87 research outputs found
Human herpes simplex virus keratitis: the pathogenesis revisted
The aim of this thesis is to elucidate pathogenic
mechanisms of different forms of
human HSV keratitis. HSV infection of the
corneal epithelium causes a classical dendritic
shaped lesion. Many studies could explain the
development and growth in dendritic keratitis,
but none of these found the anatomical substrate
for the linear branching pattern. The
most obvious explanation would be, that the
shape of dendritic ulcers corresponds with the
anatomical pattern of innervating nerves of
the cornea. In chapter 2 a relationship between
the shape of dendritic ulcers in infectious
epithelial keratitis and the subbasal nerve
plexus of the corneal epithelium is postulated.
Recurrence of HSV keratitis is a common
complication after PKP for corneal opacities
resulting from HSV infection. After PKP,
for reasons unrelated to HSV keratitis, epithelial
defects may still be caused by HSV. In
chapter 3 the incidence of newly acquired HSV
keratitis after PKP is determined and possible
contributing factors are assessed. Several possibilities
as to the origin of the infecting HSV
exist. These include reactivation oflatent virus
in the trigeminal ganglion, horizontal spread,
or transmission through the donor cornea. To
test the assumption of graft-to-host transmission
of HSV by PKP, surplus corneal material
was examined for the presence of HSV DNA. Because the amount of viral DNA available
could be very limited, a new method independent
of viral culture, was developed to
allow distinction between different virus
strains. The newly developed technique was
used to test our hypothesis that graft-to-host
transmission of HSV is possible. This new
method was used to determine the incidence
of HSV-1 superinfection in patients with
recurrent HSV keratitis.
Although HSK has been studied
extensively in the mouse model, it is not clear
what triggers the immune response and to
what extent the mouse data correlate with findings
in human keratitis. The most logical idea,
that virus-derived proteins are the eliciting factor
for the immune response, has been ruled
out in the experimental HSK mouse model.
Alternative sources of the keratogenic antigens,
like auto-antigens, have been suggested.
Data on the pathogenesis of human HSK are
limited. Therefore, in chapter 4 the antigenspecificity
of corneal T cells in HSK patients
was investigated. Besides this, corneas of
patients with HSK were examined for the
presence of corneal antigen reactive T cells
(auto-reactive T cells).
Chapter 5 provides a concise summary
of the data generated in the framework of this
thesis, and concludes with an overall discussion
of the data and their possible impact on
current ophthalmologic practice
Herpes simplex virus-specific T cells infiltrate the cornea of patients with herpetic stromal keratitis: no evidence for autoreactive T cells
PURPOSE: Herpetic stromal keratitis (HSK) is a T-cell-mediated
inflammatory disease initiated by a herpes simplex virus (HSV) infection
of the cornea. Recently, studies in the HSK mouse model have shown that
the immunopathogenic T cells are directed against the HSV protein UL6
cross-reacting with an unknown corneal autoantigen. Whether this type of
autoimmunity plays a role in human HSK was analyzed. METHODS: T-cell lines
(TCLs) were generated from corneal buttons of 12 patients with different
clinical stages of HSV-induced necrotizing stromal keratitis (n = 9) or
immune stromal keratitis (n = 3). The initiating virus was identified by
polymerase chain reaction and immunohistology performed on the corneal
buttons. Peripheral blood mononuclear cells (PBMCs) were isolated, and B
cell lines (BLCLs) were generated by transformation with Epstein-Barr
virus. Proliferative responses of these intracorneal TCLs were determined
by culturing T cells with autologous BLCLs infected with HSV-1, HSV-2,
wild-type vaccinia virus (VV-WT), or VV expressing HSV-1 UL6 (rVV-UL6).
Alternatively, T cells were incubated with PBMCs pulsed with human cornea
protein extract. RESULTS: Irrespective of clinical diagnosis or treatment,
T cells were recovered from the corneal buttons of all the 12 HSK
patients. The intracorneal TCLs of 9 of the 12 HSK patients showed
HSV-specific T-cell reactivity. In none of the TCLs, T-cell reactivity
against HSV-1 UL6 or human corneal antigens was detected. CONCLUSIONS:
These data suggest that the potentially immunopathogenic intracorneal
T-cell response in HSK patients is directed to the initiating virus and
not to a human corneal autoantigen or HSV-1 UL6
Corneal herpes simplex virus type 1 superinfection in patients with recrudescent herpetic keratitis
PURPOSE: Herpetic keratitis is a common sequel of a corneal infection with herpes simplex virus (HSV)-1. Recrudescent herpetic keratitis (RHK) may result in irreversible damage to the cornea. Recurrences may be caused by reactivation of endogenous HSV-1 or reinfection with exogenous HSV-1. The objective of this study was to determine the incidence and risk factors involved of HSV-1 superinfection in patients with RHK. METHODS: From 30 patients with RHK, sequential corneal HSV-1 isolates were genotyped by PCR amplification of the hypervariable regions located within the HSV-1 genes US1, US10/11, and US12. The clinical data from the patients obtained retrospectively were: ophthalmologic history, clinical picture during recurrences, number and time points of penetrating keratoplasty (PKP), and steroid or acyclovir treatment. RESULTS: Whereas the sequential corneal HSV-1 isolates of 19 (63%) of 30 patients had the same genotype (designated as group 1), the sequential isolates of 11 patients (37%) were genetically different (designated as group 2). Among the clinical data analyzed, only the time point of PKP was significantly different between the patient groups. A
Whole-cell metabolic control analysis
Since its conception some fifty years ago, metabolic control analysis (MCA) aims to understand how cells control their metabolism by adjusting the activity of their enzymes. Here we extend its scope to a whole-cell context. We consider metabolism in the evolutionary context of growth-rate maximisation by optimisation of protein concentrations. This framework allows for the prediction of flux control coefficients from proteomics data or stoichiometric modelling. Since genes compete for finite biosynthetic resources, we treat all protein concentrations as interdependent. We show that elementary flux modes (EFMs) emerge naturally as the optimal metabolic networks in the whole-cell context and we derive their control properties. In the evolutionary optimum, the number of expressed EFMs is determined by the number of protein-concentration constraints that limit growth rate. We use published glucose-limited chemostat data of S. cerevisiae to illustrate that it uses only two EFMs prior to the onset of fermentation and that it uses four EFMs during fermentation. We discuss published enzyme-titration data to show that S. cerevisiae and E. coli indeed can express proteins at growth-rate maximising concentrations. Accordingly, we extend MCA to elementary flux modes operating at an optimal state. We find that the expression of growth-unassociated proteins changes results from classical metabolic control analysis. Finally, we show how flux control coefficients can be estimated from proteomics and ribosome-profiling data. We analyse published proteomics data of E. coli to provide a whole-cell perspective of the control of metabolic enzymes on growth rate. We hope that this paper stimulates a renewed interest in metabolic control analysis, so that it can serve again the purpose it once had: to identify general principles that emerge from the biochemistry of the cell and are conserved across biological species
Amplification of reiterated sequences of herpes simplex virus type 1 (HSV-1) genome to discriminate between clinical HSV-1 isolates.
Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections
Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia
Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates
Partial volume correction strategies for quantitative FDG PET in oncology
Purpose: Quantitative accuracy of positron emission tomography (PET) is affected by partial volume effects resulting in increased underestimation of the standardized uptake value (SUV) with decreasing tumour volume. The purpose of the present study was to assess accuracy and precision of different partial volume correction (PVC) methods. Methods: Three methods for PVC were evaluated: (1) inclusion of the point spread function (PSF) within the reconstruction, (2) iterative deconvolution of PET images and (3) calculation of spill-in and spill-out factors based on tumour masks. Simulations were based on a mathematical phantom with tumours of different sizes and shapes. Phantom experiments were performed in 2-D mode using the National Electrical Manufacturers Association (NEMA) NU2 image quality phantom containing six differently sized spheres. Clinical studies (2-D mode) included a test-retest study consisting of 10 patients with stage IIIB and IV non-small cell lung cancer and a response monitoring study consisting of 15 female breast cancer patients. In all studies tumour or sphere volumes of interest (VOI) were generated using VOI based on adaptive relative thresholds. Results: Simulations and experiments provided similar results. All methods were able to accurately recover true SUV within 10% for spheres equal to and larger than 1 ml. Reconstruction-based recovery, however, provided up to twofold better precision than image-based methods. Cl
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