Infection-Induced Porcine Ex Vivo Corneal Wound Model to Study the Efficacy of Herpes Simplex Virus-1 Entry and Replication Inhibitors.

Corneal infections by viruses and bacteria can result in ocular surface defects, ulcers, or wounds. Herpes simplex virus type-1 (HSV-1) is a human virus with global seroprevalence in the range of 60-90%. While the virus more commonly causes mucocutaneous lesions including ulcers on the face and mouth, it is also a leading cause of infection-associated blindness. In this chapter, we discuss an in-depth protocol required to evaluate corneal damage due to HSV-1 infection using porcine models of ex vivo infection. Our methods can be adapted to study similar infections caused by other viruses and bacteria.

An M2 Rather than a TH2 Response Contributes to Better Protection against Latency Reactivation following Ocular Infection of Naive Mice with a Recombinant Herpes Simplex Virus 1 Expressing Murine Interleukin-4.

We found previously that altering macrophage polarization toward M2 responses by injection of colony-stimulating factor 1 (CSF-1) was more effective in reducing both primary and latent infections in mice ocularly infected with herpes simplex virus 1 (HSV-1) than M1 polarization by gamma interferon (IFN-γ) injection. Cytokines can coordinately regulate macrophage and T helper (TH) responses, with interleukin-4 (IL-4) inducing type 2 TH (TH2) as well as M2 responses and IFN-γ inducing TH1 as well as M1 responses. We have now differentiated the contributions of these immune compartments to protection against latency reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency-associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN-γ (HSV-IFN-γ) gene using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN-γ gene did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than in those infected with HSV-IFN-γ or parental virus. Levels of primary virus replication in the eyes and trigeminal ganglia (TG) were similar in the three groups of mice, but the numbers of gC+ cells were lower on day 5 postinfection in the eyes of HSV-IL-4-infected mice than in those infected with HSV-IFN-γ or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- and HSV-IFN-γ-infected mice than in those infected with parental virus, with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1, and IFN-γ mRNA, while reduced latency and T-cell exhaustion correlated with lower gC+ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group greater than 3,000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1, responses in HSV-1 infection would improve protection against establishment of latency, reactivation, and eye disease.IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the United States and a major cause of virus-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency reactivation is a major therapeutic goal. It is well established that absence of latency-associated transcripts (LATs) reduces latency reactivation. Here we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of TH2/M2 responses) or IFN-γ (an inducer of TH1/M1 responses) in place of LAT further reduced latency, with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages toward an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency reactivation.

The outcome of penetrating keratoplasty for corneal scarring due to herpes simplex keratitis.

PURPOSE:: To determine the outcomes of penetrating keratoplasty (PK) for treatment of corneal scarring caused by Herpes simplex virus (HSV) keratitis, and whether the corneal scar type affects treatment outcome. METHODS:: A retrospective analysis of patients who underwent PK for HSV-related corneal scarring between January 2008 and July 2011 was performed. The patients were categorized into two groups. Group 1 consisted of patients with a quiescent herpetic corneal scar and group 2 consisted of patients who developed a corneal descemetocele or perforation secondary to persistent epithelial defects with no active stromal inflammation. The mean follow-up was 21.30 ± 14.59 months. The main parameters evaluated were recurrence of herpetic keratitis, graft rejection, graft failure, visual acuity, and graft survival rate. RESULTS:: There were 42 patients in group 1 and 13 in group 2. Preoperative BCVA varied from hand movements to 0.7 logMAR. Postoperatively, 34 patients (61.8%) achieved visual acuity of 0.6 logMAR or more. Recurrence of HSV keratitis was noted in 12 (28.57%) eyes in group 1 and 4 (30.76%) eyes in group 2 (p=0.40). Graft rejection occurred in 4 eyes (9.52%) in group 1 and in 3 (23.07%) eyes in group 2 (p=0.58). The 1-, 2-, and 3-year graft survival rates were 91.9%, 76.0%, and 65.1% in group 1, and 89.5%, 76.0%, and 63.6% in group 2 (p=0.91), respectively. CONCLUSIONS:: Although there were different recurrence and graft rejection rates for two groups, the graft survival rates at 3 years were similar. According to our results, without inflammation, corneal herpetic scarring with a descemetocele or perforation achieved similar graft survival rates with quiescent herpetic corneal scars.

The outcome of penetrating keratoplasty for corneal scarring due to herpes simplex keratitis / O resultado da ceratoplastia penetrante para cicatriz de córnea consequente à ceratite por herpes simplex

ABSTRACT Purpose: To determine the outcomes of penetrating keratoplasty (PK) for treatment of corneal scarring caused by Herpes simplex virus (HSV) keratitis, and whether the corneal scar type affects treatment outcome. Methods: A retrospective analysis of patients who underwent PK for HSV-related corneal scarring between January 2008 and July 2011 was performed. The patients were categorized into two groups. Group 1 consisted of patients with a quiescent herpetic corneal scar and group 2 consisted of patients who developed a corneal descemetocele or perforation secondary to persistent epithelial defects with no active stromal inflammation. The mean follow-up was 21.30 ± 14.59 months. The main parameters evaluated were recurrence of herpetic keratitis, graft rejection, graft failure, visual acuity, and graft survival rate. Results: There were 42 patients in group 1 and 13 in group 2. Preoperative BCVA varied from hand movements to 0.7 logMAR. Postoperatively, 34 patients (61.8%) achieved visual acuity of 0.6 logMAR or more. Recurrence of HSV keratitis was noted in 12 (28.57%) eyes in group 1 and 4 (30.76%) eyes in group 2 (p=0.40). Graft rejection occurred in 4 eyes (9.52%) in group 1 and in 3 (23.07%) eyes in group 2 (p=0.58). The 1-, 2-, and 3-year graft survival rates were 91.9%, 76.0%, and 65.1% in group 1, and 89.5%, 76.0%, and 63.6% in group 2 (p=0.91), respectively. Conclusions: Although there were different recurrence and graft rejection rates for two groups, the graft survival rates at 3 years were similar. According to our results, without inflammation, corneal herpetic scarring with a descemetocele or perforation achieved similar graft survival rates with quiescent herpetic corneal scars.

RESUMO Objetivo: Determinar os resultados da ceratoplastia penetrante (PK) para o tratamento da cicatriz da córnea consequente à ceratite por Herpes simplex vírus (HSV), e se o tipo de cicatriz na córnea afeta o resultado cirúrgico. Métodos: Foi realizada análise retrospectiva dos pacientes, submetidos à PK para a cicatriz da córnea relacionados com o HSV entre janeiro de 2008 e julho de 2011. Os pacientes foram divididos em dois grupos. Grupo 1 consistiu de pacientes que tiveram cicatriz corneana herpética quiescente e grupo 2 consistiu de pacientes que desenvolveram descemetocele ou perfuração córnea secundária a defeitos epiteliais persistentes sem inflamação estromal ativa. O seguimento médio foi de 21,30 ± 14,59 meses. Os principais parâmetros avaliados foram recorrência de ceratite herpética, rejeição de enxerto, falência do enxerto, acuidade visual e taxa de sobrevida do enxerto. Resultados: Foram avaliados 42 pacientes do grupo 1 e 13 doentes do grupo 2. Acuidade visual pré-operatória variou de movimentos das mãos (HM) para 0,7 logMAR. No pós-operatório, 34 pacientes (61,8%) atingiram acuidade visual de 0,6 logMAR ou melhor. Doze olhos (28,57%) tiveram recorrência de HSV ceratite no grupo 1, e quatro olhos (30,76%) tiveram recorrência no grupo 2 (p=0,40). A rejeição do enxerto ocorreu em 4 olhos (9,52%) no grupo 1, e em 3 olhos do grupo 2 (23,07%; p=0,58), taxa de sobrevivência do enxerto foi de 91,9% a 1 ano, 76,0% aos 2 anos e 65,1% aos 3 anos no grupo 1, e 89,5% a 1 ano, 76,0% aos 2 anos e 63,6% aos 3 anos no grupo 2 (p=0,91). Conclusões: Embora diferentes taxas de recorrência e de rejeição do enxerto foram encontradas nos dois grupos, a taxa de sobrevida do enxerto em 3 anos foi semelhantes nos dois grupos. De acordo com nossos resultados, em casos sem inflamação, a cicatriz herpética da córnea com descemetocele ou perfuração demonstra as taxas de sobrevivência do enxerto semelhantes às da cicatriz corneana herpética quiescente.