Two candidate gene markers (GFI1 and TNFRSF11A) assessed in this study were identified from a previous study led by the The Cancer Genome Atlas (TCGA) Network, and analysis was performed on 112 consecutively collected, archival FFPE colorectal cancer tumour samples. Consistent with the TCGA Network study, we found reduced GFI1 expression was associated with high-grade and left-sided tumours, and reduced TNFRSF11A expression was associated with metastasis and high nodal involvement. RNAscope® combined with image analysis also enabled quantification of GFI1 and TNFRSF11A mRNA expression levels at the single cell level, allowing cell-type determination. These data showed that reduced mRNA transcript abundance measured in patients with poorer prognosis occurred in carcinoma cells, and not lymphocytes, stromal cells or normal epithelial cells. To our knowledge, this is the first study to assess the intra-tumoural expression patterns of GFI1 and TNFRSF11A and to validate their microarray expression profiles using RNAscope. We also demonstrate the utility of RNAscope® technology to show that expression differences are derived from carcinoma cells rather than from cells located in the tumour microenvironment.Genome-wide expression studies using microarrays and RNAseq have increased our understanding of colorectal cancer development. Translating potential gene biomarkers from these studies for clinical utility has typically relied on PCR-based technology and immunohistochemistry. Results from these techniques are limited by tumour sample heterogeneity and the lack of correlation between mRNA transcript abundance and corresponding protein levels. The aim of this research was to investigate the clinical utility of the RNA  The diagnosis of colorectal cancer occurs primarily by histological examination of tissue specimens obtained during an endoscopic procedure, or at the time of surgical resection . AccuratOur understanding of the molecular basis of colorectal cancer has increased with the advent of molecular technologies that enable genome-wide expression analysis, such as microarrays and massively parallel sequencing , 6. In 2®  is one such method that can satisfy these requirements.The inconsistencies seen may be due to the confounding effect of tumour heterogeneity combined with limitations in PCR-based technology and immunohistochemistry. The quantitative PCR-based method might be considered the ‘gold standard’ for measuring gene expression because of its high sensitivity and specificity. However, the sensitivity of this method is limited if applied to heterogeneous multicellular tumour samples, and it is prone to interference from non-cancerous cells. Immunohistochemistry has proven to be an integral method for measuring protein expression levels in tissue samples for diagnostic laboratories, however it is less quantitative than other molecular methods . Further® is an RNA in situ hybridisation method that enables single-molecule detection while preserving cellular and tissue morphology [® allows mRNA expression measurements for individual or multiple genes from archival tumour specimens to be obtained in a diagnostic setting [® and quantitative PCR [RNAscoperphology . This merphology , and hasrphology –19; stemrphology –22 and crphology , 24. RNA setting . Furthertive PCR .® for measuring mRNA expression levels of genes previously associated with poor prognostic outcomes in colorectal cancer, and determine mRNA transcript localisation and expression in different cell types within the tumour.The purpose of this study was to investigate the utility of RNAscopeTNFRSF11A and/or GFI1 can be affected by CNA, analysis of mRNA expression levels and CNA was performed using TCGA data. From a total of 629 patients, 218 had DNA copy number and gene expression data. GFI1 and TNFRSF11A, showed a significant association between predicted gene CNA and expression levels for both GFI1  and TNFRSF11A  with a trend of CNA deletions being associated with mRNA downregulation  with no background staining or staining artefacts  than 43 cases that presented a heterogeneous expression with MLH1 probe signals observed in localised areas of the tumour.Expression of pression , 21, so Immunohistochemistry was successfully performed with the majority of cases demonstrating positive staining of MLH1 within carcinoma cells. However, staining intensities did vary between cases.® and immunohistochemistry in 73.7% (56/76) of cases whilst 29 cases were excluded . A significant positive correlation between mRNA and protein expression was observed in 75.0% (48/64) of cases (P = 0.018). A negative mRNA expression correlated with a negative protein expression in 66.6% (8/12) of cases. Investigation of the discrepant cases found 16 cases with positive MLH1 protein expression had no detectable MLH1 mRNA expression present. Conversely, four cases with negative MLH1 protein expression, showed positive MLH1 mRNA expression. Such differences highlight potential variations in half-life between molecules and/or sensitivities between technologies. We were not able to evaluate these findings in relation to MLH1 mutation or methylation status, however, the results further demonstrate the utility of RNAscope® for measuring gene mRNA expression changes in archival FFPE tissue.Comparison of results from these technologies correlated in 55.0% 62/112) of cases , grade , left-sided tumours , and rectal tumours  were observed by univariate analysis , and AJCC Nodal Stage involvement   was available for this study.Analysis of GFI1 and TNFRSF11A, we used RNAscope® to quantify mRNA levels across 10 whole tumour sections according to tumour cell type , including normal epithelial cells, stromal fibroblasts and lymphocytes . The mean expression levels for TNFRSF11A in carcinoma cells was 7-fold higher than surrounding cell types . No statistical significance was observed for differences in mean mRNA expression when comparing amongst other cell types.To better characterise the intra-tumoural expression patterns of e Figure . This ans Figure . The meain situ hybridisation can overcome these limitations by measuring mRNA expression at the single cell level.Candidate markers from large expression profiling studies have made little clinical impact to date, possibly due to the confounding effect of heterogeneous tumour samples. RNA in situ hybridisation (RNAscope®) as a method for assessing mRNA expression on archival FFPE material in a similar manner to immunohistochemistry for assessing protein expression. This study then utilized RNAscope® to quantify mRNA expression of candidate prognostic markers GFI1 and TNFRSF11A, and analysed their association with clinicopathological features from FFPE colorectal tumour tissue. Furthermore, this study quantified intra-tumoural expression of GFI and TNFRSF11A in different cell types present within the colorectal tumour tissue.This study successfully established RNA ® data with immunohistochemistry showed a correlation between RNA and protein levels for MLH1 demonstrating the utility of RNAscope® against an established diagnostic tool. However, despite the use of a positive control gene to ensure RNA integrity in each core, some discrepancies between detection of mRNA and protein expression were encountered. Possible explanations include variable immunohistochemical staining intensities being misinterpreted; the likelihood that not all MLH1 mRNA is translated into protein due to post-translational modifications, or that protein degradation is occurring. Additionally, tumour stage-related changes may also affect mRNA expression levels during tumour progression [MLH1 immunohistochemistry is used, in conjunction with other genes, as a diagnostic assay to ascertain whether a colorectal cancer patient has a deficient mismatch repair system (microsatellite instability) . Comparigression . Interesgression , 27.® as a technical comparable method to immunohistochemistry, this study successfully utilized RNA in situ hybridisation (RNAscope®) to quantify mRNA expression of candidate prognostic markers GFI1 and TNFRSF11A from FFPE colorectal tumour tissue. Data from RNAscope® showed associations for reduced GFI1 and TNFRSF11A mRNA expression to poor prognostic features, similar to those identified using microarray expression data from a large TCGA study [Having established RNAscopeGA study .GFI1 was significantly associated with left sided and rectal (primary site) tumours. This is in contrast to the observation that left-sided colorectal cases have a better prognosis than right-sided tumours, even when adjusting for possible differences in screening practices and treatment [Reduced expression of reatment . Our res® data also showed good agreement with the TCGA study [TNFRSF11A and tumour features consistent with poor prognosis, including high nodal stage and metastatic disease. The role of TNFRSF11A in colorectal cancer tumorigenesis remains to be elucidated. There are limited studies reporting TNFRSF11A mRNA expression in colorectal cancer cases [et al., reported TNFRSF11A overexpression in primary tumours of colorectal cancer cases and their associated metastases to the bone [TNFRSF11A mRNA expression is associated with poor prognosis. A possible explanation is that reduced TNFRSF11A mRNA expression alters the TNFRSF11A /TNFSF11/TNFRSF11B signalling axis in cells. Reduced TNFRSF11A expression leads to reduced availability of TNFSF11, promoting more TNFRSF11B to be available. TNFRSF11B acts as a decoy receptor for TRAIL resulting in decreased TRAIL induced apoptosis, thus allowing cells to survive for longer and proliferate [TNFRSF11A mRNA expression may be associated with a specific molecular subtype(s) of colorectal cancer. A limitation with this study is that molecular subtyping information of cases was not available. Further work is required to determine the role of TNFRSF11A in colorectal tumorigenesis.Our RNAscopeGA study  for reduer cases . Santinithe bone . In contthe bone , show reliferate . Furtherliferate . AlternaGFI1 and lymphatic invasion, fraction of positive lymph nodes , tumour stage and distant metastasis. Additionally, we did not observe an association between TNFRSF11A and tumour stage or distant metastasis. These differences may be due to the level of power associated with the smaller cohort used in this study. This limitation affected our ability to perform multivariate analysis due to the small proportion of cases within certain categories.Unlike the TCGA study , we did ® and the TCGA study [GFI1 was selected for its high statistical significance ranking for mRNA expression levels and association with prognostic features. Similarly, TNFRSF11A was selected for its high statistical significance ranking of mRNA expression and importantly for the first gene to be known to be affected by CNA status. Analysis of the data downloaded from the cBioPortal demonstrated a statistically significant association between negative mRNA expression and gene copy number loss for both GFI1 and TNFRSF11A gene. These results suggest that copy number loss may explain the reduced expression of these genes seen in some TCGA tumours. Reduced CNA status may be driving reduced expression within our study, especially for TNFRSF11A which was selected based on this premise. Future application of DNA targeted in situ technologies will complement RNAscope® for profiling gene copy number and expression at the single cell level.Differences between RNAscopeGA study  may also® to provide a standardised, objective method to quantify mRNA expression levels  for GFI1 and TNFRSF11A. Our data showed GFI1 and TNFRSF11A were expressed at a significantly higher level in carcinoma cells compared to non-carcinoma cells . Thus, by analysing tumours at the cellular level we were able to demonstrate that reduced mRNA expression levels measured in patients with poorer prognosis were specifically due to carcinoma cells within the tumour and not due to contamination of tumour samples with non-carcinoma cells. It is therefore possible that reduced expression of these genes in patients with poor prognosis found by the TCGA study [Assessment of intra-tumoural heterogeneity within our study was achieved by combining an image analysis tool with RNAscopeGA study  was due in situ hybridisation include potential issues surrounding RNA recovery and degradation in FFPE material, which have been well documented and encompass pre-analytical variables of specimen fixation, processing and storage prior to TMA construction [® probes are specifically designed to overcome these issues [Limitations with using RNA truction –34. Thestruction . Howevere issues .In our study, archival FFPE material ranging between 10–15 years old was used. Despite the age of FFPE material, quality mRNA was present in the majority of the TMA cores. However, negative or low expression for positive control mRNA did exist in a minority of cases indicating sub-quality, lowly abundant RNA. No information regarding pre-analytical variables  was available that could help account for these observations.in situ hybridisation technique (RNAscope®) for validating potential gene expression biomarkers from published studies, circumventing the potential limitations associated with qPCR and immunohistochemistry. This study demonstrates RNAscope® as a promising method to visualise and quantify mRNA expression of candidate biomarkers on archival FFPE colorectal cancer cases. This will allow researchers to carry-out retrospective studies on archival FFPE material, which have a wealth of pathological, clinical and follow-up information available, to investigate and validate candidate biomarkers. Our selection of candidate biomarkers using copy number changes provides a level of standardisation that can be implemented across different patient cohorts when investigating biomarkers to overcome the impacts of tumour heterogeneity in gene expression studies. This study found significant associations for altered mRNA expression for the genes, GFI1 and TNFRSF11A, with a selection of poor prognostic features that were consistent with those identified by a large TCGA study. To our knowledge, this is the first study to assess the intercellular expression patterns of these candidate prognostic markers in colorectal tumours. Quantitative data generated by RNAscope® overcame potential affects from tumour heterogeneity, permitting direct association of carcinoma specific mRNA expression level changes with clinicopathological outcomes. Future larger studies are required to confirm the link between GFI1 and TNFRSF11A and patient outcome, and to determine the role of these genes in colorectal cancer development.In conclusion, this study shows the advantages of an mRNA A cohort of 112 primary colorectal adenocarcinoma cases of varying histology, grade, age and gender were obtained from the Christchurch Cancer Society Tissue Bank  . Before GFI1 and TNFRSF11A, from this study that were highly ranked  among the genes associated with tumour aggressiveness, respectively (see Table The Cancer Genome Atlas (TCGA) Network published a comprehensive study of gene expression and genomic changes in a series of colon and rectal tumours . This anGFI1 is a transcription repressor previously associated with intestinal epithelial cell differentiation [TNFRSF11A is a member of the TNF receptor super family, and plays a major role in bone remodelling and immunity [TNFRSF11A was selected based on the observation that both reduced copy number (18q deletion) and reduced expression were associated with aggressive tumours. Candidate prognostic gene markers showing a correlation between copy number alterations (CNA) and expression are more likely to be evident across a significant proportion of the tumour, and thus circumvent potential non-reproducibility associated with tumour heterogeneity [ntiation . TNFRSF1immunity . In addihttps://cancergenome.nih.gov/; Download: 10 August 2016) and analysed. We also downloaded and compiled microarray expression data for GFI1 and TNFRSF11A, and putative copy number calls for GFI1 and TNFRSF11A, using the cBioPortal for Cancer Genomics  [Expression data, DNA copy number data and clinicopathological data for 218 colorectal adenocarcinoma patients was downloaded from The Cancer Genome Atlas (TCGA) network (st 2016) , 38. Tesst 2016) .GFI1, TNFRSF11A and MLH1 were investigated using the RNAscope® 2.0 Assay  [GFI1, TNFRSF11A and MLH1 were selected from the ACD RNAscope® probe catalogue for human species. The assay was optimized and performed according to manufacturer's instructions. Briefly, for both individual tissue and TMA specimens, sections were cut at 4μm thickness and placed on SuperFrost Plus glass slides, before deparaffinising in a series of xylene and 100% ethanol steps. Each section was then subjected to a series of pre-treatment steps before progressing onto hybridisation with target probes for their respective gene. Hybridisation involved placing 4–6 drops from a Ready-To-Use bottle (approximately 150 μL) of the individual target probes onto the slide, enough to cover tissue sections. Positive (PPIB) and negative (DAB) control probes were hybridised to additional sections. Slides were then covered in a HybEZ™ Humidity Control Tray and placed in the HybEZ™ Oven and incubated at 40° C for 2 hours. After this time, a horseradish peroxidase-based signal amplification system was applied to consecutively hybridise pre-amplifier and several amplifiers to the target probes before colour development using diaminobenzedine (DAB). Slides were then counterstained with Gill's Haematoxylin, dehydrated and cleared before being mounted, coverslipped and assessed microscopically. Slides were determined to be positive for mRNA expression if brown punctuate dots could be seen within cells.The mRNA expression levels for ard, CA) . The proImmunohistochemistry was performed using the Roche VENTANA BenchMark ULTRA System . For both individual tissue and TMA specimens, 4 μm sections were cut, placed onto SuperFrost Plus glass slides, deparaffinised in xylene and placed in 100% ethanol. Slides were loaded onto the BenchMark ULTRA System and subjected to heat induced epitope retrieval using “Cell Conditioner 1” at 95° C for 64 minutes, followed by incubation with one drop of the VENTANA pre-dilute MLH1 (M1) mouse monoclonal primary antibody  for 16 minutes at 36° C. Detection was achieved with the ultraView Universal DAB Detection Kit, before each section was counterstained with haematoxylin, coverslipped, and assessed microscopically.Images were captured using a Zeiss Apotome Microscope and associated software . Three representative images of carcinoma cells, normal epithelial cells, lymphocytes and stromal fibroblasts were captured for each tumour case for each individual probe at 40x resolution.® score of >2 for the positive control gene (PPIB) is indicative of abundant, good quality RNA. Assessment and scoring of MLH1 immunohistochemistry staining in specimens was carried out by a pathologist (MRW). Positive staining for MLH1 was considered when carcinoma cell nuclei displayed any traces of brown positive staining. A negative staining was considered when carcinoma cell nuclei were negative while other types of cell nuclei were positive. ImageJ software [The mRNA expression levels for all three probes were visually assessed by a cytologist (AM-B) and a pathologist (MRW), blinded to clinicopathological data. Slides exhibiting positive mRNA expression were semi-quantitatively and manually assessed for the number of probe signals per cell for each case using the manufacturers scoring system . The scosoftware  was utilt-test with Satterthwaite's adjustment for unequal variances was used to test for differences in means between gene copy number and mRNA mean expression levels. Fisher's exact test was used to test homogeneity between RNAscope® and immunohistochemistry. Logistic regression was used to test for associations between semi quantitative gene expression levels and clinicopathological features for RNAscope® data. All tests were two sided with a p-value of ≤ 0.05 considered to be significant. ANOVA with Tukey post-hoc t-tests was used to investigate differences in mean mRNA expression levels and cell types. Analysis was performed in R 3.3.1 .The two-sample


Bacillus anthracis edema toxin (EdTx) as an adjuvant, we previously showed that innate responses triggered after sublingual immunization could limit generation of IgA responses. We tested whether co-administration of a neutrophil elastase inhibitor (NEI) could rescue the ability of EdTx to induce broad antibody responses, including mucosal IgA. NEI supplementation of sublingual vaccines containing EdTx promoted antigen-specific serum IgA responses but also enhanced serum IgG1, and IgG2b responses. This enhancing effect of NEI did not extend to all antibody isotypes and IgG sublclasses, since NEI  reduced serum IgE responses and did not affect IgG2a/c and IgG3 responses. NEI supplementation also promoted anti-Bacillus anthracis protective antigen (PA) neutralizing antibodies and enhanced high affinity IgG1 and IgA antibodies. In addition to serum IgA, NEI supplementation stimulated antigen-specific mucosal IgA responses in the GI tract, and enhanced antigen-specific IgG responses in vaginal washes. Analysis of CD4+ T helper cell responses revealed that co-administration of NEI broadened the profile of cytokine responses, by stimulating Th1, Th2, Th17, and Tfh cytokines. We also noted that NEI had a higher stimulatory effect on IL-5, IL-10, IL-17 responses.Sublingual immunization is emerging as an alternative to nasal immunization and induction of mucosal IgA responses. Using  Secretory IgA (SIgA) represents the hallmark of immune responses at mucosal surfaces. The high resistance of these polymeric immunoglobulins to degradation in the harsh environment of mucosal surfaces, including the lumen of the gastrointestinal tract, allow them to provide frontline protection at the portal of entry of most infectious microbes2. While mucosally delivered subunit-vaccines have the potential of stimulating broad mucosal and systemic immune responses, their ability to trigger mucosal IgA relies on the addition of effective vaccine adjuvants. Stimulation of inductive site immune responses in different mucosal sites  imprints the expression of discrete mucosal homing receptors and adressins which allow effector B and T cells to home in distinct mucosal tissues. For example, intranasal delivery of vaccines containing appropriate mucosal adjuvants can promote specific immune responses in the airways. However, safety issues were reported following intranasal application of  a ganglioside-binding toxin as adjuvant. Sublingual immunization is now being considered as an alternative to the intranasal route of vaccination. Nonetheless, a major challenge for the development of sublingual vaccines is the identification of appropriate antigen-adjuvant formulations3.Needle-free vaccines delivered via mucosal surface have the potential of being better-accepted by the most vulnerable and commonly vaccinated population of children. They also present higher likelihood to generate the necessary B and T cell responses for optimal protection at the portal of entry of infectious agents, in addition to promoting the levels of systemic immunity generally achieved by conventional injected vaccinesBacillus anthracis edema toxin (EdTx) is an effective adjuvant capable of promoting both systemic immunity and mucosal SIgA responses against nasally co-administered vaccine antigens5. However, when EdTx was tested as adjuvant for sublingual vaccination, it promoted antigen-specific IgG responses in the bloodstream but failed to elicit IgA responses in the serum or mucosal secretions6. This lack of IgA responses was not due to the route of immunization itself, since sublingual immunization could induce these responses when vaccines were administered with a range of adjuvants including bacterial enterotoxins, toll-like receptor ligands, and STING ligands11. Interestingly, the lack of IgA response correlated with the recruitment of neutrophils after sublingual administration of EdTx, and partial depletion of neutrophils before sublingual immunization restored the adjuvant activity of EdTx for IgA responses6. Neutrophils represent the largest population of myeloid cells in the bloodstream and characterize the initial response to inflammatory events through their own degranulation and cytokine production12. Unfortunately, depletion of neutrophils prior to immunization is not a feasible approach and thus, new strategies are needed to improve the efficacy of EdTx-based, and possibly other, sublingual vaccines.We have previously shown that 13. Neutrophil elastase inhibitors (NEI) are a class of serine protease inhibitors that target the neutrophil granule protein elastase, commonly implicated in chronic lung inflammation16.Neutrophils are recruited by inflammatory cytokines, including IL-6, IL-1β, and TNFα, and were more recently recognized to contribute to the chemotaxis of other myeloid cells through the products released after neutrophil degranulationWe first asked whether supplementation with a NEI could affects IgG responses induced by a model sublingual vaccine containing EdTx as adjuvant. Vaccines targeting two or more pathogens can reduce the schedule of vaccination. We choose to use Ovalbumin (OVA) plus Bacillus anthracis protective antigen (PA) as a combinatorial antigen to test the ability of NEI to regulate the immune response to two different antigens. OVA is a well-studied model antigen which allow a more in-depth analysis of immune responses to vaccination thanks to the large number of reagents available to study OVA-specific B and T cell responses. On the other hand, the use of PA as antigen allowed us to address the biological significance of the antibody responses through the assessment of anti-PA antibodies (Abs) ability to neutralize anthrax lethal toxin (LeTx). Analysis of OVA-specific IgG1 responses revealed that NEI increased the magnitude of responses induced by EdTx, and this effect was evident as early as day 14 after the first immunization Fig. . We alsoSince the presence of NEI differentially regulated IgG subclass responses, we next addressed its potential effects on other immunoglobulin isotypes. Sublingual immunization with EdTx as adjuvant induced OVA-specific IgE responses which peaked 2 weeks after the first immunization (day 14) Fig. . Co-admi6, sublingual immunization with EdTx as adjuvant failed to induce OVA-specific serum IgA responses  and were further increased one week later (day 28)  Fig. . To dete28) Fig. . However28) Fig. . These d+ T cells upon in vitro restimulation with OVA to elucidate the profile of T helper cytokines that supported the antibody responses induced by supplementation with NEI. Spleen cells from mice immunized with antigens plus NEI exhibited the same frequencies of Th1 cells (IFNγ+CD4+ T cells and TNFα+CD4+ T cells) as spleen cells from mice that received antigen plus EdTx . Sublingual vaccination is theoretically very simple as it involves application of liquid or rapidly dissolving tablets to the floor of the oral cavity. Like vaccines delivered at other mucosal sites, sublingual vaccines have the potential of producing mucosal SIgA in addition to IgG responses in the general bloodstream. Because sublingually administered vaccines are less likely to reach central nervous system tissues, they are potentially safer than nasal vaccines. Despite these advantages, identification of appropriate adjuvant formulations remains a requirement for the development of sublingual vaccines. Here we show that supplementation of the EdTx adjuvant with a chemical inhibitor of a neutrophil function broadens B and T cell responses induced after sublingual immunization and promotes antigen-specific SIgA Abs.6. While this work was the first to establish an inverse relationship between the presence of neutrophils and production of IgA, the mechanisms underlying the suppressive effect of neutrophils on IgA production remained elusive. It was previously shown that neutrophil peptide defensins enhance IgG, but not IgA responses against nasally co-administered vaccine antigen17. The primary (or azurophilic) granules of neutrophils contain defensins, myeloperoxidase, lysozymes, and three serine proteases: neutrophil elastase, cathepsin G and protease 318. We now show that inhibition of a single function of neutrophils can rescue the adjuvant activity of EdTx and promote IgA responses both in the blood stream and in mucosal tissues. This finding is significant because the use of a serine protease inhibitor might be a more realistic alternative to neutrophil depletion as a strategy for promoting IgA responses. It also raises the question of the mechanism behind neutrophil elastase limiting IgA production. Neutrophil elastase and the related serine proteases cathepsin G and proteinase 3, were shown to regulate cytokine responses through activation or degradation of cytokines, cytokine receptors, or toll-like receptor19. Our separate studies have shown that NEI induce the expression of IL-10 mRNA but also AID and BAFF responses by spleen cells in vitro20. Because IL-10 facilitates Ig class switching for production of IgA21 and regulate the expression of AID for induction of Ig CSR22, this may be a mechanism for enhanced IgA responses in mice sublingually immunized with EdTx in the presence of NEI.We have shown that the breath of antibody responses induced by EdTx as a sublingual adjuvant is limited by the recruitment of neutrophils in mucosal inductive sites10 and the genitourinary tract10. However, neither CpG nor the STING ligand 3′33-cGAMP as sublingual adjuvant induced SIgA in the secretions of the GI tract 10. We now show that when added to an EdTx-based sublingual vaccine, NEI primarily promoted SIgA responses in mucosal secretions of the GI tract. No significant levels of IgA were measured in other mucosal secretions although IgG responses were significantly increased in vaginal washes. The best-described factors that regulate homing of immune cells into the gut are CCR9 and gut homing receptors α4β724. These factors are induced through signals provided by retinoid acid, a product of myeloid cells in mucosal tissues23. To our knowledge, no study has examined whether neutrophils regulate expression of gut homing receptors. But it is worth noting that in conditions of normal homeostasis, neutrophils are not present in mucosal tissues where the concentration of IgA is the highest in the body. The observation that NEI primarily promotes SIgA in the gut suggests that it either directly stimulated retinoic acid production or stimulated cytokines such as IL-5, IL-6 and IL-21, which promote or enhance expression of gut homing receptors26.Sublingual immunization of mice with STING ligands as adjuvant induces IgA responses in mucosal tissues of the airways27. Thus, the presence of NEI may have promoted the maturation of dendritic cells and their expression of BAFF and APRIL, and perhaps costimulatory molecules , for enhanced antibody production. Another important point revealed in the present study is that the presence of NEI improved the quality of antibody responses. Indeed, NEI increased the titers of high affinity IgG and IgA antibodies, an effect that translated into higher titers of anti-PA neutralizing antibodies. Our findings are in line with the recent report that neutrophils regulate germinal center formation in secondary lymphoid organs and production of autoreactive plasma cells in lupus28. Analysis of antigen-specific T helper cytokine responses provided some clues on how NEI improved the breath, the magnitude and the quality of antibody responses in mice immunized via the sublingual route with EdTx as adjuvant. We found that addition of NEI resulted in higher frequencies of Th1 cells producing IFN-γ, Th2 cells producing IL-5 and IL-10, Th17 cells producing IL-17, and Tfh cells producing IL-21. This broad profile of Th cell cytokine responses has most likely contributed to increase B cell survival via signals from IL-5 and IL-1733. It is also consistent with the observation by others that neutrophils preferentially accumulate in proximity of T cells in secondary lymphoid organs during the early stage of lupus and that neutrophil depletion accelerate germinal center formation28. In our study, the NEI may have played the same role as neutrophil depletion and enhanced development of germinal centers and production of high affinity antibodies by facilitating differentiation of Tfh producing IL-21.IgA was not the only immunoglobulin isotype affected by supplementation of the EdTx-based sublingual vaccine with NEI. In fact, NEI also enhanced IgG1 and IgG2b responses. This is consistent with the previous report that elastase inhibits the maturation and function of dendritic cells, including expression of the costimulatory molecules CD40, CD80 and CD86manuscript in preparation). It is also worth noting that NEI alone exhibited some adjuvant activity and enhanced IgG1 and IgA responses against the sublingually co-administered vaccine antigens. This effect does not appear to extend to other immunoglobulin isotypes/subclasses. Previous studies have established that after oral administration, the plasma concentration of the NEI Alvelestat peaked by 05–1.5 hrs and that it was eliminated thereafter34. Thus, the NEI is believed to act quickly after sublingual immunization and to be eliminated within 24 hrs. Future studies will investigate the underlying mechanisms and whether NEI can be used as a universal strategy for improving immune responses to sublingual vaccines regardless of the adjuvant or delivery system.Altogether, our results show that supplementation of sublingual vaccine by co-administration of a NEI can improve the kinetic, breath and quality of antibody responses both in the bloodstream and in mucosal tissues. These findings raise the question of can NEI display similar adjuvant activity for vaccines given through other mucosal routes and, possibly non-mucosal vaccines? Our separate studies indicate that this is in fact the case and that co-administration of NEI modulates immune responses induced by intranasal and injected vaccines  at The Ohio State University approved all protocols. All efforts were made to avoid unnecessary pain and distress and to minimize animal suffering during the course of these studies.Female C57BL/6J mice were obtained from The Jackson Laboratory . Mice were maintained in a specific pathogen-free environment and used at 10 ± 2wks of age. All the experiments were approved by and performed in accordance with the guidelines of NIH and The Ohio State University IACUC.10 by administration of 10–13 μl of a PBS solution to mice anesthetized by intraperitoneal injection of ketamine/xylazine (0.1 ml of a 20 mg/ml ketamine/20 gm body weight). Each anesthetized mouse received antigen  in PBS. To assess the adjuvant activity of Bacillus anthracis edema toxin . To address the effect of NEI supplementation, groups of mice were given antigens plus 100 μM of Alvelestat  , or antigen plus EF plus Alvelestat. Mice that received the NEI showed no change in their vitality, food consumption or  body weight. All groups were immunized at weekly intervals for 3 consecutive weeks . Serum, fecal pellet and vaginal wash samples were collected weekly, and nasal washes were collected at day 28.Sublingual immunization was performed as previously described36 Briefly, microtiter plates were coated with OVA (1 mg/ml) or PA (15 μg/ml). For detection of OVA- or PA-specific IgG and IgA Abs, serum or fecal material extracts were serially diluted in PBS 1% BSA, added to the plates and the binding antibodies were detected with HRP-conjugated anti-mouse γ- or α-heavy chain-specific antisera . Biotin-conjugated rat anti-mouse IgG1, IgG2a/c, IgG2b or IgG3 monoclonal Abs and HRP-conjugated streptavidin  were used to measure IgG subclass responses. The reactions were revealed by addition of the water-soluble HRP substrate ABTS  and the Ab titers were determined as the last dilutions of samples with an absorbance of >0.1 above that of control samples from naïve mice.To determine OVA-specific and PA-specific antibody titers, ELISA were performed with antigen-coated plates as described previouslyFor assessment of IgA levels in the intestinal secretions, freshly emitted fecal pellets were normalized by homogenization in PBS (1 ml per 0.1 g feces). After centrifugation, dilutions of supernatants were used for evaluation of antigen-specific IgA levels as described above.37. After washing, detection antibodies were added and the remaining steps of the ELISA conducted as described above.High affinity antibody responses were measured by ELISA as described above with a minor modification. Briefly, plates were coated with PA and incubated with dilutions of the samples. Urea (4 mM) was then added and the plates incubated for 30 min at room temperature to remove antibodies that bound to the antigen with low affinity6. Briefly, sample dilutions were added to J774 macrophages cultured in cultured in RPMI supplemented with 10% fetal calf serum. Bacillus anthracis lethal toxin (LeTx)  was then added to the plates. After overnight incubation, MTT −2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added to assess the viability of macrophages as a function of redox potential. Toxin neutralizing antibody titers were determined as the lowest concentrations of sera  that protect macrophages from the cytotoxicity of LeTx.Toxin neutralization assay was performed as previously describedin vitro restimulation and intracellular staining with cytokine-specific fluorescent antibodies. Briefly, splenocytes and cervical lymph nodes were collected on day 28 after the first immunization and restimulated with antigen  in vitro as previously described6. After 5 days culture, cells were subjected to extracellular staining with lineage-specific antibodies  and, after fixation, to intracellular staining with Th1, Th2, Th17, and Tfh cytokine-specific antibodies (Biolegend). Labeled cells were then analyzed with an Attune NxT flow cytometer .Antigen-specific T helper cell cytokine responses were analyzed by flow cytometry after Results are expressed as mean ±one standard deviation. Statistical significance was determined by one-way ANOVA, followed by Tukey post-hoc test. All statistical analyses were performed with the StataSE 12.0 software  and Prism 7 software .


In the visual system, retinal ganglion cells (RGCs) of various subtypes encode preprocessed photoreceptor signals into a spike output which is then transmitted towards the brain through parallel feature pathways. Spike timing determines how each feature signal contributes to the output of downstream neurons in visual brain centers, thereby influencing efficiency in visual perception. In this study, we demonstrate a marked population-wide variability in RGC response latency that is independent of trial-to-trial variability and recording approach. RGC response latencies to simple visual stimuli vary considerably in a heterogenous cell population but remain reliable when RGCs of a single subtype are compared. This subtype specificity, however, vanishes when the retinal circuitry is bypassed via direct RGC electrical stimulation. This suggests that latency is primarily determined by the signaling speed through retinal pathways that provide subtype specific inputs to RGCs. In addition, response latency is significantly altered when GABA inhibition or gap junction signaling is disturbed, which further supports the key role of retinal microcircuits in latency tuning. Finally, modulation of stimulus parameters affects individual RGC response delays considerably. Based on these findings, we hypothesize that retinal microcircuits fine-tune RGC response latency, which in turn determines the context-dependent weighing of each signal and its contribution to visual perception. There is much evidence showing that RGCs detect stimuli with various time delays13, resulting in an uneven launch of signals through various visual pathways. Moreover, the dissimilar RGC axon calibers may result in additional differences in signaling speed towards the brain as well14. It has been found earlier that our visual system processes information about moving objects more rapidly than flashed objects and due to this latency difference, we are prone to well-known visual illusions17. These latter observations reflect that certain visual feature pathways operate faster than others and dissimilar RGC response latencies might provide the backbone for this phenomenon. A recent work presented evidence in the retina of the cold-blooded salamander that the RGC latency difference can be exploited by the visual system to encode certain spatio-temporal features of the scene18. These facts indicate that RGC response latency is a non-negligible factor in understanding the fundamentals of visual processing. However, some important questions yet remain unanswered: (i) how great is the latency range across the entire RGC population; (ii) is the observed range based on imprecise responding of RGCs or summated by RGC subtype specific subranges; (iii) are latency differences due to dissimilar RGC membrane properties and/or determined by the signaling speed through the upstream retinal circuitry and (iv) how does the visual system exploit differences in response latency to perfect mammalian vision?In the mammalian retina, various features of the visual scene are encoded by dedicated retinal ganglion cell (RGC) subtypes and their spike signals are streamed towards the brain through parallel visual pathways. These separate information avenues inform the brain about object- and/or background motionvia multiple recording paradigms while stimulus parameters were modulated or pharmacological interventions were carried out. Simple full-field photopic light stimuli exerted RGC responses with rather different delays, with most of them ranging between 40 and 240 ms. Our results showed that response delays were RGC subtype specific and varied extensively when stimulus parameters were altered. Trial-to-trial variability of RGC light responses were also subtype dependent but contributed little to the observed population-wide latency range. Results of this study thus indicate that key components of a latency control mechanism are embedded in retinal microcircuits that fine-tune RGC response latency, thereby adjusting the weighing of signals in generating postsynaptic effects.To answer some of these cardinal questions here, light-evoked responses of mouse RGCs were recorded Pvalbtm1(cre)Arbr (JAX #017320) mice with the Ai95(RCL-GCaMP6f)-D (C57BL/6J) (JAX #028865) animals. Mice were deeply anesthetized with the inhalation of Forane  and then sacrificed using cervical dislocation. Eyes and retinas were removed under dim red illumination and hemisected anterior to the ora-serrata. Cornea, lens, vitreous humor, as well as the pigment epithelium were isolated, and the resultant isolated retina was attached to a filter paper (Millipore). Specimen then were placed in a superfusion chamber mounted in a light-tight Faraday cage and superfused with an oxygenated  and heated (34 °C) mammalian Ringer solution (pH = 7.4). Animal handling, housing, and experimental procedures were reviewed and approved by the ethical committee of the University of Pécs . All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All efforts were made to minimize pain and discomfort during the experiments and all procedures were done by obeying the 3R law.Adult (P30-90) C57BL/6J and Thy1-GCamP3 (JAX #029860) mice were maintained in a 12/12 hours dark/light cycle; all experiments were carried out during the day with dissections between 10–12 AM and after 12 hours of dark-adaptation prior to experiments. For one set of experiments a PV-GCaMP6f hybrid mouse line was utilized as well. This line was obtained by crossing ++-imaging experiments were carried out in the Thy-1 GCaMP3 transgenic mouse line, in which the Ca++-indicator is expressed by all RGCs. In the PV-GCaMP6f line, GCaMP was expressed only by a subset (6 cell types19) of RGCs. A modified upright Nikon FN1 microscope equipped with 4x, 10x, 40x and 60x objectives  was used for imaging. RGC responses were captured upon full-field photopic light stimulation  and sampled at 33.3 Hz. We measured the RGC response delay and trial-to-trial variability. Excitation light was generated by Polychrome V monochromator  and image sequences were captured by a Retiga 2000DC digital camera (QImaging) while the acquisition was performed with Live Acquisition software . Alternatively, an Intensilight light source (Nikon) and a Sony A6300 camera were used for the image sequence acquisition driven by TTL-pulses, which were generated in the WinWCP  acquisition software. In this latter case, a magnetic switch modulated a LED-light source and the sampling frequency was 60 Hz with constant ISO and filter settings, in Full HD (1080p) resolution. In most cases, input/output signals were synchronized by an Instrument Control Unit . Image sequences were first handled in FIJI ; region of interests (ROI) for examined RGCs were selected here by the ROI manager. Image analyses were performed in Matlab and NeuroCa20 integrated framework. Temporal ROI sequences of a network burst were implemented, nonparametric statistical analyses were performed and temporal orders were determined. Ca++-transients (spikes) were collected for all RGCs in the examined frame and initiation times, amplitudes and widths were determined by using fluorescence changes (ΔF/F0) in each ROI20. Some analyses on acquired imaging data were performed in Microsoft Excel and/or Origin2018 .CaUnit extracellular recordings were performed from RGCs with carbon microelectrodes  attached to an AC differential amplifier  and digitized with an analog-to-digital board . RGC action potentials were recorded with Axoscope  with a sampling rate of 20 kHz. In some experiments, multielectrode extracellular electrophysiology was also performed by utilizing either a 60 or a 120 channel MEA system  that allowed for recording simultaneously from many RGCs. Analyses were performed offline using Spike 2 , Off-line Sorter  and NeuroExplorer 5  softwares. Histograms and graphs were generated in Origin2018 . Collected spikes were timestamped (bin size 10 ms) to generate peristimulus-time histograms . PSTHs then served to obtain response peak positions and delays. Response fadeout time (PSTHτ) and trial-to-trial variability were calculated in Microsoft© Office Excel, further data- and statistical analyses were performed in Origin 2018 .Patch clamp recordings were performed with an Axopatch 200B Patch clamp (PC) amplifier (Axon Instruments) and ECS filled PC pipettes  in a cell attached configuration (Voltage Clamp mode). Signals were digitized with a Digidata 1440A ADC (Axon Instruments) and acquired with WinWCP software . Electrodes used for dye staining and stimulation were filled with ICS . Electrodes were pulled with a P-87 micropipette puller (Sutter Instruments). ICS contained (in mM) 125 potassium gluconate, 8 NaCl, 0.1 CaCl2, 0.6 MgCl2, 1 EGTA, 10 HEPES, 2 Mg-ATP and 0.4 Na-GTP at pH 7.3 (KOH-adjusted) and supplemented with 0.5% A568-hydrazide and 2% Neurobiotin to fill up target cells upon successful membrane-breach.∗/rod/s). In order to obtain these values, light intensity was first measured with a radiometer  and then converted assuming an average rod density of 437,000 rods/mm2 21 and quantum efficiency of 0.6722. In experiments where intensity response functions of RGCs were generated, stimulus intensity varied from 10−2 to 104 Rh*/rod/s by using an LED driver (with adjustable DC output); in the rest of the experiments photopic stimuli (>102 Rh*/rod/s) were delivered to the preparation. For spectral sensitivity recordings, a 100 W tungsten-halogen lamp and a light bench was used to deliver full-field white light. Light intensity was adjusted by neutral density filters  and spectral components were determined by a color bandpass filter set .Full-field light stimuli were delivered by a green (λ = 525 nm) LED and light beams were directly focused on the surface of the retina. Stimulus intensity was expressed as the time-averaged rate of photoisomerizations per rod  first and then switching to containers holding either 40 µM meclofenamic acid (MFA) or 50 µM picrotoxin (PTX) to block GJs and GABA mediated signaling, respectively. Recordings were repeated under drug administration and then wash-back incubations and recordings were attempted.After Neurobiotin filling, samples were incubated for minimum 30 minutes then tissues were fixed in 4% PFA for 15–25 minutes, washed with PBS, blocked with BTA , then incubated in streptavidin-Cy3 or -Cy5 (Thermo Fisher Scientific) in BTA for overnight. Washed retina samples were placed on slides mounted in Vectashield (Vector laboratories) and cover-slipped for microscopy.Retinal samples were scanned with a Zeiss LSM710 confocal microscope with 20x  and 63x objectives  in high resolution and normalized laser intensity. Minor manipulations of brightness and contrast of images were performed in .To examine the time course of RGC signaling in the mouse retina, light stimulus evoked responses were recorded. In order to minimize potential methodology related artefacts , we performed spike recordings by utilizing either a Tungsten extracellular electrode or a multi electrode array  and combined extracellular data were used for the following analysis. In a first set of experiments, RGCs were presented with six consecutive photopic  full-field TTL light flashes  and light evoked spike activity was recorded. Responses with either ON or OFF polarity appeared to show a wide range of variability in their latencies Fig. . To inveIt seemed unlikely that the observed response latency variability stems from the dissimilar times currents require to travel from the recorded soma to the electrode because Tungsten electrodes maintained a direct contact with recorded somata and they showed results comparable to those of MEA recordings. To gain further proof of this, the soma-to-electrode travel time was calculated based on selected MEA recordings in which multiple electrodes (with different soma-to-electrode distances) recorded the spiking activity of the same cell (n = 7). We found that signal delays and electrode distances showed no clear sign of correlation, indicating that a variety in electrode distance is a negligible factor in our measurements  were determined based on the timing of the first spike following the stimulus onset (the stimulus onset was used as a reference). We found that first spike time values in general were lower  than those of corresponding PSTH delays. However, delay distribution histograms appeared very similar in terms of the delay ranges for the two methods  upon stimulus onset may carry information about the stimulus24. To examine this possibility, we determined the variability of light evoked response delays for a cohort of randomly selected RGCs (n = 94). The trial-to-trial variability of responses varied across the RGC population  as well as the characteristic tracer coupling pattern . Only cells with clear OFF-alpha morphology were used in the following analysis  but rather reflected the heterogeneous nature of our dataset comprising many different RGCs with subtype specific response delays. To test this hypothesis, we targeted OFF-alpha RGCs in the mouse retina that could be readily discerned in the DIC view based on their large spherical or somewhat elongated soma and the rather eccentric nucleus positionsis Fig. . As expesis Fig. . Furthersis Fig. . In addisis Fig.  with stavia the presynaptic retinal network. To this end, we recorded the RGC spike output (n = 5) first by stimulating retinas with a full-field photopic light flash (I = 7000Rh*/rod/s) and then we maintained the preparation in dark and switched to a direct electrical stimulation of the target RGC soma. To standardize electrical stimulations, recordings of this experiment were carried out by glass micropipettes in a cell-attached mode (R = 6 MΩ). RGC macro-seals were maintained on a stable membrane potential (Vm = −60 mV) and stimulating impulses were applied through the recording pipette , their dynamic response range has been reported as adequate under natural stimulus conditions34. Therefore, we examined the range of response speeds by utilizing this methodology as well. Note that slow responses were not the results of the slow kinetics of the GCamP3 construct as the faster GCamP6 indicator resulted in both slow and fast responses when parvalbumin specific GCamP6 expressing RGCs were examined  are already heterogenous in terms of their latencies, thus they spread out in time similar to PSTH time-to-peak values. In fact, we found that examined RGCs displayed Ca++-transients with a broad range of delays  and that was clearly distinguishable from the trial-to-trial response variation range detected for individual cells (mean = 73.46 +/− 36.8 ms (SD); Fig. Another set of supporting results was obtained in our Caned Fig. ; n = 102via parallel retinal pathways. To see if this variety in signaling speeds is due to active fine-tuning performed by retinal interneuron circuits, we performed both spike recordings and Ca++-imaging measurements during pharmacological blockade of specific circuit elements including GABA-ergic inhibition and signaling through gap junctions (GJs). GJ blockade was carried out by the addition of meclofenamic acid  into the superfusate and light evoked RGC activity was recorded prior to, and following application. The GJ blockade induced changes in the response delay for all examined RGCs, either by decreasing or increasing response speeds. This dichotomy in latency change was observed in both spike recordings and Ca++-transient signals and it was as high as 47% and 200%, respectively  was achieved by incubating retinas in 50 μM picrotoxin (PTX) and recordings were performed both under control conditions and following drug application. Similar to results obtained under GJ blockade, GABA receptor inactivation resulted in either an increase or a decrease of response latency for most examined RGCs, irrespective of the experimental approach Fig. . Howevernts Fig. .37. Here, we tested how further non-spatiotemporal stimulus parameters, including stimulus strength as well as chromatic features, affect RGC response delays. First, the effects of stimulus intensity changes were tested by performing RGC (n = 16) recordings while retinas were excited with a series of full-field light stimulus steps with increasing strengths . This iths Fig. . This waths Fig. . In thisNext, effects of chromatic changes of the stimulating light in RGC response delays were tested. Light evoked responses of randomly selected RGCs (n = 34) were recorded and compared while the wavelength of the stimulating full-field light beam was gradually increased through 12 wavelength steps from 330 nm to 674 nm  or the freshness of the preparation (RGCs were recorded for up to 6 hours with no change in response characteristics). Therefore, as for the validity of our measurements, we are confident that the observed great variety in response delays is not deceptive and unrelated to experimental circumstances. In fact, similar latency differences have been reported extensively in retinas of various species35 and thus the data presented here further support these previous findings.One of the critical issues of the present work is the accuracy of latency measurements and whether their errors distort the interpretation of the data. As most experiments were carried out without visual targeting, recorded RGCs formed a heterogenous population in terms of their response latencies. In fact, all samples contained neurons with brisk-, sluggish- and intermediary responses as well as cells with no preference for any response phenotype regardless of the hour of the day, the age of the mouse  are collected to obtain PSTHs to determine latency values. In this study, we utilized a 10 ms bin size to generate PSTHs, which inherently create a +/−5 ms error in the dataset. However, this is negligible when compared to the observed latency range (~40–240 ms) across the mouse RGC population. Therefore, measurement error could not contribute considerably to the detected latency range.39; reviewed by35. Here we showed evidence that first spike latencies and PSTH peak latencies of the same RGC population, though they differed in their actual values, displayed comparably great ranges. In addition, we also showed that the observed broad response latency range was present in our experiments regardless of the data acquisition method  in terms of the code sent towards visual brain centers. In fact, there has been a long debate concerning the nature of the neuronal code utilized by the brain to perform a variety of tasks. The spiking rate as a parameter that the brain might utilize to decode incoming information from sensory organs is just one of the several possible variables40 have reported recently that foveal photopic signaling of midget RGCs endures a 30 ms delay compared to peripheral counterpart midget cells in the primate retina. This phenomenon was accounted to structural differences of foveal vs. peripheral cone photoreceptors. As the mouse retina holds no retinal specializations  and displays only little centro-peripheral structural differences41, retinal locations of recorded RGCs could not be the basis for latency differences observed in this study. Note that the mouse retina displays a dorso-ventral asymmetry in terms of cone photoreceptor chromatic sensitivity44. However, we recorded from both slow and fast responding RGCs in all retinal locations, ruling out the possibility that this dorso-ventral difference plays a role in the observed distinction. Moreover, the finding in the primate retina was based on responses of one particular cell type, the midget RGC, whereas we presented recordings from many different RGC subtypes in the mouse . In addition, the latency range observed here was about an order of magnitude broader than those reported for primate midget cells. Therefore, the basis for our findings must reside elsewhere in the mouse retinal circuitry.We found that RGC signal latencies varied on a rather broad range of 40–240 ms Fig. . Sinha aWe presented evidence showing that the RGC trial-to-trial response variability may contribute up to 10% (~24 ms) to the observed broad latency range. However, much of the observed variability must be accounted for by other mechanisms. We also showed through two example RGC subtypes that their responses covered only narrow latency subranges. This indicates that each mouse RGC subtype contributes with a narrow subrange to the detected population range. Such subtype specific response latency differences can be defined by dissimilar RGC membrane properties or by distinct signaling speeds of intraretinal circuits targeting each of the RGC subtypes. In a series of experiments using the dual electrical/light stimulating paradigm, we showed that RGCs in fact respond to extracellular electrical stimuli within the first PSTH bin (<10 ms), regardless of the recorded RGC subtype. Moreover, the range of response latencies dropped from ~200 to ~10 ms when light stimulation was replaced with electrical stimuli. Thus, bypassing the retinal circuitry diminished the broad range entirely, indicating that the population-wide broad latency range is determined primarily by the various speeds of RGC targeting intraretinal circuits.17. It is possible that vital visual features, such as movement (e.g. image of a moving predator), are carried faster to induce the necessary visually driven behavior well before contours, color or other features of the same object perceived.Certainly, some RGCs studied here are subtypes that send output to non-image forming brain centers in the midbrain and thus the latency by which they operate does not necessarily have to match those that project to the dorsal lateral geniculate nucleus and eventually the visual cortex. However, we suspect that most of our recorded cells do contribute to visual image formation and thus certain visual features travel faster to the cortex than others. In fact, psychophysiological experiments showed that object movement is perceived faster than object contour, contrary to the coincidence, or even the opposite order of the two types of stimuli++-imaging measurements. Although transient Ca++-responses were several times slower than the PSTH time-to-peak latency values, their latency range was similarly broad  are already heterogenous regarding their latencies, further suggesting that the upstream presynaptic circuitry provides inputs with heterogenous speeds to RGC subtypes. We found in both electrophysiological and imaging experiments that blocking either inhibition or electrical synapse-mediated signaling alters RGC response latencies. While the blockade of these circuit elements resulted in a mixture of effects (either a decrease or an increase) on response kinetics of individual cells, the population wide RGC latency range shrank in both cases . It has been reported that the dynamics of postsynaptic integration are in fact strongly dependent on incoming spike times50. This, together with the evidence of ~2–9 ms time constant measured for cortical pyramidal cells in the cat51, suggests the existence of a strong temporal filter in the visual cortex. This filter utilizes afferent signals correlated on a narrow temporal scale (<10 ms) to generate the spike output and dumps the rest, thereby filtering out non-adequate signal components. Based on the above converging evidence, we hypothesize that the ability to slightly alter response latency is a tool exploited by the visual system to weigh incoming signals and fine-tune visual perception. Moreover, key neuronal and synaptic components of this latency modulating mechanism are embedded in the retinal hyper-circuitry.It has been previously reported that the visual system informs the brain about different visual aspects through varying signaling speedsSupplemental Figures 1 and 2


Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC. Breast cancer is composed of several distinct subtypes that exhibit markedly different outcomes ,2. TriplAmongst available therapeutic options, anti-angiogenic agents have been reported to inhibit breast tumor growth . However+) and carbon dioxide (CO2), contribute to the perturbation of intracellular pH (pHi) homeostasis, a situation with potentially lethal consequences  and tissues were incubated at 4 °C for 2 h. Subsequently, tissues were washed and incubated in modified blocking buffer (0.15% blocking solution) for 1 h at 4 °C, followed by a solution change and continued incubation in modified blocking buffer O/N at 4 °C. Finally, tissues were mounted with Vectashield mounting medium .For the imaging of whole mount tissue slices, Z stacks were acquired from randomly chosen fields of view using a Nikon Ti confocal microscope  equipped with a 20x objective lens. A 3D maximum projection was generated for each Z stack for downstream analysis and quantification.+ blood vessels were enumerated using the cell counter plugin for ImageJ. For the purposes of analysis, each side branch stemming from a larger vascular structure was counted as an individual vessel. The amount of extravasated dextran was determined by subtracting the total dextran signal in a given image from the dextran signal colocalizing with staining for CD31 using Photoshop. For each quantified parameter, the number of images analyzed for each group is reported in the appropriate Figure legend.The percent area of each image positively stained for a given marker was quantified using ImageJ or Adobe Photoshop CS5 . 3D maximum projection images were used for quantification. CD31Tissues labeled in vivo with Lysine-fixable tetramethylrhodamine dextran (LRD) having a molecular weight of 70 kDa were whole mount immunostained as described above with antibodies targeting CD31  for detection of tumor blood vessels. Tissue slices were imaged as Z stacks by a Nikon Ti confocal microscope equipped with a 20x objective lens  and 3D maximum projections were produced for downstream analysis. Extravasation of LRD was quantified from randomized fields (20x magnification) of each group using ImageJ software .Conditioned media collected from the indicated cell lines was analyzed using the Human Angiogenesis Antibody Array  according to the manufacturer’s instructions. Positive signals were quantified using ImageJ software . The mean signal of duplicate spots representing each angiogenesis-related protein was normalized to the mean value of the reference signals present on the array.qPCR for CAIX and VEGFA expression was carried out as previously described . Brieflyp < 0.05. Statistical analyses were performed using GraphPad Prism 7 . Outliers were identified using Grubb’s test  and excluded from further analysis. Data are reported as the mean ± standard error of the mean (SEM). For comparison of three or more data sets, a one-way analysis of variance (ANOVA) was performed and a Holms-Sidak’s test was used to correct of multiple comparisons. Statistical significance was set at In conclusion, we have generated robust data demonstrating that the use of pharmacologic inhibitors of CAIX activity in combination with anti-angiogenic agents results in the inhibition of tumor growth and reduced lung metastasis in TNBC. Our findings suggest that targeting angiogenesis and hypoxia in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.


Tobacco exposure correlated significantly (p < 0.001) with decreased CD8 infiltration in p16+ OPSCC tumors. Our findings demonstrate that the HPV+ OPSCC clinical outcomes are strongly correlated with the TIME, which is potentially modulated by tobacco exposure. Immunomodulatory strategies targeting this disease in smokers must take into consideration the potential modifying effects of tobacco exposure on treatment effectiveness and clinical outcomes.Oropharyngeal squamous cell carcinoma (OPSCC) incidence is increasing at a nearly epidemic rate, largely driven by the human papillomavirus (HPV). Despite the generally favorable clinical outcomes of patients with HPV driven (HPV+) OPSCC, a significant subset of HPV tumors associated with tobacco exposure have diminished treatment response and worse survival. The tumor immune microenvironment (TIME) has been shown to be a critical driver of treatment response and oncologic outcomes in OPSCC generally and HPV+ OPSCC more specifically. However, the impact of tobacco exposure on the TIME in OPSCC patients remains unclear. We analyzed the relationship between TIME, tobacco exposure and clinical outcomes in OPSCC patients (n = 143) with extensive tobacco exposure (median pack-years = 40). P16 overexpression, a surrogate marker of HPV association, was a strong predictor of relapse-free (RFS) and overall survival (OS)  regardless of tobacco exposure and associated strongly with differential infiltration of the tumor by both CD3 and CD8 lymphocytes measured via immunohistochemistry . CD3 and CD8 infiltration was a strong predictor of RFS and OS and associated strongly with disease stage (AJCC 8 Virally mediated malignancies such as HPV+ OPSCC demonstrate increased tumor immunity, which is thought to contribute to differential treatment response10. This may also lead to improved response to immunomodulatory strategies in this HNSCC subtype, which is currently under investigation in a number of national and international clinical trials.Tumor immunity has been shown to be a critical driver of tumorigenesis, treatment response and clinical outcomes in multiple solid tumor types including head and neck squamous cell carcinoma (HNSCC). Among HNSCC sites, SCC of the oropharynx, associated with the human papillomavirus (HPV+ OPSCC) is increasing at a nearly epidemic rate across the United States6. In these patients, clinical outcomes are worse compared to those of non-smoking OPSCC patients12. The precise mechanisms which underlie the differential clinical outcomes of patients with a history of tobacco exposure remain unclear and may in fact be multi-factorial given the complex effects of tobacco exposure17. However, there is reason to believe that tobacco exposure may be an important modulator of the tumor immune microenvironment (TIME), a critical driver of treatment response and survival in patients with solid tumors19.Although a majority of new OPSCC diagnoses are thought to be driven by HPV, a significant number of patients also demonstrate a history of significant tobacco exposure, a known carcinogenic risk factor for OPSCC and a critical modulator of overall patient health23. The interaction between tobacco and TIME in HPV+ OSCC therefore remains an open question. CD8 lymphocytes are a critical component of TIME and a critical mediator of checkpoint inhibition10. Previous studies have shown that CD8 infiltration is high in HPV+ OPSCC10. In the current study we evaluated the relationship between aspects of the TIME (CD3 and CD8 lymphocytes) and clinical outcomes in intermediate risk OPSCC patients (HPV+ smokers).Previous literature has shown that tobacco has profound and widespread immunomodulatory effects which remain somewhat unclear. Some of this evidence suggests a potentially inflammatory effect, some a potentially immunosuppressive effectth Edition) staging system; staging was also recalculated using the 8th Edition of the Staging Manual in order to more appropriately account for the impact of p16/HPV status and reflect current staging algorithms24. Results of diagnostic procedures including imaging results, biopsies, and fine needle aspirations as well as the treatments provided and the associated dates were recorded.Following approval from Baylor College of Medicine and the Michael E. Debakey Veteran’s Administration (MEDVAMC) Institutional Review Boards, we reviewed the records of Veterans with previous untreated oropharyngeal squamous cell carcinoma (SCC) between January 1, 2000 and January 1, 2012. Oropharyngeal sites included: tonsil, base of tongue, pharyngeal wall, glossopharyngeal sulcus and soft palate. Waiver of consent was obtained from the IRBs; informed consent cannot be obtained from patients who are deceased and thus our normal IRB protocol is to provide a waiver of consent for retrospective analysis of previously collected tissue specimens. Data collection and analysis were performed in a manner consistent with existing standards for clinical research . Inclusion criteria included primary oropharyngeal SCC, tissue diagnosis at the MEDVAMC, sufficient pathologic tissue for immunohistochemical analysis, and treatment delivery at the MEDVAMC. Exclusion criteria included treatment at an outside institution and recurrent disease at presentation. Demographic information was recorded including age, gender, race, smoking history and alcohol consumption. Smoking history is collected at our institution at the time of initial diagnosis as “pack-years” consistent with general practice. Clinical-pathologic features were collected including clinical stage according to the American Joint Commission on Cancer  biopsy tissue blocks were retrieved from the archive maintained at the MEDVAMC Department of Pathology under BCM IRB and MEDVAMC R&D approved protocols. Sections were cut from each FFPE block at 5 µm thickness and mounted on positively charged slides. One section was stained with Hematoxylin & Eosin and reviewed by a surgical pathologist to confirm the original histopathological diagnosis and to ensure tumor adequacy. Slides were deparaffinized in Bond Dewax Solution , and rehydrated in descending grades of 100%, 90% and 70% ethanol. Endogenous peroxide activity was blocked by pretreatment with 3% hydrogen peroxide for 10 minutes, and antigen recovery was achieved by 20 minutes of heat-induced epitope retrieval. Immunohistochemical (IHC) stains were performed on an automated tissue-staining system using the Bond Polymer Refine Detection . Serial tissue sections were stained with monoclonal antibodies: CD3, clone LN10; CD8, clone 4B11; CD68, clone 514H12 ; p16, clone E6H4 . Detection was done using polymer reagent conjugated with horseradish peroxidase followed by the 3,3′-Diaminobenzidine (DAB) Chromogen Kit . Appropriate positive and negative controls were performed.25. Data for CD3 and CD8 were calculated as a % of positively stained nuclei compared to total nuclei.Low-power images were capture with 4X/0.16 objective using Vectra 3 multispectral camera. High powered images were selected using PhenoChart software . Up to 10 fields of view were selected from each low-power image and were captured using 20X/0.75 objective. Germinal centers were excluded from high-power imaging. inForm software  was used to analyze the high-power images. The software was trained on representative images for tissue and cell segmentation. Lymphocytes were scored using the Phenotyping feature to determine positively stained cells and cell counts were performed. P16 staining was performed as previously described by our group and classified as either positive or negative. A subgroup was defined by high-nuclear p16 staining consistent with our previous analysisth & 8th edition, p16 status ascertained via immunohistochemistry, treatment modalities, and tobacco exposure) was done. For all statistics, p-values were considered to be statistically significant if below a threshold of 0.05 (two-sided). Statistical calculations were performed with JMP Pro statistical software .Endpoints of interest included time to recurrence and death, all reported in months. Imaging was used as a surrogate in the absence of a pathological report documenting recurrence whether local, regional or distant. ‘Relapse-free survival (RFS)’ was defined as time from date of primary diagnosis to date of diagnosis of loco-regional and/or distant recurrence (i.e. whichever was diagnosed first); ‘Overall survival (OS)’ was defined  as time from date of primary diagnosis to date of death or last recorded follow-up in hospital notes. Patients suspected of recurrence were restaged using clinical exam and imaging. Patients who did not receive curative intent (n = 16) were removed from the RFS analysis; all patients were included in the OS analysis. Actuarial survival rates were generated using the Kaplan-Meier method, and comparisons between groups were made using log-rank statistics. Chi-square test was used to report likelihood of association between categorical variables. Univariate analysis was performed using Cox regression. All variables with p ≤ 0.05 on univariate analysis were used for multivariate analysis. We applied recursive partitioning analysis (RPA) to quantify a threshold for CD3 and CD8 infiltration -as surrogates for TIME- that is significantly associated with overall survival (p < 0.05). For multivariable analysis, RFS and OS modeling using standard clinical variables  consisted of 83 p16-positive (p16+) and 60 p16-negative (p16−), newly diagnosed, oropharyngeal carcinomas. All patients were male and the mean age at presentation was comparable between the two subgroups. Mean and median follow-up times for the p16+ cohort were 41.5 and 30.8 months respectively. Mean and median follow-up times for the p16− cohort were 14.9 and 7.8 months respectively. A majority of patients in both subgroups were treated with curative intent external beam radiation therapy (EBRT) based treatment though the proportion of patients receiving such treatment was higher in the p16+ compared to the p16− cohort . The full cohort had a high prevalence of tobacco exposure (91%) with all p16-negative patients having used tobacco products in their lifetime , and P16 status were significantly associated with RFS while T-classification, N-classification, M-classification, AJCC stage (8th edition) p16, and tobacco exposure (in pack-years), were significantly correlated with OS , AJCC  assessing RFS and OS as a function of mean CD3 and CD8 infiltration treated as continuous variables, detected a statistically significant effect on RFS and OS at a CD3 threshold of 33% and a CD8 threshold of 7% Fig. . Based oth Edition staging  or even decreased (larynx), survival for patients with OPSCC continues to improve, driven primarily by a shift in the epidemiology of the disease toward HPV mediated cancers29. However, this shift in survival outcomes for patients with OPSCC has been largely uneven across different segments of the US population30. One prime culprit for this differential response is tobacco exposure. Multiple datasets have shown that HPV status and tobacco exposure interact strongly to modulate treatment response and patient survival1. A recent analysis demonstrated a deleterious effect of tobacco exposure on HPV+ OPSCC outcomes of more than 20%, a dramatic effect that compares favorably to several known prognostic indicators such as positive cervical nodes, extranodal extension and the addition of chemotherapy to adjuvant radiation32. This is consistent with post-hoc analyses of several prospective clinical trials including RTOG0129 and RTOG900315. A parallel analysis by our group has identified a similar pattern of inferior oncologic outcomes in HPV+ OPSCC patients with extensive tobacco exposure (≥30 pack-years) (manuscript submitted). We previously showed that in Veterans with heavy tobacco exposure (a majority)33, survival for HPV+ and HPV− disease were in line with the rates reported by Ang et al. for intermediate-risk and high-risk disease34. This effect was consistent across racial groups35, and matches long term results from both RTOG0129 and RTOG052211.Modern day oncologic outcomes for HPV+ OPSCC are remarkable, considering the relative lack of clinical progress in other HNSCC disease sites40. Several clinical trials, including CheckMate 141 have now demonstrated substantial effectiveness of immunomodulatory agents both in the single agent setting and when combined with chemotherapy with a potentially more pronounced effect in p16+ tumors38. More recently pembrolizumab was shown to increase survival in the first line setting for recurrent or metastatic HNSCC either alone in the PD-L1 combined positive score (CPS) positive population or combined with chemotherapy in the CPS negative population when compared to cetuximab and chemotherapy combination therapy41. It is very likely that TIME will be a critical driver of differential efficacy for immunomodulatory strategies. Therefore, modifiers of TIME are likely to impact relative efficacy and will need to be considered during regimen design and trial implementation.The introduction of immunomodulatory agents for OPSCC represents both an opportunity to improve clinical outcomes and a challenge in terms of stratifying patients to the appropriate treatment regimen and intensity42, including direct modulation of immunocyte gene expression and function23. These effects are broad based and are not completely consistent across solid tumor types as shown in a recent TCGA analysis of head and neck and lung tumors13. A recent analysis of several datasets from oral cavity SCC (OCSCC) demonstrated that changes in the tumor immune microenvironment were the primary effect noted secondary to variable tobacco exposure43. The current analysis extends these previous datasets in two overlapping ways. First, we demonstrated that in OPSCC patients with extensive tobacco exposure, RFS strongly correlates with CD3 tumor infiltration on both univariable and multivariate analysis and with CD8 on univariable analysis. Additionally, OS strongly correlates with CD8 tumor infiltration on both univariable and multivariate analysis and with CD3 on univariable analysis. These correlations hold in the context of p16 positivity, especially for patients who demonstrate high-nuclear p16 staining, suggesting that in patients in which p16 staining correlates with putative functional implications (regulation of cellular processes) TIME may be a critical driver of overall tumor biology and treatment response. Overall, the correlation between TIME and survival in a cohort enriched for intermediate-risk patients (smokers) is encouraging, since it indicates that potentially immunomodulatory strategies targeting tumor lymphocyte infiltration and activation might achieve a favorable effect even in patients with extensive tobacco exposure. This has significant implications for planned and ongoing clinical trials which include both low-risk and intermediate-risk OPSCC in the context of immune modulation. Second, we demonstrated that tobacco exposure significantly and substantially reduces CD8 infiltration in the context of HPV+ OPSCC. Patients with p16+ tumors demonstrated approximately a 2-fold increase in CD3 and CD8 infiltrates compared to patients with p16− tumors. By way of comparison, patients with extensive tobacco exposure demonstrated nearly a 40% reduction in CD8 infiltration despite maintaining p16 positivity. Therefore, the potential effect size of tobacco-based modulation of TIME is substantial. Based on data from other tumor types and preclinical models, this would be expected to impact the effectiveness of immunomodulatory strategies targeting infiltrating lymphocytes. Clinical trials which incorporate immunomodulatory strategies as part of a de-escalation approach may be inappropriate for intermediate-risk OPSCC patients and should consider very carefully criteria for inclusion of patients11.Tobacco exposure has potent effects on anti-tumor and systemic immunity and inflammationThe current study has several limitations. First, the analysis is limited to a small number of immunohistochemistry markers, which is less comprehensive than high-throughput molecular approaches . However, immunohistochemistry also has the higher translational potential which is an advantage for replication of the data and integration into clinical practice. Second, we analyzed a patient cohort with extensive tobacco exposure which may not be representative of patients with minimal tobacco exposure. Although true, this also provides us with the additional power to detect a tobacco effect which was the primary goal of this study. Finally, our analysis cannot differentiate between direct effects of tobacco exposure on TIME and indirect effects driven by changes in tobacco induced genomic, epigenetic and proteomic changes at the level of tumors cells which secondarily impact TIME.Differential lymphocyte infiltration of HPV+ OPSCC is associated with significant differences in survival and is impacted by tobacco exposure. Mechanistic studies are required to better elucidate the mechanisms by which tobacco exposure can modulate HPV+ OPSCC TIME and may impact response to immunomodulatory strategies.Supplementary Data.