http://repub.eur.nl/res/aut/24606/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/24626/ 2009-11-01T00:00:00Z Background: The incidence and clinical significance of herpes simplex virus type 1 (HSV-1) acyclovir resistance were determined in patients with recurrent herpetic keratitis (RHK). Methods: Sequential corneal isolates (np39) from 15 immunocompetent patients with RHK were assayed for acyclovir susceptibility and genotyped by analyzing the hypervariable regions of the HSV-1 genes US1 and US12. The thymidine kinase (TK) gene of each isolate was sequenced, and the proportion of acyclovir-resistant viruses within isolates was determined. Results: Uniform acyclovir-resistant or acyclovir-sensitive sequential isolates were identified in 4 and 2 patients, respectively. Notably, the acyclovir susceptibility of sequential isolates changed from acyclovir sensitive to acyclovir resistant (5 patients) or from acyclovir resistant to acyclovir sensitive (3 patients). The acyclovir-resistant phenotype of the isolates correlated with the patients unresponsiveness to acyclovir therapy. Combined analyses of the TK gene and genotype of sequential isolates showed that acyclovir-sensitive isolates contained multiple acyclovirresistant variants of the same virus and that an identical acyclovir-resistant HSV-1 strain reappeared in the patients cornea during RHK episodes. Conclusions: Corneal HSV-1 isolates are mixtures of acyclovir-sensitive and acyclovir-resistant viruses that share the same genotype but have different TK sequences. Recovery of the same acyclovir-resistant virus during consecutive herpetic keratitis episodes suggests that acyclovir-resistant HSV-1 establishes latency and reactivates intermittently to cause acyclovir-refractory RHK. 2009 by the Infectious Diseases Society of America. http://repub.eur.nl/res/pub/24624/ 2009-07-01T00:00:00Z Background. We determined the prevalence and clinical consequences of herpes simplex virus (HSV) type 1 (HSV-1), HSV type 2 (HSV-2), and varicella-zoster virus (VZV) in cornea tissues obtained after penetrating keratoplasty (PKP) was performed. Methods. The excised corneas of 83 patients with a history of herpetic keratitis (HK; hereafter referred to as "patients with HK") and 367 patients without a history of HK (hereafter referred to "patients without HK") were analyzed by real-time polymerase chain reaction (PCR) and virus culture for the presence of HSV-1, HSV-2, and VZV In addition, 273 post-PKP donor corneoscleral rims were analyzed. The medical records of the transplant patients were reviewed to determine the risk factors influencing intracorneal viral load and graft survival. Results. HSV-1 was the most prevalent herpesvirus. Both the prevalence of HSV-1 and the HSV-1 DNA load were higher in the corneas of patients with HK than in those of patients without HK. The HSV-1 DNA load in the corneas of patients with HK correlated with age, the recurrence-free interval, cornea neovascularization, steroid treatment before PKP, and disease severity. Herpesvirus DNA was detected in 2 of 273 corneoscleral rims. Graft survival was inversely correlated with the corneal HSV-1 DNA load in patients with HK. Conclusions. The data presented in this study argue for the implementation of real-time HSV-1 PCR to analyze the excised corneas of patients with HK, to improve post-PKP diagnosis and therapy. Screening of donor corneal tissues for herpesviruses is redundant to prevent newly acquired post-PKP HK. http://repub.eur.nl/res/pub/20467/ 2009-06-30T00:00:00Z Our first knowledge of human herpes can be traced back to the ancient Greeks, who coined the phrase: ‘herpes’. Hippocrates used this term to describe lesions that appeared to creep or crawl along the skin. The virus that causes this condition, herpes simplex virus (HSV), has been described in more detail over the past 3 decades. The spectrum of herpetic disease continues to expand. Nowadays, the structure of this virus is well documented. Herpesviruses are linear double-stranded DNA viruses, consisting of an envelope, a tegument, a nucleocapsid, and a core. The size of herpesvirus virions varies from 125-260 nm, and the shape varies from spherical to pleomorphic. Virus-encoded glycoproteins, exhibited as spikes are embedded in the envelope, which wraps the capsid. Herpesviruses encode a large group of enzymes involved in nucleic acid metabolism, DNA synthesis, and assembling capsid into the cell nucleus. Virions are processed in the cytoplasm. Production of infectious virus accompanies the destruction of the infected cells. All herpesviruses are able to remain latent in the host. More than 100 different herpesviruses are known to infect vertebrates. Only 8 of them can infect humans: human herpesvirus 1 to 8 (HHV1-HHV8). They are classified into three subfamilies based on their biological properties and DNA sequence homology: alpha-, beta-, gamma-herpesvirinae. http://repub.eur.nl/res/pub/28953/ 2008-09-01T00:00:00Z Aim: Recent phylogenetic analyses on the herpes simplex virus type 1 (HSV-1) genes US4, encoding glycoprotein G (gG) and US7, encoding gl, of clinical HSV-1 isolates have led to the classification of HSV-1 into three genotypes, arbitrarily designated as A, B and C. The prevalence of the HSV-1 gG and gl genotypes and their potential disease association was determined in a large cohort of patients with herpetic keratitis (HK). Methods: Primary corneal HSV-1 isolates of 178 HK patients were genotyped by a PCR-based restriction fragment length polymorphism method targeting the viral genes US4 and US7. Results: Genotype B was more frequently expressed by the corneal HSV-1 isolates compared with genotypes A and C. Fifty-five of 178 corneal isolates (31%) had different genotypes in both loci. No clinically relevant associations were observed between the HSV-1 genotypes and disease outcome in the HK patients studied. Conclusions: The data presented demonstrate a high frequency of recombinant corneal HSV-1 isolates and suggest that clinical outcome of HSV-1-induced keratitis is independent of a gG or gl genotype. http://repub.eur.nl/res/pub/29144/ 2008-09-01T00:00:00Z The prevalence and molecular characteristics of isolates from 173 immunocompetent patients with herpetic keratitis (HK) whowere infected with acyclovir (ACV)-resistant(ACVR) corneal herpes simplex virus (HSV)-1 was determined. Isolates from 11 (6.4%) of the patients were ACVR, and 9 of these 11 patients were refractory to therapy with ACV; the ACVRisolates from 5 and 1 of these 9 patients were cross-resistant to gancyclovir and to both gancyclovir and foscarnet, respectively. Of the 11 ACVRisolates, 10 had, in the thymidine kinase gene, mutations that presumably conferred the ACVRphenotype. These data demonstrate a relatively high prevalence of corneal HSV-1 ACVRisolates in patients with HK, which emphasizes the need to monitor for ACV susceptibility in patients with HK who are refractory to therapy with ACV. http://repub.eur.nl/res/pub/35865/ 2007-01-01T00:00:00Z PURPOSE. Granulocyte macrophage colony-stimulating factor (GM-CSF) is thought to play a key role in chronic inflammatory diseases by governing the survival and function of infiltrating neutrophils. The objective of this study was to determine the putative role of GM-CSF in the pathogenesis of human herpetic stromal keratitis (HSK). METHODS. Primary human corneal fibroblast (HCF) cultures and a telomerase-immortalized human corneal epithelial (HCE) cell line representative of native HCE were stimulated with the known HSK-inducing cytokines interferon (IFN)-γ, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α. Alternatively, the T-cell cytokine IL-17 was added solely or simultaneously. Human neutrophils were incubated with conditioned medium (CM) of the HCF and HCE stimulated with the aforementioned cytokines, or recombinant GM-CSF, and their viability or activation status was determined by flow cytometry. GM-CSF and IL-8 secretion levels in the CM were determined by ELISA. The antibody-dependent cellular cytotoxicity (ADCC) of neutrophils toward herpes simplex virus (HSV)-infected HCFs was determined by flow cytometry. The expression of GM-CSF was determined in HSK and control corneal buttons by real-time RT-PCR and immunohistology. RESULTS. Compared with IFN-γ, CM of either cell type stimulated with IL-1β, or in the case of HCE cells, stimulated with TNF-α or IL-17, delayed neutrophil apoptosis significantly. Only in HCFs did IL-17 exhibit a synergistic effect with TNF-α. The antiapoptotic activity was attributable in part to the GM-CSF secreted by the activated HCFs and HCE cells. GM-CSF stimulation of neutrophils induced their activation and the secretion of IL-8. GM-CSF did not increase significantly the ADCC reaction of neutrophils toward HSV-infected HCFs. Finally, GM-CSF was expressed in corneas of the patients with HSK but not in control subjects. CONCLUSIONS. The data suggest that GM-CSF, expressed by cornea-resident cells such as HCFs and HCE cells, may play a role in the immunopathogenesis of HSK by prolonging the survival and modulating the effector function of corneal infiltrating neutrophils. Copyright