Fighting Pancreatic Cancer with a Vaccine-Based Winning Combination: Hope or Reality?

Pancreatic adenocarcinoma (PDA) represents the fourth leading cause of cancer-related mortality in the USA. Only 20% of patients present surgically resectable and potentially curable tumors at diagnosis, while 80% are destined for poor survival and palliative chemotherapy. Accordingly, the advancement of innovative and effective therapeutic strategies represents a pivotal medical imperative. It has been demonstrated that targeting the immune system represents an effective approach against several solid tumors. The immunotherapy approach encompasses a range of strategies, including the administration of antibodies targeting checkpoint molecules (immune checkpoint inhibitors, ICIs) to disrupt tumor suppression mechanisms and active immunization approaches that aim to stimulate the host's immune system. While vaccines have proved effective against infectious agents, vaccines for cancer remain an unfulfilled promise. Vaccine-based therapy targeting tumor antigens has the potential to be a highly effective strategy for initiating and maintaining T cell recognition, enhancing the immune response, and ultimately promoting cancer treatment success. In this review, we examined the most recent clinical trials that employed diverse vaccine types to stimulate PDA patients' immune systems, either independently or in combination with chemotherapy, radiotherapy, ICIs, and monoclonal antibodies with the aim of ameliorating PDA patients' quality of life and extend their survival.

In pancreatic cancer patients, chemotherapy reshapes the gene expression profile and antigen receptor repertoire of T lymphocytes and enhances their effector response to tumor-associated antigens.

Introduction: Pancreatic Ductal Adenocarcinoma (PDA) is one of the most aggressive malignancies with a 5-year survival rate of 13%. Less than 20% of patients have a resectable tumor at diagnosis due to the lack of distinctive symptoms and reliable biomarkers. PDA is resistant to chemotherapy (CT) and understanding how to gain an anti-tumor effector response following stimulation is, therefore, critical for setting up an effective immunotherapy. Methods: Proliferation, and cytokine release and TCRB repertoire of from PDA patient peripheral T lymphocytes, before and after CT, were analyzed in vitro in response to four tumor-associated antigens (TAA), namely ENO1, FUBP1, GAPDH and K2C8. Transcriptional state of PDA patient PBMC was investigated using RNA-Seq before and after CT. Results: CT increased the number of TAA recognized by T lymphocytes, which positively correlated with patient survival, and high IFN-γ production TAA-induced responses were significantly increased after CT. We found that some ENO1-stimulated T cell clonotypes from CT-treated patients were expanded or de-novo induced, and that some clonotypes were reduced or even disappeared after CT. Patients that showed a higher number of effector responses to TAA (high IFN-γ/IL-10 ratio) after CT expressed increased fatty acid-related transcriptional signature. Conversely, patients that showed a higher number of regulatory responses to TAA (low IFN-γ/IL-10 ratio) after CT significantly expressed an increased IRAK1/IL1R axis-related transcriptional signature. Conclusion: These data suggest that the expression of fatty acid or IRAK1/IL1Rrelated genes predicts T lymphocyte effector or regulatory responses to TAA in patients that undergo CT. These findings are a springboard to set up precision immunotherapies in PDA based on the TAA vaccination in combination with CT.

PI3Kγ inhibition combined with DNA vaccination unleashes a B-cell-dependent antitumor immunity that hampers pancreatic cancer.

Phosphoinositide-3-kinase γ (PI3Kγ) plays a critical role in pancreatic ductal adenocarcinoma (PDA) by driving the recruitment of myeloid-derived suppressor cells (MDSC) into tumor tissues, leading to tumor growth and metastasis. MDSC also impair the efficacy of immunotherapy. In this study we verify the hypothesis that MDSC targeting, via PI3Kγ inhibition, synergizes with α-enolase (ENO1) DNA vaccination in counteracting tumor growth.Mice that received ENO1 vaccination followed by PI3Kγ inhibition had significantly smaller tumors compared to those treated with ENO1 alone or the control group, and correlated with i) increased circulating anti-ENO1 specific IgG and IFNγ secretion by T cells, ii) increased tumor infiltration of CD8+ T cells and M1-like macrophages, as well as up-modulation of T cell activation and M1-like related transcripts, iii) decreased infiltration of Treg FoxP3+ T cells, endothelial cells and pericytes, and down-modulation of the stromal compartment and T cell exhaustion gene transcription, iv) reduction of mature and neo-formed vessels, v) increased follicular helper T cell activation and vi) increased "antigen spreading", as many other tumor-associated antigens were recognized by IgG2c "cytotoxic" antibodies. PDA mouse models genetically devoid of PI3Kγ showed an increased survival and a pattern of transcripts in the tumor area similar to that of pharmacologically-inhibited PI3Kγ-proficient mice. Notably, tumor reduction was abrogated in ENO1 + PI3Kγ inhibition-treated mice in which B cells were depleted.These data highlight a novel role of PI3Kγ in B cell-dependent immunity, suggesting that PI3Kγ depletion strengthens the anti-tumor response elicited by the ENO1 DNA vaccine.

HLA-DPB1*13:01 associates with enhanced, and KIR2DS4*001 with diminished protection from developing severe COVID-19.

Extreme polymorphism of HLA and killer-cell immunoglobulin-like receptors (KIR) differentiates immune responses across individuals. Additional to T cell receptor interactions, subsets of HLA class I act as ligands for inhibitory and activating KIR, allowing natural killer (NK) cells to detect and kill infected cells. We investigated the impact of HLA and KIR polymorphism on the severity of COVID-19. High resolution HLA class I and II and KIR genotypes were determined from 403 non-hospitalized and 1575 hospitalized SARS-CoV-2 infected patients from Italy collected in 2020. We observed that possession of the activating KIR2DS4*001 allotype is associated with severe disease, requiring hospitalization (OR = 1.48, 95% CI 1.20-1.85, pc = 0.017), and this effect is greater in individuals homozygous for KIR2DS4*001 (OR = 3.74, 95% CI 1.75-9.29, pc = 0.003). We also observed the HLA class II allotype, HLA-DPB1*13:01 protects SARS-CoV-2 infected patients from severe disease (OR = 0.49, 95% CI 0.33-0.74, pc = 0.019). These association analyses were replicated using logistic regression with sex and age as covariates. Autoantibodies against IFN-α associated with COVID-19 severity were detected in 26% of 156 hospitalized patients tested. HLA-C*08:02 was more frequent in patients with IFN-α autoantibodies than those without, and KIR3DL1*01502 was only present in patients lacking IFN-α antibodies. These findings suggest that KIR and HLA polymorphism is integral in determining the clinical outcome following SARS-CoV-2 infection, by influencing the course both of innate and adaptive immunity.

Circulating autoantibodies to alpha-enolase (ENO1) and far upstream element-binding protein 1 (FUBP1) are negative prognostic factors for pancreatic cancer patient survival.

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis due to a lack of early diagnostic markers and effective therapy. In PDA patients, the glycolytic enzyme and plasminogen receptor alpha-enolase (ENO1) and the transcription factor far upstream element-binding protein 1 (FUBP1) are upregulated and elicit the production of autoantibodies (aAb) that discriminate healthy subjects from PDA patients, with the latter mostly directed to post-translational phosphorylated isoforms. Here, the correlation of prognosis with circulating ENO1 and FUBP1aAb, and their protein tissue expression was analyzed in PDA patients. Circulating ENO1 and FUBP1 aAb was analyzed in two cohorts of PDA patients by ELISA (n = 470), while tissues expression was observed by immunohistochemistry (n = 45). Overall survival (OS) was estimated using the Kaplan-Meier method, while the Cox model was used to estimate the hazard ratios (HR) adjusted for the main prognostic factors. Logistic models were applied to assess associations between death and its risk indicators. All statistical analyses were performed with Stata version 15. Unlike ENO1 aAb, there was a significant correlation between FUBP1 aAb and FUBP1 expression in tumors (p = 0.0268). In addition, we found that high ENO1 (p = 0.016) and intermediate FUBP1 aAb levels (p = 0.013) were unfavorable prognostic factors. Notably, it was found that high anti-FUBP1 aAb level is a good prognostic marker for tail-body PDA (p = 0.016). Our results suggest that different levels of circulating aAb to ENO1 and FUBP1 predict a poor outcome in PDA patients and can be used to improve therapeutic strategies.

The Glycolytic Pathway as a Target for Novel Onco-Immunology Therapies in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal forms of human cancer, characterized by unrestrained progression, invasiveness and treatment resistance. To date, there are limited curative options, with surgical resection as the only effective strategy, hence the urgent need to discover novel therapies. A platform of onco-immunology targets is represented by molecules that play a role in the reprogrammed cellular metabolism as one hallmark of cancer. Due to the hypoxic tumor microenvironment (TME), PDA cells display an altered glucose metabolism-resulting in its increased uptake-and a higher glycolytic rate, which leads to lactate accumulation and them acting as fuel for cancer cells. The consequent acidification of the TME results in immunosuppression, which impairs the antitumor immunity. This review analyzes the genetic background and the emerging glycolytic enzymes that are involved in tumor progression, development and metastasis, and how this represents feasible therapeutic targets to counteract PDA. In particular, as the overexpressed or mutated glycolytic enzymes stimulate both humoral and cellular immune responses, we will discuss their possible exploitation as immunological targets in anti-PDA therapeutic strategies.

IL17A critically shapes the transcriptional program of fibroblasts in pancreatic cancer and switches on their protumorigenic functions.

A hallmark of cancer, including pancreatic ductal adenocarcinoma (PDA), is a massive stromal and inflammatory reaction. Many efforts have been made to identify the anti- or protumoral role of cytokines and immune subpopulations within the stroma. Here, we investigated the role of interleukin-17A (IL17A) and its effect on tumor fibroblasts and the tumor microenvironment. We used a spontaneous PDA mouse model (KPC) crossed to IL17A knockout mice to show an extensive desmoplastic reaction, without impaired immune infiltration. Macrophages, especially CD80+ and T cells, were more abundant at the earlier time point. In T cells, a decrease in FoxP3+ cells and an increase in CD8+ T cells were observed in KPC/IL17A-/- mice. Fibroblasts isolated from IL17A+/+ and IL17A-/- KPC mice revealed very different messenger RNA (mRNA) and protein profiles. IL17A-/- fibroblasts displayed the ability to restrain tumor cell invasion by producing factors involved in extracellular matrix remodeling, increasing T cell recruitment, and producing higher levels of cytokines and chemokines favoring T helper 1 cell recruitment and activation and lower levels of those recruiting myeloid/granulocytic immune cells. Single-cell quantitative PCR on isolated fibroblasts confirmed a very divergent profile of IL17A-proficient and -deficient cells. All these features can be ascribed to increased levels of IL17F observed in the sera of IL17A-/- mice, and to the higher expression of its cognate receptor (IL17RC) specifically in IL17A-/- cancer-associated fibroblasts (CAFs). In addition to the known effects on neoplastic cell transformation, the IL17 cytokine family uniquely affects fibroblasts, representing a suitable candidate target for combinatorial immune-based therapies in PDA.

Enhanced Expression of miR-181b in B Cells of CLL Improves the Anti-Tumor Cytotoxic T Cell Response.

The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40-CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.

In pancreatic cancer, chemotherapy increases antitumor responses to tumor-associated antigens and potentiates DNA vaccination.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is an almost incurable tumor that is mostly resistant to chemotherapy (CT). Adaptive immune responses to tumor-associated antigens (TAA) have been reported, but immunotherapy (IT) clinical trials have not yet achieved any significant increase in survival, confirming the suppressive environment of PDA. As CT has immune-modulating properties, we investigated the effect of gemcitabine (GEM) in antitumor effector responses to TAA in patients with PDA. METHODS: The IgG antibody repertoire in patients with PDA before and after CT was profiled by serological proteome analysis and ELISA and their ability to activate complement-dependent cytotoxicity (CDC) was measured. Peripheral T cells were stimulated in vitro with recombinant TAA, and specific proliferation, IFN-γ/IL-10 and CD8+/Treg ratios were measured. Mice that spontaneously developed PDA were treated with GEM and inoculated with an ENO1 (α-Enolase) DNA vaccine. In some experimental groups, the effect of depleting CD4, CD8 and B cells by specific antibodies was also evaluated. RESULTS: CT increased the number of TAA recognized by IgG and their ability to activate CDC. Evaluation of the IFN-γ/IL-10 ratio and CD8+/Treg ratios revealed that CT treatment shifted T cell responses to ENO1, G3P (glyceraldheyde-3-phosphate dehydrogenase), K2C8 (keratin, type II cytoskeletal 8) and FUBP1 (far upstream binding protein 1), four of the most recognized TAA, from regulatory to effector. In PDA mice models, treatment with GEM prior to ENO1 DNA vaccination unleashed CD4 antitumor activity and strongly impaired tumor progression compared with mice that were vaccinated or GEM-treated alone. CONCLUSIONS: Overall, these data indicate that, in PDA, CT enhances immune responses to TAA and renders them suitable targets for IT.

Development, Optimization and Characterization of Eudraguard®-based Microparticles for Colon Delivery.

Development of pH-dependent systems for colon delivery of natural active ingredients is an attractive area of research in the field of nutraceutical products. This study was focused on Eudraguard® resins, that are methacrylate copolymers approved as "food grade" by European Commission and useful for the production of food supplements. In particular, Eudraguard® Biotic (EUG-B), characterized by a pH-dependent solubility and Eudraguard® Control (EUG-C), whose chemical properties support a prolonged release of the encapsulated compounds, were tested. To obtain EUG microparticles, different preparation techniques were tested, in order to optimize the preparation method and observe the effect upon drug encapsulation and specific colonic release. Unloaded microparticles were initially produced to evaluate the influence of polymer characteristics on the formulation process; subsequently microparticles loaded with quercetin (QUE) as a low solubility model drug were prepared. The characterization of microparticles in the solid-state (FT-IR spectroscopy, differential scanning calorimetry and X-ray diffractometry) indicated that QUE was uniformly dispersed in a non-crystalline state in the polymeric network, without strong signs of chemical interactions. Finally, to assess the ability of EUG-C and EUG-B to control the drug release in the gastric environment, and to allow an increased release at a colonic level, suitable in vitro release tests were carried out by simulating the pH variations along the gastro-intestinal tract. Among the evaluated preparation methods, those in which an aqueous phase was not present, and in particular the emulsion-solvent evaporation method produced the best microparticle systems. The in vitro tests showed a limited drug release at a gastric level and a good specific colon release.

Unsaturated Poly(Hydroxyalkanoates) for the Production of Nanoparticles and the Effect of Cross-Linking on Nanoparticle Features.

A biodegradable poly(3-R-hydroxyalkanoate) synthesized by Pseudomonas mediterranea was investigated as a biomaterial to obtain colloidal drug delivery systems. Using a nanoprecipitation method, nanoparticles with a mean size of 155 nm and a negative surface charge were formed. They can be freeze-dried by adding hydroxypropyl-ß-cyclodextrin as a cryoprotectant, and they have been shown to efficiently load both a hydrophilic (calcein) and a lipophilic (Nile red) model probe. Since this polymer contains terminal double bonds in the side chains, cross-linking conditions were tested. In particular, under the action of UV rays or irradiation with an incandescent yellow lamp, this polymer tended to cross-link.

Phage-Based Anti-HER2 Vaccination Can Circumvent Immune Tolerance against Breast Cancer.

Δ16HER2 is a splice variant of HER2 and defined as the transforming isoform in HER2-positive breast cancer. It has been shown that Δ16HER2 promotes breast cancer aggressiveness and drug resistance. In the present work, we used in silico modeling to identify structural differences between Δ16HER2 and the wild-type HER2 proteins. We then developed DNA vaccines specifically against the Δ16HER2 isoform and showed that these immunotherapies hampered carcinogenesis in a breast cancer transplantable model. However, the vaccines failed to elicit immune protection in Δ16HER2 transgenic mice because of tolerogenic mechanisms toward the human HER2 self-antigen, a scenario commonly seen in HER2+ patients. Thus, we engineered bacteriophages with immunogenic epitopes of Δ16HER2 exposed on their coat for use as anticancer vaccines. These phage-based vaccines were able to break immune tolerance, triggering a protective anti-Δ16HER2 humoral response. These findings provide a rationale for the use of phage-based anti-HER2/Δ16HER2 vaccination as a safe and efficacious immunotherapy against HER2-positive breast cancers.

Next Generation Immunotherapy for Pancreatic Cancer: DNA Vaccination is Seeking New Combo Partners.

Pancreatic Ductal Adenocarcinoma (PDA) is an almost incurable radio- and chemo-resistant tumor, and its microenvironment is characterized by a strong desmoplastic reaction associated with a significant infiltration of T regulatory lymphocytes and myeloid-derived suppressor cells (Tregs, MDSC). Investigating immunological targets has identified a number of metabolic and cytoskeletal related molecules, which are typically recognized by circulating antibodies. Among these molecules we have investigated alpha-enolase (ENO1), a glycolytic enzyme that also acts a plasminogen receptor. ENO1 is also recognized by T cells in PDA patients, so we developed a DNA vaccine that targets ENO1. This efficiently induces many immunological processes (antibody formation and complement-dependent cytotoxicity (CDC)-mediated tumor killing, infiltration of effector T cells, reduction of infiltration of myeloid and Treg suppressor cells), which significantly increase the survival of genetically engineered mice that spontaneously develop pancreatic cancer. Although promising, the ENO1 DNA vaccine does not completely eradicate the tumor, which, after an initial growth inhibition, returns to proliferate again, especially when Tregs and MDSC ensue in the tumor mass. This led us to develop possible strategies for combinatorial treatments aimed to broaden and sustain the antitumor immune response elicited by DNA vaccination. Based on the data we have obtained in recent years, this review will discuss the biological bases of possible combinatorial treatments (chemotherapy, PI3K inhibitors, tumor-associated macrophages, ENO1 inhibitors) that could be effective in amplifying the response induced by the immune vaccination in PDA.

In vivo and ex vivo evaluation of cell-cell interactions, adhesion and migration in ocular surface of patients undergone excimer laser refractive surgery after topical therapy with different lubricant eyedrops.

PURPOSE: To investigate, in vivo by means of in vivo confocal microscopy (IVCM) and ex vivo by impression cytology, epithelial cellular damage after excimer laser refractive surgery in patients under different topical lubricant therapies. METHODS: Two hundred eyes of 100 patients, undergone bilateral excimer laser refractive surgery for medium myopic error correction [spherical equivalent refraction from -1.75 to -3.50 dioptres (D) with refractive astigmatism under -0.75 D], have been recruited. All patients received, in addition to standard therapy for refractive surgery, high weight hyaluronic acid 0.2% eyedrops in one randomly selected eye and carboxymethylcellulose 1% eyedrop in the comparator eye (control eye) 4 times daily for 90 days. Follow-up included a baseline visit and further examination 7-, 30- and 90-day intervals [clinical evaluation with Schirmer test and tear break-up time (TBUT), IVCM and impression cytology]. RESULTS: No significant difference in Schirmer test and TBUT was observed during the follow-up period in eyes under different therapies. IVCM showed an improvement of conjunctival and corneal epithelial cells quality in eye in treatment with high weight hyaluronic acid 0.2% when compared to carboxymethylcellulose. Conjunctival impression cytology demonstrated an evident positivity for CD44 in eyes treated with both treatments in all follow-up controls. ICAM1 expression showed an increasing positivity starting at 30 days that became statistically significant after 90 days of high weight hyaluronic acid 0.2% therapy (p = 0.0167). CONCLUSIONS: In vivo and in vitro results showed the effectiveness of high weight hyaluronic acid 0.2% in facilitating cell-cell interaction, migration, cell proliferation, stabilizing epithelial barrier of the ocular surface. Moreover, use of high weight hyaluronic acid in treatment of corneal tissue damage after refractive surgery, in concordance with standard topical corticosteroids and antibiotics therapy, could be effective in promoting corneal epithelial wound healing with consequent good results in clinical outcome and patients' satisfaction.

Endogenous glutamine decrease is associated with pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its in vivo evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the in vitro glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and spontaneous mouse models of PDAC and other pancreatic-related disorders by High-Performance Liquid Chromatography (HPLC) and/or MS. Notably we observed a reduction of blood glutamine as much as the tumor progressed from pancreatic intraepithelial lesions to invasive PDAC, but was not related to chronic pancreatitis-associated inflammation or diabetes. In parallel the increased levels of branched-chain amino acids (BCAA) were observed. By contrast blood glutamine levels were stable in non-tumor bearing mice. These findings demonstrated that glutamine uptake is measurable both in vitro and in vivo. The higher in vitro avidity of PDAC cells corresponded to a lower blood glutamine level as soon as the tumor mass grew. The reduction in circulating glutamine represents a novel tool exploitable to implement other diagnostic or prognostic PDAC biomarkers.

In Vivo and Ex Vivo Evaluation of Inflammation and Apoptosis Induced after SMILE Procedures for Different Refractive Error Range.

PURPOSE: To detect corneal inflammation and apoptosis induced after small incision lenticule extraction (SMILE) at different refractive corrections for moderate to high values of myopia. METHODS: Fifty patients (50 eyes) suffering from medium to high myopia (spherical equivalent refraction from -3.75 to -10.00 diopters (D) with refractive astigmatism under -0.75 D) underwent SMILE in order to correct myopic error. In vivo evaluation was done by corneal confocal microscopy (IVCM) and ex vivo by immunohistochemistry. After surgery, all corneal lenticules were checked for regularity, entirety, and fixed in formalin for immunohistochemistry evaluation of apoptosis (TUNEL) and inflammation (CD11b) levels. Postoperative assessments took place during the first week and the first and third months after surgery. Patients returned for IVCM examination for analysis of the corneal stromal femtosecond laser treatment interfaces reflectivity. RESULTS: No correlation was observed between treated myopic refractive error and number of CD11b+ and TUNEL+ cell in all analyzed extracted lenticules. IVCM at 1 week and 1 month of follow-up showed numerous reflective particles at the laser treatment interface with a moderate light scattering. In semi-quantitative analysis of reflectivity intensity at the laser interfaces, a statistical difference was evident only between 1 week and 1 month (p = 0.0213). CONCLUSIONS: SMILE, as an innovative all-femto surgical procedure, results in a reduced tissue inflammation and apoptosis levels with a minimum tissue response, in terms of interface reflectivity, and there are no statistically significant differences among variable treated refractive error range.

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm2 UVA (group 4) and three for 9 min at 10 mW/cm2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

Immunohistochemical study of corneal inflammation after femtosecond laser clear corneal incisions or manual surgery.

PURPOSE: To use immunohistochemical staining to evaluate corneal inflammation and apoptosis induced after femtosecond laser incisions or manual incisions. SETTING: Ophthalmology Clinic, University G. d'Annunzio, Chieti, Italy. DESIGN: Experimental study. METHODS: Ninety human cadaver corneas were cut manually or with the femtosecond laser at different energies and analyzed by immunohistochemistry after 5 minutes or 4 hours. The corneas were divided into 5 groups: untreated (Group 1), cut manually (Group 2), and treated with the femtosecond laser with increasing energies (Groups 3 to 5; 3.0 µJ, 6.0 µJ, and 15.0 µJ, respectively). RESULTS: At 5 minutes, increased expression of interleukin (IL)-18 was observed in the femtosecond laser groups compared with the manual group (P < .01). Interferon gamma (IFNγ) positivity was significantly higher in Groups 4 and 5 than in Group 2 and between Groups 3 and 4 (P < .05). The terminal uridine deoxynucleotidyl nick end-labeling (TUNEL) positivity increased with higher energy (Group 2 versus Group 4 and Group 2 versus Group 5; P < .05). After 4 hours, IFNγ positivity was higher in Group 5 than in Group 2 (P = .0021) and between Group 5 and Groups 3 and 4 (P < .05). No sign of IL-18 positivity was found after 4 hours in any sample. Group 5 showed significant higher TUNEL positivity than all other groups (P < .0001). CONCLUSION: The femtosecond laser technique at high energies induced a higher corneal inflammatory response and a higher corneal cell apoptosis than the manual technique. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

In Vivo Goblet Cell Density as a Potential Indicator of Glaucoma Filtration Surgery Outcome.

PURPOSE: We analyzed the preoperative conjunctival goblet cell density (GCD), MUC5AC, and HLA-DR in glaucomatous patients undergoing trabeculectomy, using laser scanning confocal microscopy (LSCM) and impression cytology (IC). METHODS: We enrolled 57 patients undergoing trabeculectomy. At baseline LSCM and IC were performed at the site planned for surgery; LSCM was repeated after 12 months at the bleb site. The main outcomes were: GCD, mean microcyst density (MMD) and area (MMA) at LSCM, MUC5AC, and HLA-DR positivity at IC, and IOP. The relationships between baseline GCD, and 12-month IOP, MMD, and MMA were analyzed. RESULTS: Trabeculectomy was successful in 39 patients (complete success in 27, Group 1; qualified in 12, Group 2), and unsuccessful in 18 (Group 3). At baseline IOP (mm Hg) was 27.2 ± 3.12, 27.5 ± 2.23, and 27.7 ± 1.90 in Groups 1 to 3, respectively; GCD and MUC5AC positivity were higher in Group 1 compared to Groups 2 and 3 (P < 0.05); HLA-DR, MMD, and MMA were not significantly different among the groups. At 12 months, IOP reduced by 45.3%, 35.4%, and 12.8% in Groups 1 to 3, respectively. Goblet cell density did not change in Group 1, whereas it was reduced in Groups 2 and 3 (P < 0.05), with values lower in Group 3. Mean microcyst density and MMA increased in Groups 1 and 2 (P < 0.05), with values higher in Group 1 (P < 0.05). Baseline GCD positively correlated with 12-month IOP reduction (P < 0.001, r = 0.641), MMD (P < 0.05, r = 0.454), and MMA (P < 0.001, r = 0.541). CONCLUSIONS: Goblet cells positively affect the filtration ability after trabeculectomy; therefore, preoperative GCD could be considered as a potential in vivo biomarker of surgical success.

Intradermal DNA Electroporation Induces Cellular and Humoral Immune Response and Confers Protection against HER2/neu Tumor.

Skin represents an attractive target for DNA vaccine delivery because of its natural richness in APCs, whose targeting may potentiate the effect of vaccination. Nevertheless, intramuscular electroporation is the most common delivery method for ECTM vaccination. In this study we assessed whether intradermal administration could deliver the vaccine into different cell types and we analyzed the evolution of tissue infiltrate elicited by the vaccination protocol. Intradermal electroporation (EP) vaccination resulted in transfection of different skin layers, as well as mononuclear cells. Additionally, we observed a marked recruitment of reactive infiltrates mainly 6-24 hours after treatment and inflammatory cells included CD11c(+). Moreover, we tested the efficacy of intradermal vaccination against Her2/neu antigen in cellular and humoral response induction and consequent protection from a Her2/neu tumor challenge in Her2/neu nontolerant and tolerant mice. A significant delay in transplantable tumor onset was observed in both BALB/c (p ≤ 0,0003) and BALB-neuT mice (p = 0,003). Moreover, BALB-neuT mice displayed slow tumor growth as compared to control group (p < 0,0016). In addition, while in vivo cytotoxic response was observed only in BALB/c mice, a significant antibody response was achieved in both mouse models. Our results identify intradermal EP vaccination as a promising method for delivering Her2/neu DNA vaccine.