The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters

In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

BACKGROUND: This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the (51)Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. METHODS: We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard (51)Cr-release assays were made using human LAK cells. RESULTS: Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca(2+), thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. CONCLUSIONS: Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the (51)Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-γ ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses

A modified human ELISPOT assay to detect specific responses to primary tumor cell targets

BACKGROUND: The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific). METHODS: Towards this goal, we adapted the IFN-γ ELISPOT assay to measure immune responses against autologous primary tumor cells in vaccinated cancer patients. As a model system to develop the assay, we utilized peripheral blood mononuclear cells (PBMC) directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype protein. RESULTS: After optimizing several variables, we demonstrated that the modified IFN-γ ELISPOT assay could be used to reliably and reproducibly determine the tumor-reactive T cell frequency in the PBMC of these patients. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. CONCLUSIONS: These results demonstrate the feasibility of quantitating tumor-specific T cell responses when autologous, primary tumor cells are available as targets

Cortical atrophy predicts visual performance in long-term central retinal disease; GCL, pRNFL and cortical thickness are key biomarkers

Purpose: The aim of this study was to assess both retinal and cortical structure in a cohort of patients with long-term acquired central retinal disease in order to identify potential disease biomarkers and to explore the relationship between the anterior and posterior visual pathways. Methods: Fourteen participants diagnosed with long-term central retinal disease underwent structural assessments of the retina using spectral-domain optical coherence tomography, including macular ganglion cell layer (GCL) and peripapillary retinal nerve fiber layer (pRNFL) thickness. Structural magnetic resonance imaging was used to measure visual cortex, including cortical volume of the entire occipital lobe and cortical thickness of the occipital pole and calcarine sulcus, representing the central and peripheral retina, respectively. Results: Mean thickness was significantly reduced in both the macular GCL and the inferior temporal pRNFL across patients. Cortical thickness was significantly reduced in both the occipital pole and calcarine sulcus, representing the central and peripheral retina, respectively. Disease duration significantly correlated with GCL thickness with a large effect size, whereas a medium effect size suggests the possibility that cortical thickness in the occipital pole may correlate with visual acuity. Conclusions: Long-term central retinal disease is associated with significant structural changes to both the retina and the brain. Exploratory analysis suggests that monitoring GCL thickness may be a sensitive biomarker of disease progression and reductions in visual cortical thickness may be associated with reduced visual acuity. Although this study is limited by its heterogeneous population, larger cohort studies would be needed to better establish some of the relationships detected between disease dependent structural properties of the anterior and posterior visual pathway given the effect sizes reported in our exploratory analysis

Assessing functional reorganization in visual cortex with simulated retinal lesions

Macular degeneration (MD) causes central vision loss, removing input to corresponding representations in the primary visual cortex. There is disagreement concerning whether the cortical regions deprived of input can remain responsive, and the source of reported cortical responses is still debated. To simulate MD in controls, normally sighted participants viewed a bright central disk to adapt the retina, creating a transient ‘retinal lesion’ during a functional MRI experiment. Participants viewed blocks of faces, scrambled faces and uniform gray stimuli, either passively or whilst performing a one-back task. To assess the impact of the simulated lesion, participants repeated the paradigm using a more conventional mean luminance simulated scotoma without adaptation. Our results suggest our attempt to create a more realistic simulation of a lesion did not impact on responses in the representation of the simulated lesion. While most participants showed no evidence of stimulus-driven activation within the lesion representation, a few individuals (22%) exhibited responses similar to a participant with juvenile MD who completed the same paradigm (without adaptation). Reliability analysis showed that responses in the representation of the lesion were generally consistent irrespective of whether positive or negative. We provide some evidence that peripheral visual stimulation can also produce responses in central representations in controls while performing a task. This suggests that the ‘signature of reorganization of visual processing’, is not found solely in patients with retinal lesions, consistent with the idea that activity may be driven by unmasked top-down feedback

Population Receptive Field Dynamics in Human Visual Cortex

Seminal work in the early nineties revealed that the visual receptive field of neurons in cat primary visual cortex can change in location and size when artificial scotomas are applied. Recent work now suggests that these single neuron receptive field dynamics also pertain to the neuronal population receptive field (pRF) that can be measured in humans with functional magnetic resonance imaging (fMRI). To examine this further, we estimated the pRF in twelve healthy participants while masking the central portion of the visual field. We found that the pRF changes in location and size for two differently sized artificial scotomas, and that these pRF dynamics are most likely due to a combination of the neuronal receptive field position and size scatter as well as modulatory feedback signals from extrastriate visual areas

Following the status of visual cortex over time in patients with macular degeneration reveals atrophy of visually deprived brain regions

Purpose: Previous research has shown atrophy of visual cortex can occur in retinotopic representations of retinal lesions resulting from eye disease. However, the time course of atrophy cannot be established from these cross-sectional studies, which included patients with long-standing disease of varying severity. Our aim therefore was to measure visual cortical structure over time in participants after onset of unilateral visual loss resulting from age-related macular degeneration (AMD). Methods: Inclusion criteria were onset of acute unilateral neovascular AMD with bilateral dry-AMD based on clinical examination. Therefore, substantial loss of unilateral visual input to cortex was relatively well-defined in time. Changes in cortical anatomy were assessed in the occipital lobe as a whole, and in cortical representations of the lesion and intact retina, the lesion and intact projection zones, respectively. Whole brain, T1-weighted MRI was taken at diagnosis (before anti-angiogenic treatment to stabilise the retina), during the 3-4-month initial treatment period, with a long-term follow-up ~5 (range 3.8 – 6.1 years) years later. Results: Significant cortical atrophy was detected at long-term follow-up only, with a reduction in mean cortical volume across the whole occipital lobe. Importantly, this reduction was explained by cortical thinning of the lesion projection zone, which suggests additional changes to those associated with normal ageing. Over the period of study, anti-angiogenic treatment stabilised visual acuity and central retinal thickness, suggesting that the atrophy detected was most likely governed by long-term decreased visual input. Conclusions: Our results indicate that consequences of eye disease on visual cortex are atrophic and retinotopic. Our work also raises the potential to follow the status of visual cortex in individuals over time to inform on how best to treat patients, particularly with restorative techniques

Assessing functional reorganization in visual cortex with simulated retinal lesions

Macular degeneration (MD) causes central vision loss, removing input to corresponding representations in the primary visual cortex. There is disagreement concerning whether the cortical regions deprived of input can remain responsive, and the source of reported cortical responses is still debated. To simulate MD in controls, normally sighted participants viewed a bright central disk to adapt the retina, creating a transient ‘retinal lesion’ during a functional MRI experiment. Participants viewed blocks of faces, scrambled faces and uniform grey stimuli, either passively or whilst performing a one-back task. To assess the impact of the simulated lesion, participants repeated the paradigm using a more conventional mean luminance simulated scotoma without adaptation. Our results suggest our attempt to create a more realistic simulation of a lesion did not impact on responses in the representation of the simulated lesion. While most participants showed no evidence of stimulus-driven activation within the lesion representation, a few individuals (22%) exhibited responses similar to a participant with juvenile MD who completed the same paradigm (without adaptation). Reliability analysis showed that responses in the representation of the lesion were generally consistent irrespective of whether positive or negative. We provide some evidence that peripheral visual stimulation can also produce responses in central representations in controls while performing a task. This suggests that the ‘signature of reorganization of visual processing’, is not found solely in patients with retinal lesions, consistent with the idea that activity may be driven by unmasked top–down feedback

Structural changes to primary visual cortex in the congenital absence of cone input in achromatopsia

Autosomal recessive Achromatopsia (ACHM) is a rare inherited disorder associated with dysfunctional cone photoreceptors resulting in a congenital absence of cone input to visual cortex. This might lead to distinct changes in cortical architecture with a negative impact on the success of gene augmentation therapies. To investigate the status of the visual cortex in these patients, we performed a multi-centre study focusing on the cortical structure of regions that normally receive predominantly cone input. Using high-resolution T1-weighted MRI scans and surface-based morphometry, we compared cortical thickness, surface area and grey matter volume in foveal, parafoveal and paracentral representations of primary visual cortex in 15 individuals with ACHM and 42 normally sighted, healthy controls (HC). In ACHM, surface area was reduced in all tested representations, while thickening of the cortex was found highly localized to the most central representation. These results were comparable to more widespread changes in brain structure reported in congenitally blind individuals, suggesting similar developmental processes, i.e., irrespective of the underlying cause and extent of vision loss. The cortical differences we report here could limit the success of treatment of ACHM in adulthood. Interventions earlier in life when cortical structure is not different from normal would likely offer better visual outcomes for those with ACHM

Electronic retinal prosthesis for severe loss of vision in geographic atrophy in age-related macular degeneration : First-in-human use

BACKGROUND: To date there are yet no available approved therapies for Geographic Atrophy (GA) secondary to age-related macular degeneration (AMD). METHODS: Single site, non-randomized safety and efficacy study presenting the preliminary results in a cohort of five late stage AMD (GA) patients successfully implanted with the Argus II Retinal Prosthesis System (Second Sight Medical Products Inc., Sylmar, CA, USA). Extensive fundus imaging including retinal photographs from which the GA area was measured. A combination of custom and traditional tests designed for very low vision subjects assessed visual function in study subjects. A Functional Low-Vision Observer Rated Assessment was carried out to evaluate the impact of the system on the subject's daily life. In addition, a study to evaluate structural characteristics of the visual cortex of the brain was performed in one subject using magnetic resonance imaging. RESULTS: Seven device-related adverse events were reported, four of which were classed as serious adverse events. Retinal detachment was reported in three patients and was successfully treated within 12 months of onset. Testing showed an improvement in visual function in three of five patients with the system turned on. Magnetic resonance imaging assessed in one patient after implantation indicates a selective increase in cortical myelin and thickness in visual brain regions 1 year post implantation. CONCLUSIONS: Epiretinal prostheses can successfully be implanted in those affected by GA secondary to late-stage AMD and can elicit visual percepts by electrical stimulation of residual neuroretinal elements and improve basic visual function in those affected