A case series of hypersensitivity reactions to ventriculoperitoneal shunt material.

Hypersensitivity reactions to ventriculoperitoneal (VP) or lumboperitoneal (LP) shunts are rare. Symptoms often resolve following shunt replacement with a silicone-free hypoallergenic shunt. We describe novel cases of allergies to both standard and hypoallergenic shunts and highlight the utility of patch testing. Patient 1, a 24-year-old female with Chiari I malformation, developed diarrhea, abdominal pain, and rash along the LP shunt tract. Patch testing was positive. The shunt was replaced with a hypoallergenic VP shunt with symptom improvement. Five weeks later, she developed a new rash. Subsequent patch testing to the hypoallergenic shunt was positive. Patient 2, a 37-year-old female with Chiari I malformation, developed pruritus along the VP shunt tract. Patch testing to the standard shunt was positive. The shunt was replaced with a hypoallergenic shunt, with symptomatic improvement. One month later, she developed neck pain, headache, and pruritis. Patch testing to the hypoallergenic shunt was positive. The development of a pruritic rash along the shunt tract with or without gastrointestinal symptoms should prompt shunt allergy evaluation and consideration of patch testing to the shunt material.

Responses of cultured human keratocytes and myofibroblasts to ethyl pyruvate: a microarray analysis of gene expression.

PURPOSE: Ethyl pyruvate (EP) has pharmacologic effects that remediate cellular stress. In the organ-cultured murine lens, EP ameliorates oxidative stress, and in a rat cataract model, it attenuates cataract formation. However, corneal responses to EP have not been elucidated. In this study, the potential of EP as a therapeutic agent in corneal wound healing was determined by examining its effects on the transition of quiescent corneal stromal keratocytes into contractile myofibroblasts. METHODS: Three independent preparations of cultured human keratocytes were treated with TGF-beta1, to elicit a phenotypic transition to myofibroblasts in the presence or absence of 10 or 15 mM EP. Gene expression profiles of the 12 samples (keratocytes +/- EP +/- TGF-beta1 for three preparations) were produced by using gene microarrays. RESULTS: TGF-beta1-driven twofold changes in at least two of three experiments defined a group of 1961 genes. Genes showing twofold modulation by EP in at least two experiments appeared exclusively in myofibroblasts (857 genes), exclusively in keratocytes (409 genes), or in both phenotypes (252 genes). Analysis of these three EP-modulated groups showed that EP (1) inhibited myofibroblast proliferation with concomitant modulation of some cell cycle genes, (2) augmented the NRF2-mediated antioxidant response in both keratocytes and myofibroblasts, and (3) modified the TGF-beta1-driven transition of keratocytes to myofibroblasts by inhibiting the upregulation of a subset of profibrotic genes. CONCLUSIONS: These EP-induced phenotypic changes in myofibroblasts indicate the potential of EP as a therapeutic agent in corneal wound healing.

Rho-mediated regulation of TGF-beta1- and FGF-2-induced activation of corneal stromal keratocytes.

PURPOSE: To investigate the role of Rho GTPase signaling in FGF-2- and TGF-beta1-induced activation of corneal keratocytes. METHODS: Keratocytes isolated from rabbit corneal stroma and plated in a serum-free medium were treated with FGF-2/heparin or TGF-beta1 in the presence or absence of Rho inhibitor (C3 exoenzyme) or ROCK (Rho kinase) inhibitor (Y27632). Specific phenotypic changes were analyzed by immunocytochemistry and Western blot analysis, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR. RESULTS: TGF-beta1-induced expression of alpha-SMA and transcription of alpha-SMA mRNA in activated keratocytes were reduced by Rho or ROCK inhibition during the activation. In nonactivated keratocytes, the expression of alpha3(IV) collagen was downregulated by Rho-inhibition. TGF-beta1- or FGF-2-induced downregulation of the expression of alpha3(IV) collagen and its mRNA was not significantly altered by Rho or ROCK inhibition. TGF-beta1- and FGF-2-induced decreases in cell-associated and secreted KS, and lumican mRNA levels were prevented by Rho or ROCK inhibition. However, FGF-2-induced decreases in keratocan mRNA levels were prevented by Rho inhibition but not by ROCK inhibition. Whereas Rho inhibition downregulated both TGF-beta1- and FGF-2-induced tenascin-C expression, ROCK inhibition was found to downregulate only TGF-beta1-induced expression. CONCLUSIONS: Rho signaling has a significant role in the activation of keratocytes. Rho, via ROCK-independent and/or -dependent pathways differentially regulates the TGF-beta1-induced expression of alpha-SMA and TGF-beta1- and FGF-2-induced de novo expression of tenascin-C and the downregulation of alpha3(IV) collagen and KSPGs, lumican and keratocan.

Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

PURPOSE: The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. METHODS: Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. RESULTS: ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. CONCLUSIONS: ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation.

PURPOSE: To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation. METHODS: Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents. RESULTS: Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels. CONCLUSIONS: The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.