Genetic and Epigenetic Factors at <i>COL2A1</i> and <i>ABCA4</i> Influence Clinical Outcome in Congenital Toxoplasmosis

Background: Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings: In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions: These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.</p

Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

Background: Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute.Methods and Findings: In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting.Conclusions: These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite

Decreased Vision in Multiple Sclerosis is Associated With Novel Foveal Features Visualized Using Adaptive Optics

Multifocal electroretinography has demonstrated dysfunction of non-ganglion cell retinal components in people with MS not associated with structural abnormalities on OCT, which raises the possibility of previously unidentified retinal pathology in MS. The objective of this study was to identify structural pathologies in the foveal avascular zone of people with MS that may account for these functional changes

Corneal dysfunction precedes the onset of hyperglycemia in a mouse model of diet-induced obesity.

PurposeThe purpose of this study was to use a mouse model of diet-induced obesity to determine if corneal dysfunction begins prior to the onset of sustained hyperglycemia and if the dysfunction is ameliorated by diet reversal.MethodsSix-week-old male C57BL/6 mice were fed a high fat diet (HFD) or a normal diet (ND) for 5-15 weeks. Diet reversal (DiR) mice were fed a HFD for 5 weeks, followed by a ND for 5 or 10 weeks. Corneal sensitivity was determined using aesthesiometry. Corneal cytokine expression was analyzed using a 32-plex Luminex assay. Excised corneas were prepared for immunofluorescence microscopy to evaluate diet-induced changes and wound healing. For wounding studies, mice were fed a HFD or a ND for 10 days prior to receiving a central 2mm corneal abrasion.ResultsAfter 10 days of HFD consumption, corneal sensitivity declined. By 10 weeks, expression of corneal inflammatory mediators increased and nerve density declined. While diet reversal restored nerve density and sensitivity, the corneas remained in a heightened inflammatory state. After 10 days on the HFD, corneal circadian rhythms (limbal neutrophil accumulation, epithelial cell division and Rev-erbα expression) were blunted. Similarly, leukocyte recruitment after wounding was dysregulated and accompanied by delays in wound closure and nerve recovery.ConclusionIn the mouse, obesogenic diet consumption results in corneal dysfunction that precedes the onset of sustained hyperglycemia. Diet reversal only partially ameliorated this dysfunction, suggesting a HFD diet may have a lasting negative impact on corneal health that is resistant to dietary therapeutic intervention

Serial block-face scanning electron microscopy reveals neuronal-epithelial cell fusion in the mouse cornea.

The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8-10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ≤ 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation

Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils, and Mast Cell Degranulation

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the β2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation

P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages

The P2X₇R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X₇R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X₇R knockout mice (P2X₇R^−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X₇R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production

Evidence for associations between the purinergic receptor P2X7 (P2RX7) and toxoplasmosis

Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong proinflammatory responses. Ligation of ATP by purinergic receptor P2X 7, encoded by P2RX7, stimulates proinflammatory cytokines and can lead directly to killing of intracellular pathogens. To determine whether P2X7 has a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z-scores ± 2.429; P=0.015) between the derived C(+)G(-) allele (f=0.68; OR=2.06; 95% CI: 1.14-3.75) at single-nucleotide polymorphism (SNP) rs1718119 (1068T>C; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical subgroups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0-4.25; 0.004<P<0.009) when hydrocephalus was removed from the analysis. Association with toxoplasmic retinochoroiditis was replicated (FBAT Z-scores ± 3.089; P=0.002) in a small family-based study (60 families; 68 affected offspring) of acquired infection in Brazil, where the ancestral T(+) allele (f=0.296) at SNP rs1718119 was strongly protective (OR=0.27; 95% CI: 0.09-0.80)