedral scleral flap. Thermocautery was used to complete the cyclo-iridectomy (case 1) and partial iridectomy (case 2) to excise the mass en bloc. Histopathology revealed a completely excised iridociliary adenoma (case 1) and iris melanocytoma (case 2). The surgery sites healed without complication. Mild uveitis (cases 1 and 2), scant vitreal hemorrhage (case 1), and mild hyphema (case 2) were present three days postoperatively but had resolved ten days postoperatively. The patients remain visual twenty-two months (case 1) and seven months (case 2) postoperatively with a normal intraocular examination other than an iridal defect and mild dorsonasal lens capsular opacities. Conclusions The surgical approach described in these cases is utilized in physician-based medicine. This approach and the use of thermocautery provide a viable surgical option for excision of large iridociliary tumors in dogs.

The ethics of heritable genome editing (HGE), or germline engineering, are currently being debated vigorously among scientists and bioethicists, and the Catholic Church has declared the procedure to be morally illicit. While these judgments are based mostly on the justice and consequences of the act, a fruitful approach is to consider HGE from the perspective of the virtuous Christian. This article examines participation in HGE according to the virtues of charity, justice, hope, faith, fortitude, temperance, and prudence. HGE does not appear to be consonant with the virtuous life of a Christian person. Summary: The article evaluates heritable genome editing (HGE or genetic engineering of embryos) according to the Christian virtues of charity, justice, hope, faith, fortitude, temperance, and prudence. HGE does not seem to be consonant with the virtuous life of a Christian person.

This study evaluated cataracts in wild boar exposed to chronic low-dose radiation. We examined wild boar from within and outside the Fukushima Exclusion Zone for nuclear, cortical, and posterior subcapsular (PSC) cataracts in vivo and photographically. Plausible upper-bound, lifetime radiation dose for each boar was estimated from radioactivity levels in each animal’s home range combined with tissue concentrations of 134+137Cesium. Fifteen exposed and twenty control boar were evaluated. There were no significant differences in overall prevalence or score for cortical or PSC cataracts between exposed and control animals. Nuclear (centrally located) cataracts were significantly more prevalent in exposed boar (p

B lymphocytes play an important role in the pathophysiology of many autoimmune disorders by producing autoantibodies, secreting cytokines, and presenting antigens. B cells undergo extreme physiological changes as they develop and differentiate. Aberrant function in tolerogenic checkpoints and the metabolic state of B cells might be the contributing factors to the dysfunctionality of autoimmune B cells. Understanding B-cell metabolism in autoimmunity is important as it can give rise to new treatments. Recent investigations have revealed that alterations in metabolism occur in the activation of B cells. Several reports have suggested that germinal center (GC) B cells of individuals with systemic lupus erythematosus (SLE) have altered metabolic function. GCs are unique microenvironments in which the delicate and complex process of B-cell affinity maturation occurs through somatic hypermutation (SHM) and class switching recombination (CSR) and where Bcl6 tightly regulates B-cell differentiation into memory B-cells or plasma cells. GC B cells rely heavily on glucose, fatty acids, and oxidative phosphorylation (OXPHOS) for their energy requirements. However, the complicated association between GC B cells and their metabolism is still not clearly understood. Here, we review several studies of B-cell metabolism, highlighting the significant transformations that occur in GC progression, and suggest possible approaches that may be investigated to more precisely target aberrant B-cell metabolism in SLE. Published version

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors.We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation. This work was supported by internal funding from Virginia-Maryland College of Veterinary Medicine and National Institutes of Health Grants AR067418 and AR073240 (X.M.L.). Published version

Physically enhancing brain–machine interfaces communicate elec­tronically with the patient’s mind in both directions. They present significant opportunities to improve a patient’s health and to restore his or her physical function, but they also present problems for the patient’s sense of agency and self. This is exacerbated by notions of extension and enhancement that are not grounded in an authentic human anthropology that describes the inherently dignified person as an integral union of body and soul.

Embryo adoption, when oriented to the rescue of a dignified human person, is a merciful and morally licit response to an evil consequence of in vitro fertilization and the freezing of embryos. Those who object to embryo adoption not only misconstrue the relevant moral reasoning but exhibit confusion among the object, intention, and circumstances and between two very different potential objects. Because the mercy and charity effected through embryo adoption are at the very heart of moral action, juridical arguments that undermine people’s confidence in these priorities have far-reaching, harmful implications.

Squamous cell carcinoma (SCC) is the most common periocular cancer in horses and the second most common tumor of the horse overall. A missense mutation in damage-specific DNA-binding protein 2 (DDB2, c.1012 C>T, p.Thr338Met) was previously found to be strongly associated with ocular SCC in Haflinger and Belgian horses, explaining 76% of cases across both breeds. To determine if this same variant in DDB2 contributes to risk for ocular SCC in the Arabian, Appaloosa, and Percheron breeds and to determine if the variant contributes to risk for oral or urogenital SCC, histologically confirmed SCC cases were genotyped for the DDB2 variant and associations were investigated. Horses with urogenital SCC that were heterozygous for the DDB2 risk allele were identified in the Appaloosa breed, but a significant association between the DDB2 variant and SCC occurring at any location in this breed was not detected. The risk allele was not identified in Arabians, and no Percherons were homozygous for the risk allele. High-throughput sequencing data from six Haflingers were analyzed to ascertain if any other variant from the previously associated 483 kb locus on ECA12 was more concordant with the SCC phenotype than the DDB2 variant. Sixty polymorphisms were prioritized for evaluation, and no other variant from this locus explained the genetic risk better than the DDB2 allele (P=3.39×10−17, n=118). These data provide further support of the DDB2 variant contributing to risk for ocular SCC, specifically in the Haflinger and Belgian breeds.

BACKGROUND: Belgian horses are commonly affected with ocular squamous cell carcinoma (SCC), the most common cancer of the equine eye. A missense mutation in damage‐specific DNA binding protein 2 (DDB2 c.1013C>T, p.Thr338Met) has been established as a recessive genetic risk factor for ocular SCC in the Haflinger breed. A sample of Belgian horses with unknown SCC phenotype was shown to possess this variant at a similar frequency to the Haflinger breed. Retrospective studies indicate that chestnut coat colour may predispose to the development of SCC. OBJECTIVES: To determine if DDB2 c.1013C>T is a risk factor for ocular SCC in a strictly phenotyped sample of Belgian horses. To investigate associations between coat colour loci genotypes and ocular SCC. STUDY DESIGN: Retrospective and prospective case identification, genetic investigation. METHODS: Genomic DNA was isolated from blood, hair or formalin‐fixed paraffin‐embedded tissue from 25 Belgian horses with histologically confirmed ocular SCC and 18 unaffected Belgian horses. Association testing of 34 single nucleotide variants from 11 genomic loci and genotyping for DDB2 c.1013C>T and coat colour alleles were performed. Exons of DDB2 were sequenced in four cases and two controls. Associations were analysed by Chi‐square or Fisher's exact tests and relative risk was calculated. RESULTS: Homozygosity for DDB2 c.1013C>T was significantly associated with ocular SCC (P = 7.4 × 10⁻⁷). Seventy‐six per cent of affected horses were homozygous for the variant. Relative risk for homozygous horses developing SCC was 4.0 (P = 1.0 × 10⁻⁴). Sequencing DDB2 did not identify a variant more concordant with disease phenotype. An association between disease and coat colour loci was not identified. MAIN LIMITATIONS: Phenotyping was determined at a single timepoint. Each included horse genotyped as chestnut, so association with this MC1R variant could not be investigated. CONCLUSIONS: A missense variant, DDB2 c.1013C>T, p.Thr338Met, is a risk factor for ocular SCC in Belgian horses. A genetic risk test is commercially available.

Objective To report onset and progression of clinical signs of a neuroendocrine neoplasm (NEN) presumed metastatic to the choroid in a dog. Animals studied A 7.5-year-old female spayed German shepherd dog mix referred for advanced imaging and evaluation of a subretinal mass in the right eye. Procedures Procedures performed included general physical and ophthalmic examinations; ocular, orbital, and abdominal ultrasonography; thoracic radiographs; cranial magnetic resonance imaging; serologic testing for infectious agents; analysis of hematologic as well as serum and urine biochemical parameters; echocardiography; electrocardiography; cytologic assessment of lymph nodes; and histopathology and immunohistochemistry of the enucleated globe. Results Examination and imaging identified a pigmented mass within and expanding the superior choroid. Following enucleation, a choroidal NEN with tumor emboli in scleral blood vessels was diagnosed by histopathologic assessment and confirmed by immunohistochemical labelling. Despite extensive and repeated diagnostic testing over many months, a putative primary site was not identified until 19 months after the initial ocular signs were noted. At that time, a heart-base mass and congestive heart failure were highly suggestive of a chemodectoma. Conclusion This comprehensive report of a NEN presumed metastatic to the choroid in a dog suggests that ocular disease can be a very early and solitary sign of NEN in the dog.

Significance Infants are born without an established gut microbiota, which develops rapidly after birth and is shaped by the maternal microbiota. However, how the maternal microbiota, through shaping the neonatal microbiota, would affect the establishment of a strong immune system in neonates remains unclear. Here, we show mechanistically how the maternal microbiota regulates the de novo production of neonatal IgA., Infants are prone to enteric infections due to an underdeveloped immune system. The maternal microbiota, through shaping the neonatal microbiota, helps establish a strong immune system in infants. We and others have observed the phenomenon of enhanced early neonatal immunoglobulin A (IgA) production in preweaning immunocompetent mice nursed by immunodeficient dams. Here, we show that this enhancement of IgA in neonates results from maternally derived microbiota. In addition, we have found that the neonatal IgA production can be induced by Lactobacillus reuteri, which is enriched in the milk of immunodeficient dams. Moreover, we show that while the production of neonatal IgA is dependent on neonatal T cells, the immunodeficient maternal microbiota-mediated enhancement of neonatal IgA has a T cell–independent component. Indeed, this enhancement may be dependent on type 3 innate lymphoid cells in the neonatal small intestinal lamina propria. Interestingly, maternal microbiota-induced neonatal IgA does not cross-react with common enteric pathogens. Future investigations will determine the functional consequences of having this extra IgA.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive inflammation and production of pathogenic antibodies. Histone deacetylase 6 (HDAC6) is a class IIb histone deacetylase. It has been reported that selective HDAC6 inhibition decreases inflammation in lupus mice models. C57BL/6 mice develop SLE-like symptoms following pristane injection. In this study, sex and age-matched wild type and HDAC6−/− mice on the C57BL/6 background were administered 0.5 ml pristane or PBS intraperitoneally (i.p.) at 8–12 weeks-of-age and were euthanized 10 days later. At sacrifice, body weight and spleen weight were measured, sera were collected, splenocytes and peritoneal cells were harvested for flow cytometry. We found pristane administration increased the spleen weight with no difference between WT mice and HDAC6−/− mice. Flow cytometry results showed that there was no difference in T cell or B cell populations in the spleen. Pristane administration promoted the population of CD11b+ Ly6Chigh inflammatory monocytes and CD11b+Ly6G+ neutrophils. Peritoneal recruitment of these inflammatory monocytes and neutrophils in HDAC6−/− mice was significantly decreased compared to the WT mice. Pristane administration also induced the interferon (IFN) signature genes, like Mx1, Oas1a, Irf7, Irf9, Cxcl10, Isg15. qRT-PCR revealed that these IFN signature genes were decreased in HDAC6−/− mice compared to the WT mice. In summary, our results show that HDAC6 knockout inhibits the recruitment of inflammatory monocytes and neutrophils in the peritoneum in early inflammation response to pristane. HDAC6 deletion also inhibited the expression of IFN signature genes with pristine stimulation.

Transcription factor early growth response protein 2 (EGR2) prevents the development of lupus-like autoimmune disease in C57BL/6 (B6) mice by negatively regulating T cell activation, inflammatory cytokine IFNγ and IL-17 production and by suppressing humoral responses. However, increased EGR2 expression has been observed in lupus and also other autoimmune disorders, which contradicts the autoimmune suppressive role of EGR2. Here, we derived conditional EGR2−/−B6/lpr and EGR2−/−B6 mice to investigate and compare the immune regulatory role of EGR2 in autoinflammation (B6/lpr) versus normal physiological (B6) conditions. Significantly, we found that in B6/lpr mice, but not in B6 mice, EGR2 deletion dramatically suppressed serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. We further demonstrated that while EGR2 deletion promoted germinal center B (GCB) cell development in both B6 and B6/lpr mice, it significantly reduced the differentiation of plasma and plasmablasts cells in B6/lpr mice. EGR2 deletion promoted splenomegaly and T cell activation in both B6 and B6/lpr mice, but it differentially regulated the profiles of immune cell subpopulations in the spleens of B6 and B6/lpr mice. Especially, EGR2 deletion dramatically suppressed the development of double negative T cells in B6/lpr mice. Moreover, we found that EGR2 had an opposite role in regulating IL-17 production in in vitro activated splenocytes from B6/lpr and B6 mice, although EGR2 had a similar suppressive role for IFNγ production. Together, our data strongly suggest that the immune and autoimmune regulatory roles of EGR2 are context dependent, and should not be generalized in normal physiology and different pathological conditions. Supported by VMCVM-VCOM Center for One Heath Research Seed Grant Program

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. The cause of SLE is not only genetic; in fact, environmental factors may play a more important role in disease development. The MRL/lpr mouse model lupus-like symptoms due to multiple SLE susceptible loci in the MRL background, and offers an accelerated model compared to the MRL parent strain due to the Faslpr mutation. Recently, our laboratory witnessed a loss of disease phenotype in our in-house colony of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX; Stock Number 000485) to our in-house mice. The single-nucleotide polymorphism (SNP) analysis showed no genetic drift, suggesting that environmental factors could be triggering a phenotypic drift. Surprisingly, the newly purchased JAX mice also had attenuation of glomerulonephritis. Even though JAX mice manifested similar attenuation of lupus nephritis, our in-house colony showed differences in organ weights. Furthermore, males showed a significantly higher level of anti-double stranded DNA auto-IgG consistent with germinal center maturation. In addition, in-house males had significantly higher levels of microRNA-21 and microRNA-183 explaining spleen size difference. Moreover, the composition of gut microbiota was different between in-house and new JAX mice at early age, with many groups of bacteria differing at later time points, which might explain some of the phenotypic differences. These results suggest that microRNAs and gut microbiota might be responsible for the phenotypic differences of MRL/lpr mice in JAX and our colonies as they were genetically identical. On the other hand, the attenuation of nephritis in both groups requires further investigation. Supported by R01AR073240

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Autoimmune diseases, such as systemic lupus erythematosus, are characterized by excessive inflammation in response to self-antigens. Loss of appropriate immunoregulatory mechanisms contribute to disease exacerbation. We previously showed the suppressive effect of vancomycin treatment during the "active-disease" stage of lupus. In this study, we sought to understand the effect of the same treatment given before disease onset. To develop a model in which to test the regulatory role of the gut microbiota in modifying autoimmunity, we treated lupus-prone mice with vancomycin in the period before disease development (3-8 weeks of age). We found that administration of vancomycin to female MRL/lpr mice early, only during the pre-disease period but not from 3 to 15 weeks of age, led to disease exacerbation. Early vancomycin administration also reduced splenic regulatory B (Breg) cell numbers, as well as reduced circulating IL-10 and IL-35 in 8-week old mice. Further, we found that during the pre-disease period, administration of activated IL-10 producing Breg cells to mice treated with vancomycin suppressed lupus initiation, and that bacterial DNA from the gut microbiota was an inducer of Breg function. Oral gavage of bacterial DNA to mice treated with vancomycin increased Breg cells in the spleen and mesenteric lymph node at 8 weeks of age and reduced autoimmune disease severity at 15 weeks. This work suggests that a form of oral tolerance induced by bacterial DNA-mediated expansion of Breg cells suppress disease onset in the autoimmune-prone MRL/lpr mouse model. Future studies are warranted to further define the mechanism behind bacterial DNA promoting Breg cells. NIAMSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Arthritis & Musculoskeletal & Skin Diseases (NIAMS) [AR073240, AR067418] NIAMS AR073240, AR067418 (XL).

OBJECTIVE:(a) To evaluate the epidemiology of equine eosinophilic keratoconjunctivitis (EK) in the western United States, (b) to ascertain the efficacy of keratectomy and diamond burr debridement vs medical management alone, (c) to determine the efficacy of various medical therapies, and (d) to further characterize the histopathologic findings of the disease in horses. ANIMALS STUDIED:Twenty-nine horses (47 eyes) diagnosed with EK from 1993 to 2017. PROCEDURE:Retrospective medical record review; owner questionnaire. RESULTS:Average age of presentation was 11±4years. Warmbloods were significantly overrepresented (P=0.024). Twenty horses were treated with medical therapy alone, five were treated with superficial lamellar keratectomy, and four were treated with diamond burr debridement. Follow-up data were available for 38 eyes of 23 horses. Median time to resolution for horses treated with either superficial keratectomy or diamond burr debridement (62days) was not statistically significantly different from those that underwent medical therapy alone (46days; P=0.33). Eyes treated with topical steroids had a statistically significant longer median time to resolution (61days) compared to those that did not receive topical steroid (44days; P=0.023). Common histopathologic findings in keratectomy samples included the presence of eosinophils, vascularization, and an eosinophilic membrane spanning areas of ulceration. CONCLUSION:In this population, time to EK resolution was similar for horses treated with medical and surgical management. The use of topical steroids was associated with a prolonged time to resolution. Keratectomy samples from horses with EK had similar findings to those reported in other species.

This report describes post-traumatic ocular lymphoma in 3 companion rabbits; 2 rabbits with unilateral disease and 1 with bilateral disease. Historical findings suggestive of a traumatic event included either external unilateral ocular trauma or bilateral phacoemulsification. Severe corneal changes, presence of an anterior chamber mass(es), low intraocular pressures, and ocular discomfort were noted on ophthalmic examinations. All eyes were treated for variable courses with standard ophthalmic topical medications (antibiotic, anti-inflammatories, and steroid) and systemic anti-inflammatories. Based upon progression of disease, all affected eyes were ultimately enucleated; lenticular capsular rupture and a round cell neoplasm effacing normal structures with variable mitotic indices were noted on histopathology. Neoplastic lymphocytes strongly expressed CD79a via immunohistochemistry and lacked expression for CD3, indicating B lymphocyte lineage and not of T cell lineage. A single animal had evidence of local metastasis to a regional lymph node. Post-traumatic sarcomas have been reported in this species previously, however, these cases are the first reports of this novel round cell variant, named post-traumatic ocular lymphoma due to the B cell lineage confirmed through immunohistochemistry. Clinicians should be aware of this clinical presentation and the possibility of metastasis when evaluating ocular pathology in this species.

The ethical implications of brain–machine interface (BMI) are mind-boggling. Connecting a person’s brain to a computer or other machine and successfully transmitting thoughts and instructions bidirectionally has enormous potential for therapeutic applications in health care; but great harm can come from meddling with fragile human brains, succumbing to the temptations of cognitive enhancement, and exposing vulnerable individuals to the power relations formed among owners, generators, users, and sellers of what was once considered the private realm of human thoughts.

Purpose To evaluate the recurrence frequency and the time to recurrence of canine palpebral masses treated with debulking and cryotherapy. Methods Medical records of dogs that underwent debulking and cryotherapy for palpebral masses from December 2010 to November 2015 at the University of Minnesota's Veterinary Medical Center were reviewed. A telephone survey with owners was used to determine recurrence and time to recurrence. Results Forty-six masses (31 dogs) were included in this retrospective study. Included palpebral masses were as follows: adenomas 17/46 (37%), epitheliomas 8/46 (17.4%), papillomas 6/46 (13%), inflammatory masses 6/46 (13%), mixed adenoma-papillomas 6/46 (13%), mixed epithelioma-papillomas 2/46 (4.4%), and a melanocytoma 1/46 (2.2%). Of the seven masses that were noted to recur (7/46, 15.2%), three were mixed adenoma-papillomas (three of six adenoma-papillomas, 50%), two were epitheliomas (two of eight epitheliomas, 25%), one was a papilloma (one of six papillomas, 16.7%), and one was a melanocytoma (one of one melanocytoma, 100%). Average recurrence time was 367.9 days [range: 30-904 days]. A significant association between tumor type and tumor recurrence was found (P = .009). Adenomas were significantly less likely to recur (P = .017), and mixed adenoma-papillomas were significantly more likely to recur (P = .001). Conclusions Debulking and cryotherapy is an effective method of treatment for eyelid masses in dogs with a low study recurrence rate of 15.2% and average recurrence time of 367.9 days. Results also suggest that mixed adenoma-papilloma masses have a significantly higher recurrence rate than other eyelid masses, whereas adenomas had no recurrence in our study population.

Squamous cell carcinoma (SCC) is the most common cancer affecting the equine eye, with a higher incidence documented in Haflinger horses. Recently, a missense variant in the gene damage specific DNA binding protein 2 (DDB2, p.Thr338Met) on ECA12 was identified as a risk factor for the development of limbal SCC in Haflinger horses. SCC also occurs on the nictitating membrane; therefore, investigating the role of this missense variant in nictitating membrane SCC is warranted. In this study, a common ancestor was identified among Haflinger horses affected with limbal SCC or with nictitating membrane SCC, thus supporting a recessive risk factor for the development of cancer at both ocular locations. Analysis of genotype data from Haflinger horses with and without nictitating membrane SCC revealed that the same region on ECA12 associated with limbal SCC was also associated with nictitating membrane SCC (P T, p.Thr338Met, is a risk factor for nictitating membrane SCC, as 88% of our cases were homozygous for this variant and no other polymorphism was more strongly associated (P = 4.13 × 10-14 ). These data indicate that the genetic risk is the same for the development of both limbal and nictitating membrane SCC in Haflinger horses and validates utilization of genetic testing of the DDB2 variant for both clinical management and the guidance of mating decisions.

An 18-year-old American Miniature Horse mare was presented with a complaint of a scleral swelling affecting the right eye and a history of suspected trauma 6 weeks prior to evaluation. Clinical findings included severe blepharospasm, a bulbous swelling of the dorsotemporal bulbar conjunctiva, and phthisis bulbi. Ocular ultrasound was recommended but declined. Enucleation was elected for the blind, painful eye and was performed standing. Gross and histopathologic examinations of the globe were consistent with extrusion of the lens to the episcleral space, which is classified as a traumatic phacocele when associated with naturally occurring trauma. The location of lens entrapment suggested globe rupture occurred at the limbus, which is described as one of the weakest points of the equine globe. Subconjunctival dislocation of the lens and development of a traumatic phacocele should be considered as a differential diagnosis for horses presenting with subconjunctival masses, apparent aphakia, and historical trauma.