134 research outputs found
SOCS and Herpesviruses, With Emphasis on Cytomegalovirus Retinitis
Suppressor of cytokine signaling (SOCS) proteins provide selective negative feedback to prevent pathogeneses caused by overstimulation of the immune system. Of the eight known SOCS proteins, SOCS1 and SOCS3 are the best studied, and systemic deletion of either gene causes early lethality in mice. Many viruses, including herpesviruses such as herpes simplex virus and cytomegalovirus, can manipulate expression of these host proteins, with overstimulation of SOCS1 and/or SOCS3 putatively facilitating viral evasion of immune surveillance, and SOCS suppression generally exacerbating immunopathogenesis. This is particularly poignant within the eye, which contains a diverse assortment of specialized cell types working together in a tightly controlled microenvironment of immune privilege. When the immune privilege of the ocular compartment fails, inflammation causing severe immunopathogenesis and permanent, sight-threatening damage may occur, as in the case of AIDS-related human cytomegalovirus (HCMV) retinitis. Herein we review how SOCS1 and SOCS3 impact the virologic, immunologic, and/or pathologic outcomes of herpesvirus infection with particular emphasis on retinitis caused by HCMV or its mouse model experimental counterpart, murine cytomegalovirus (MCMV). The accumulated data suggests that SOCS1 and/or SOCS3 can differentially affect the severity of viral diseases in a highly cell-type-specific manner, reflecting the diversity and complexity of herpesvirus infection and the ocular compartment
Ocular complications of herpes zoster ophthalmicus
Fifty percent of patients with ophthalmic zoster develop ocular complications. These may be mild or severe, and can lead to loss of sight which, by timely and good management can be prevented in most cases. Unfortunately knowledge of the complications and their management is often poor making the design and interpretation of clinical trials difficult. 1356 cases of ophthalmic zoster were collected over fifteen years with at least 6 months follow up. Their details and complications were entered into a database. There is a generally held impression that patients developing zoster do so because of impaired immunity. Analysis of our findings showed that this was not so. The database results were analyzed to quantify the incidence of complications and their correlates. Two common corneal complications that are difficult to manage and lead to visual impairment were studied in detail including: Mucous plaque keratitis is defined as a distinct entity appearing at an early or late stage and occurred in forty seven cases. Poor management leads to visual impairment from neurotrophic ulceration, megaplaque keratitis and glaucoma. Lipid keratopathy induces diminished visual acuity and photophobia. It occurred in thirty six cases. Careful and prolonged treatment of chronic stromal keratitis with topical steroid will prevent this occurring, but when it does successful laser occlusion of corneal blood vessels halts the deposition of lipid and may actually disperse it or make the host cornea safer for keratoplasty. One hundred and seventy six patients were screened orthoptically and the incidence of extraocular muscle palsies assessed with regard to distribution and natural history. Possible pathogeneses are discussed. Overall recovery was good. Lastly iritis and iris atrophy were identified in five hundred and twenty patients and twenty three were investigated with anterior segment fluorescein angiography. This showed that they were associated with an ischaemic vasculitis
CD4 T Cell Immunity Is Critical for the Control of Simian Varicella Virus Infection in a Nonhuman Primate Model of VZV Infection
Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses
Novel Transporter Targeted Lipid Ester Prodrugs of Cidofovir and Sustained Release Formulations for Cytomegalovirus Infection
Tittle from PDF of title page, viewed on December 10, 2015Dissertation advisor: Ashim K. MitraVitaIncludes bibliographical references (pages 213-230)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2015Cidofovir (CDF) has shown potential in vitro and in vivo antiviral activity against cytomegalovirus (CMV) retinitis. However, low CDF permeability due to its hydrophilic nature limits its effectiveness. Furthermore, physicochemical properties such as high water solubility (170 mg/mL) and log p (-3.9) of CDF are unfavorable for passive transcellular absorption. Therefore, we have tested novel transporter targeted lipid prodrugs of CDF and their nanoparticle formulations to achieve sustain drug delivery for the treatment of CMV retinitis. These prodrugs contains lipid linker of different carbon chain length (e.g. C2, C6, and C12) carrying minimum two functional groups to link one end with CDF and another end with ligand (biotin) targeting sodium dependent multivitamin transporter (SMVT).
We have successfully synthesized three biotinylated (B) derivatives (B-C2-cCDF, B-C6-cCDF, and B-C12-cCDF) and two lipid ester derivatives (C6-cCDF and C12-cCDF) of cCDF. Structure and purity of all the produgs were confirmed using ³¹P NMR and LC-MS/MS analysis. All synthesized prodrugs were characterized in terms of its physicochemical properties, in vitro cellular accumulation, cytotoxicity, transporter affinity, ocular tissue stability and in vitro antiviral potency against various virus strains. Furthermore, prodrug loaded PLGA nanoparticle formulations were prepared and characterized.
Overall, improvement in physicochemical properties, lipophilicity, interaction with SMVT transporter, higher tissue-buffer partition coefficient and rapid conversion into parent molecule (in retina–choroid) were observed with higher lipid chain length (B-C12-cCDF > B-C6-cCDF > B-C2-cCDF). In vitro antiviral activity shows B-C12-cCDF as the potential prodrug candidate for future in vivo evaluation. Sustained release (~3 weeks) formulation of B-C12-cCDF loaded PLGA nanoparticles may serve as a viable intravitreal delivery approach for the treatment of CMV retinitis. Based on the above finding we hypothesized that lipid raft facilitates enhanced prodrug interaction with cell membrane and thereby assist docking of targeted ligand into the binding domain of transporter. Therefore, current novel approach combines both lipids mediated facilitate diffusion and transporter targeted delivery to generate synergistic drug effect. Overall, these findings indicate that transporter-targeted lipid analogue of CDF could be a viable strategy for the treatment of CMV retinitis.Literature review -- Novel transporter targeted lipid prodrugs of cyclic cidofoivr for treatment of cytomegalovirus infection -- Synthesis, characterization and analytical method development of transporter targeted lipid and lipid ester prodrugs of cyclic cidofovir -- Interact of transporter targeted lipid ester prodrugs of cyclic cidofovir with mdck-mdr1: for oral drug delivery -- Interaction of transporter targeted lipid ester prodrugs of cyclic cidofovir with arpe-19 cells: for ocular drug delivery -- Antiviral efficacy of transporter targeted lipid ester and lipid ester prodrugs of cyclic cidofovir -- Cidofovir and prodrug loaded poly lactide poly glycolide (PLGA) nanoparticle formulation for sustained delivery -- Summary and recommendation
Lymphocyte subpopulations and cytokine expression in uveitis
RESUMO: As subpopulações linfocitárias, e em particular as células T reguladoras (Tregs), têm sido alvo de
intensa investigação nos últimos anos. As Tregs são células reconhecidas pelo seu papel
imunorregulador e responsáveis pela expressão de citocinas anti-inflamatórias como, por exemplo, a
IL-10 e o TGF-β. A redução de nÃveis circulantes de Tregs tem sido associada a várias doenças auto imunes, particularmente em casos de doença activa. No caso da uveÃte não-infecciosa (NIU), os
resultados têm sido contraditórios, sendo necessários estudos adicionais para que se possa entender
o papel definitivo da frequência e função das subpopulações de células Tregs na sua patogénese.
O presente estudo tem como objectivo analisar as subpopulações linfocitárias, em especial a
subpopulação Treg, bem como os perfis de citocinas inflamatórias e anti-inflamatórias circulantes de
doentes com NIU activa, comparando-os com os de controlos normais. Há ainda a realçar a análise
adicional de um subgrupo de doentes com NIU após tratamento e resolução da uveÃte. Em doentes
com uveÃte infecciosa (IU) foram ainda analisadas amostras de humor aquoso (HAq), tendo em vista a
caracterização de um perfil de citocinas.
Em doentes com NIU activa não foram encontradas diferenças significativas nos nÃveis periféricos de
Tregs (incluindo naïve e de memória) relativamente aos controlos. Em doentes avaliados ao longo do
tempo, os nossos resultados mostraram um aumento das percentagens de Tregs totais e de memória,
numa primeira avaliação, mas novamente sem diferença relativamente aos controlos após o
tratamento. Ainda assim é interessante observar que este aumento inicial nas percentagens de Tregs
totais e de memória não foi acompanhado de um aumento significativo na expressão de CD39, o que
pode significar a perda da normal função supressora destas células.
Relativamente aos nÃveis séricos de citocinas em doentes com NIU e controlos, os nossos resultados
mostraram uma tendência para elevação da IL-17 no grupo com uveÃte, bem como correlações
positivas entre o ratio de citocinas inflamatórias (TNF-α + IFN-ɣ + IL-17) /anti-inflamatórias (IL-10 +
TGF-β) e o ratio IL-17/IL-10 com as contagens absolutas de Tregs de memória. Para além disso, nÃveis
séricos mais elevados de IL-17 mostraram uma associação com concentrações mais elevadas de Tregs
naïve e de memória e ainda com nÃveis séricos mais elevados de TNF-α e de IFN-É£.Após tratamento, houve uma diminuição significativa nos nÃveis circulantes de IL-17 e de TNF-α no
grupo de doentes com NIU em remissão, bem como uma redução significativa, entre avaliações, no
ratio de citocinas inflamatórias/anti-inflamatórias. Por fim, no grupo de doentes com IU, os nossos
resultados mostraram um aumento das concentrações de IL-10, TNF-α e IFN-ɣ no humor aquoso de
doentes com infecção intraocular comprovada por vÃrus varicella zoster (VVZ). Já no sangue periférico,
não se encontraram diferenças significativas entre perfis de citocinas de doentes e controlos
saudáveis.
Os nossos resultados não apoiam a utilização isolada da frequência periférica total de Tregs como um
biomarcador de uveÃte activa. Pensamos que este estudo é pioneiro na avaliação de subpopulações
Treg naïve e de memória, e sua respectiva expressão CD39, no sangue periférico de doentes com NIU.
No entanto, são necessários mais estudos para que o papel individual de cada uma destas
subpopulações Treg na patogénese e eventual tratamento da NIU possa ser esclarecido.
Relativamente aos perfis de citocinas em doentes com uveÃte de causa infecciosa e não-infecciosa, este
trabalho de investigação permite sublinhar a importância da citocina IL-17 na NIU activa e uma
eventual associação entre nÃveis intraoculares elevados de IL-10 e a uveÃte associada a infecção por
VVZ.
Estes resultados podem vir a contribuir para a identificação de biomarcadores de inflamação
intraocular activa e na investigação de novos alvos terapêuticos.ABSTRACT: Lymphocyte subpopulations, particularly regulatory T-cells (Tregs), have been extensively studied in
the last few years. Tregs are T-cells with an immunosuppressive role, responsible for the expression of
regulatory cytokines like IL-10 and TGF-β. A reduction of circulating Treg levels has been associated
with various auto-immune diseases, especially in active disease. As for non-infectious uveitis (NIU),
results have been conflicting, and further studies are needed to better understand the role of the
frequency and function of total Treg and subsets in NIU.
The present thesis aimed to analyze the lymphocyte subpopulations, especially Tregs, as well as
inflammatory and anti-inflammatory cytokines in patients with active NIU and compare them with
normal controls. One subgroup of NIU patients was further evaluated after treatment and uveitis
resolution. Later on, in infectious uveitis (IU) patients, aqueous humour (AqH) samples were also
analyzed for cytokine characterization.
In active NIU, we found no significant differences in Treg levels (including naïve and memory subsets)
between patients and controls. In NIU patients evaluated over time, our results showed an increased
percentage of both total and memory Tregs in patients with active inflammation, without significant
difference from controls after treatment. Nevertheless, it is interesting that this initial increase in total
and memory Treg percentages was not associated with an increased CD39 expression, which may lead
us to speculate whether these cells maintained their normal suppressive function.
When comparing serum cytokine levels in NIU patients and controls, our results showed a tendency
for IL-17A elevation in the NIU group and positive correlations between the inflammatory (TNF-α +
IFN-ɣ + IL-17A) /anti-inflammatory (IL-10 + TGF-β) cytokine ratio and the IL-17/IL-10 ratio with the
absolute counts of memory Tregs. We also found that higher IL-17A levels were associated with higher
serum concentrations of memory and naïve Tregs as well as higher TNF-α and IFN-ɣ levels. After
treatment, lower levels of IL-17A and TNF-α were present in patients in uveitis remission. Furthermore,
the inflammatory/anti-inflammatory ratio also showed a significant reduction between evaluations.
As for cytokine levels in IU patients, our results showed that while there were no significant differences
between patients and controls regarding serum cytokine profiles, increased concentrations of IL-10,
TNF-α, and IFN-ɣ in the AqH samples were found in patients diagnosed with varicella-zoster virus (VZV)-
associated uveitis. Our results do not support the role of total Treg frequency alone as a uveitis biomarker, and since, to
our knowledge, these are the first studies addressing the role of Treg naïve and memory subsets and
their respective CD39 expression in the peripheral blood of NIU patients, further studies should be
addressed to elucidate the possible role of each subset in NIU pathogenesis and treatment.
As for cytokine profiles in infectious and non-infectious uveitis, our results highlight the importance of
IL-17 in active NIU and the possible association between elevated intraocular IL-10 levels and VZV associated infection.
We hope that this work may contribute to finding new biomarkers for active intraocular inflammation
and new therapeutic targets for future research
Cellular Responses of the Retina to West Nile Virus Infection
Age-related macular degeneration (AMD) is the leading cause of blindness in the developing world in people aged over 60 years, manifested as a loss of central vision in one or both eyes, with significant morbidity including loss of mobility and depression. This condition involves the degeneration of the macula, and although the exact aetiology of this disease is unknown, various epidemiological studies have shown it to be multifactorial. Current research points towards the involvement of a dysregulated immune system in the pathogenesis and progression of the disease: as the body ages, the immune system increasingly adopts a more inflammatory basal state. However, not all of the aged population develops AMD and it is highly likely that an additional stimulus or stimuli is/are needed to exploit this dysregulated immune environment to initiate this disease. Given the range of pathogens that can infect the retina, we hypothesize that this breaking point could manifest as a chronic inflammation as a result of a low-level infection. West Nile Virus (WNV) is a flavivirus that has come into international prominence ever since its spread into previously WNV-free regions following the 1999 New York outbreak. As several case reports have shown that WNV is capable of infecting the retina, and given its immunopathogenic properties, we believe the virus is a useful tool to model key immune pathways and responses that may be involved in the development and progression of AMD. Of significant interest are the processes involved in the breakdown of the outer blood-retinal barrier (BRB), which is an important step in the progression of AMD from an early stage to a more severe one. Additionally, deciphering and understanding the profile and populations of leukocytes that are recruited during an immunopathic infection in an organ regarded as being immunoprivileged is of great appeal. With this in mind, we set out to investigate the effects of WNV infection on the retinal pigment epithelium (RPE), which comprises the outer BRB. Previously, our laboratory established the WNV BRB model by quantitating various parameters, such as level of infectivity, viral output by WNV-infected RPE and effects of WNV infection on RPE proliferation/migration. The effect of WNV on the extracellular matrix (ECM) production by RPE was also investigated and increases in collagen I, IV and fibronectin were noted. Global ECM production induced a lowered rate of proliferation of RPE seeded on WNV-infected RPE ECM as opposed to mock-infected ECM. A full genome microarray was also undertaken on WNV-infected RPE to analyse differentially regulated gene mRNA production, and increases in several immune genes, as well as genes involved in the stress-response pathway and the TGFβ pathway were found. This current investigation expanded upon these results, and found that WNV infection produces a predominantly CCL5 chemokine response rather than a CCL2 response. Additionally, a lack of TNF production was noted, despite a high initial upregulation of the TNF gene in WNV-infected cells. WNV attenuation was found to be predominantly IFNβ-1-driven, while induction of indoleamine 2,3 dioxygenase activity was induced in part by IFNλ-1 and -2. The effects of WNV infection on RPE barrier integrity was investigated, and an initial increase in infected cells of barrier integrity was observed. Several investigations resulted in a conclusion of a soluble-mediator as the likely mechanism behind this initial increase, and while none of the chemokines tested appeared to contribute to this change, the results suggest that it may be TLR3/RIG-I independent. Finally, establishment of a murine WNV intravitreal model was also undertaken, and several key parameters were determined, including confirmation of WNV-infection of the murine retina, effect of WNV titre on mortality, and histological analysis of the effects of WNV infection on the murine retina. Quantification of the leukocyte profile recruited into the WNV-infected murine retina and choroid revealed significant increases in inflammatory Ly6Chi monocytes, as well as significant differences between immune mice and naïve mice intravitreally infected with WNV, and differences between 2 month old and 5 month old mice. Collectively, these results highlight the importance of the interferon response in both direct and indirect anti-WNV activities and immunomodulation, the changes in outer BRB integrity and possible contributors to its degradation, and the establishment of the murine intravitreal WNV model along with identification of several key leukocytes that are recruited at the peak of infection. These results will help guide further research and highlight possible immune pathways that may contribute to dysregulated inflammatory processes that may occur during the pathogenesis of AMD
Suppressor of Cytokine Signaling (SOCS)1 and SOCS3 Stimulation during Experimental Cytomegalovirus Retinitis: Virologic, Immunologic, or Pathologic Mechanisms
AIDS-related human cytomegalovirus (HCMV) retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. Understanding the pathogenesis of this disease is essential for developing new, safe, and effective treatments for its prevention or management, yet much remains unknown about the virologic and immunologic mechanisms contributing to its pathology. To study such mechanisms, we use a well-established, reproducible, and clinically relevant animal model with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) that mimics in mice the symptoms and progression of AIDS in humans. Over 8 to 12 weeks, MAIDS mice become susceptible to experimental murine cytomegalovirus (MCMV) retinitis. We have found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling (SOCS)1 and SOCS3, host proteins which dampen immune-related signaling by cytokines, including antiviral interferons. Herein we investigated virologic and/or immunologic mechanisms involved in this stimulation and how virally-modulated SOCS1 and/or SOCS3 proteins may contribute to MCMV infection or experimental MAIDS-related MCMV retinitis. Through pursuit of two specific aims, we tested the central hypothesis that MCMV stimulates and employs SOCS1 and/or SOCS3 to induce the onset and development of MCMV retinal disease. MCMV-related SOCS1 and SOCS3 stimulation in vivo occurred with intraocular infection, was dependent on method and stage of immune suppression and severity of ocular pathology, was associated with stimulation of SOCS-inducing cytokines, and SOCS1 and SOCS3 were differentially sensitive to antiviral treatment. In vitro studies further demonstrated that SOCS1 and SOCS3 stimulation during MCMV infection occurred with expected immediate early kinetics, required viral gene expression in cell-type-dependent and virus origin-dependent patterns of expression, and displayed differential sensitivity to antiviral treatment. These data suggest that SOCS1 and SOCS3 are stimulated by divergent virologic, immunologic, and/or pathologic mechanisms during MCMV infection, and that they contribute to the pathogenesis of retinal disease, revealing new insights into the pathophysiology of AIDS-related HCMV retinitis
On interactions between alphaherpesviruses and natural killer cells
Natural killer (NK) cells are a critical component of the innate immune response to viral infection. NK cells are responsible for the early control of virus spread, cytolytically killing infected cells, as well as secreting proinflammatory cytokines to enhance immune responses. In patients with deficiencies in NK cell function, there is an extreme susceptibility to infection with herpesviruses, in particular, varicella zoster virus (VZV) and herpes simplex virus type 1 (HSV-1). These two medically important human alphaherpesviruses cause widespread disease in human hosts, with VZV being the causative agent of varicella (chickenpox) and herpes zoster (shingles), while HSV-1 causes recurrent orolabial lesions (cold sores). Both viruses have the potential to cause severe complications, such as encephalitis and debilitating nerve pain. The vital role that NK cells play in controlling VZV and HSV-1 infections denotes an intricate struggle for dominance between virus and NK cell antiviral immunity; however, research in this area has remained surprisingly limited. This thesis explored the interactions between human NK cells and alphaherpesviruses, examining NK cell recognition of infected cells, as well as investigating viral infection of NK cells and manipulation of their function. Investigation into alphaherpesvirus interactions with NK cells first focused on examining whether VZV and HSV-1 modulated the surface of infected cells to potentially regulate NK cell recognition. In vitro co-culture of NK cells with VZV infected cells revealed that NK cells did not display enhanced activation in response to the infection, suggesting that specific viral mechanisms to limit NK cell detection may be at play. Delving into this, the expression of four specific ligands (MICA, ULBP1–3) recognised by the activating NK cell receptor, NKG2D, were examined during viral infection. Comparing VZV and HSV-1, differential patterns of regulation were found between the two viruses, as well as between distinct NKG2D ligands. Given that VZV appeared to be evading NK cell recognition, the research focus then turned to investigating how VZV directly interacted with NK cells. VZV is established as a lymphotropic virus, using the infection of immune cells to disseminate virus around the body, however it has so far remained unknown whether NK cells are permissive to VZV infection. Examination of human peripheral blood NK cells revealed that VZV productively infected NK cells, facilitating transmission of infectious virus to other cells in culture. VZV preferentially infected mature NK cell populations, as well as modulating cell-surface expression of maturityassociated markers. Notably, VZV infection of NK cells led to upregulated expression of chemokine receptors implicated in trafficking to the skin, suggesting that NK cells may play a key role in VZV pathogenesis. As NK cells were permissive to productive VZV infection, the effect of VZV on NK cell function was then investigated. Assessing cytolytic function, it was found that co-culture with VZV lead to potent inhibition of NK cell responsiveness to target cell stimulation. Remarkably, not only were VZV infected NK cells impaired, but also NK cells exposed to virus were inhibited without needing to progress to full productive infection. HSV-1 had a similar capacity to paralyse NK cell cytolytic function, identifying a powerful immune evasion strategy shared by both alphaherpesviruses. In contrast, when NK cell cytokine responses were investigated, differential targeting of cytokine production was demonstrated between VZV and HSV-1. Overall, this thesis illuminates the complex interactions that occur between viral infection and the immune response. The findings presented in this thesis enhance our understanding of how viruses like VZV and HSV-1 are able to evade the immune system to establish lifelong infections, as well as furthering our understanding of how viruses can shape and manipulate the immune response
Ocular monitoring in immunosuppressed patients and quantification of immunosuppression by assessment of intracellular cytokines.
Although laboratory indices, such as drug levels, are in routine use for monitoring immunosuppression, these do not always correlate well with an individual's risk of toxicity. In practice individual organs are monitored for toxicity with a combination of laboratory and clinical methods. These methods are limited by the fact that one has to await compromise before making therapeutic changes and there are often idiosyncrasies in the way individuals respond. Ocular complications can be sight threatening and many have proposed routine ocular monitoring in immunosuppressed patients. This study prospectively monitored a cohort of patients receiving high levels of immunosuppression for the prevention of rejection of heart, lung and heart-lung transplants for the development of ocular complications and to assess ocular morbidity. Specific surrogate markers that gave more information about the level of immunosuppression in a particular patient would improve patient care. Cytokines have the advantage of being directly generated by the immune response and offer promise as surrogate markers. Various, and not always consistent, cytokine profiles have been described for a number of conditions. Flow cytometry with intracellular cytokine staining allows quantification of the amount of cytokine present. These studies used this technique to describe the cytokine profile and changes in cytokine profile seen in patients during treatment for autoimmune uveitis and in patients with human immunodeficiency virus (HIV) receiving combination anti-retroviral therapy (ART). These studies emphasized the importance of prompt and careful clinical examination in the presence of clinical symptoms but did not support routine screening of patients on high levels of immunosuppression. Accurate measurements of interleukin-2 (IL-2) and interferon-gamma (IFNgamma) in patients with uveitis did not correlate well with disease activity. In contrast patients with different HIV profiles did show measurably different Thl cytokine expression providing information on the T cell deficits that persist despite treatment with ART
Decoding the Architecture of the Varicella-Zoster Virus Transcriptome
Varicella-zoster virus (VZV), a double-stranded DNA virus, causes varicella, establishes lifelong latency in ganglionic neurons, and reactivates later in life
to cause herpes zoster, commonly associated with chronic pain. The VZV genome is
densely packed and produces multitudes of overlapping transcripts deriving from
both strands. While 71 distinct open reading frames (ORFs) have thus far been experimentally defined, the full coding potential of VZV remains unknown. Here, we
integrated multiple short-read RNA sequencing approaches with long-read direct
RNA sequencing on RNA isolated from VZV-infected cells to provide a comprehensive reannotation of the lytic VZV transcriptome architecture. Through precise mapping of transcription start sites, splice junctions, and polyadenylation
sites, we identified 136 distinct polyadenylated VZV RNAs that encode canonical
ORFs, noncanonical ORFs, and ORF fusions, as well as putative noncoding RNAs
(ncRNAs). Furthermore, we determined the kinetic class of all VZV transcri
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