The "Real R0": A Resection Margin Smaller Than 0.1 cm is Associated with a Poor Prognosis After Oncologic Esophagectomy.

BACKGROUND: Although resection margin (R) status is a widely used prognostic factor after esophagectomy, the definition of positive margins (R1) is not universal. The Royal College of Pathologists considers R1 resection to be a distance less than 0.1 cm, whereas the College of American Pathologists considers it to be a distance of 0.0 cm. This study assessed the predictive value of R status after oncologic esophagectomy, comparing survival and recurrence among patients with R0 resection (> 0.1-cm clearance), R0+ resection (≤ 0.1-cm clearance), and R1 resection (0.0-cm clearance). METHODS: The study enrolled all eligible patients undergoing curative oncologic esophagectomy between 2012 and 2018. Clinicopathologic features, survival, and recurrence were compared for R0, R0+, and R1 patients. Categorical variables were compared with the chi-square or Fisher's test, and continuous variables were compared with the analysis of variance (ANOVA) test, whereas the Kaplan-Meier method and Cox regression were used for survival analysis. RESULTS: Among the 160 patients included in this study, 113 resections (70.6%) were R0, 34 (21.3%) were R0+, and 13 (8.1%) were R1. The R0 patients had a better overall survival (OS) and disease-free survival (DFS) than the R0+ and R1 patients. The R0+ resection offered a lower long-term recurrence risk than the R1 resection, and the R status was independently associated with DFS, but not OS, in the multivariate analysis. Both the R0+ and R1 patients had significantly more adverse histologic features (lymphovascular and perineural invasion) than the R0 patients and experienced more distant and locoregional recurrence. CONCLUSIONS: Although R status is an independent predictor of DFS after oncologic esophagectomy, the < 0.1-cm definition for R1 resection seems more appropriate than the 0.0-cm definition as an indicator of poor tumor biology, long-term recurrence, and survival.

Maltodextrin-Nanoparticles as a Delivery System for Nasal Vaccines: A Review Article.

Nanoparticles are increasingly being studied as antigen delivery systems for immunization with nasal vaccines. The addition of adjuvants is still generally required in many nanoparticle formulations, which can induce potential side effects owing to mucosal reactogenicity. In contrast, maltodextrin nanoparticles do not require additional immunomodulators, and have been shown to be efficient vaccine delivery systems. In this review, the development of maltodextrin nanoparticles is presented, specifically their physico-chemical properties, their ability to load antigens and deliver them into airway mucosal cells, and the extent to which they trigger protective immune responses against bacterial, viral, and parasitic infections. We demonstrate that the addition of lipids to maltodextrin nanoparticles increases their potency as a vaccine delivery system for nasal administration.

Improved ELISPOT protocol for monitoring Th1/Th17 T-cell response following T.gondii infection.

In the monitoring of human Toxoplasma gondii infection, it is crucial to confirm the development of a specific Th1/Th17 immune response memory. The use of a simple, specific, and sensitive assay to follow the T-cell activation is thus required. Current protocols are not always specific as stimulation with peptides is Human Leukocyte Antigen (HLA)-dependent, while stimulation with total-lysis antigens tends to stimulate seronegative donors resulting to false positives. Here, an improved ELISPOT protocol is reported, using peripheral blood mononuclear cells (PBMC) of T.gondii-infected donors, incubated with the inactivated parasite. The results showed that, contrary to standard protocols, a pre-incubation step at high cell density in presence of the inactivated parasite allowed a specific Th1/Th17 response with the secretion of IFN-γ, IL-2, IL-12 and IL-17 cytokines. This protocol allows to evaluate precisely the immune response after a T.gondii infection.

Prospective evaluation of MR-TRG (Tumor Regression Grade) in esophageal cancer after neo-adjuvant therapy: Preliminary results.

PURPOSE: To develop MRI-based criteria to assess tumor response to neoadjuvant therapies (NAT) of esophageal cancers (EC) and to evaluate its diagnostic performance in predicting the pathological Tumor Regression Grade (pTRG). METHOD: From 2018 to 2022, patients with newly diagnosed locally advanced EC underwent MRI examinations for initial staging and restaging after NAT. Magnetic Resonance TRG (MR-TRG), equivalent to the Mandard and Becker classifications, were developed and independently assessed by two radiologists, blinded to pTRG, using T2W and DW-MR Images. All patients underwent surgery and benefited from a blinded pTRG evaluation by two pathologists. The agreement between readers and between MR-TRG and pTRG were assessed with Cohen's Kappa. The correlation of MR-TRG and pTRG was determined using Spearman's correlation. RESULTS: 28 patients were included. Interrater agreement was substantial between radiologists, improved when grouping grade 1 and 2 (κ = 0.78 rose to 0,84 for Mandard and 0.68 to 0,78 for Becker score). Agreement between pTRG and MR-TRG was moderate with a percentaged agreement (p) = 87.5 %, kappa (κ) = 0.54 and p = 83.3 %, κ = 0.49 for Mandard and Becker, respectively. Agreement was improved to substantial when grouping grades 1-2 for Mandard and 1a-1b for Becker with p = 89.3 %, κ = 0.65 and p = 85.2 %, κ = 0.65 respectively. Sensitivity and specificity of MR-TRG in predicting pTRG were 88.2 % and 72.7 % for Mandard system (scores 1-2 versus 3-5), and 83.3 % and 80 % for Becker system (scores 1a-1b versus 2-3). CONCLUSION: A substantial agreement between MR-TRG and pTRG was achieved when grouping grade 1-2. Hence, MR-TRG could be used as a surrogate of complete and near-complete pTRG.

Human T-cell activation with Toxoplasma gondii antigens loaded in maltodextrin nanoparticles.

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

Effective immuno-therapeutic treatment of Canine Leishmaniasis.

BACKGROUND: Canine Leishmaniasis (CanL) caused by the L. infantum species is one of the biggest threats to the health of the South American canine population. Chemotherapeutics currently used for the treatment of CanL fail to induce a total parasite clearance while inducing numerous side effects. As CanL is an immunomodulated disease, the use of immuno-treatments should strengthen the deficient immune response of infected dogs. In this study, we evaluated a nasally administered immunotherapy in dogs naturally infected with L. infantum (stage 2), with both visceral and cutaneous manifestations. Noteworthy, some of them were also infected by other parasites (E. canis, D. immitis, A. platys), what worsen their chance of survival. METHODOLOGY/PRINCIPAL FINDINGS: The treatment was based on 2 intranasal (IN.) administrations of a killed L. infantum parasite loaded into maltodextrin nanoparticles, which treatment was compared with the classical oral administration of Miltefosine (2 mg/kg) for 28 days, as well as a combination of these 2 treatments. The results showed that two IN administrations significantly reduced the serology, and were at least as efficient as the chemotherapy to reduce the skin and bone marrow parasite burden, as well as clinical scores, and that unlike Miltefosine treatments, this nasally administered nanoparticle vaccine was without side effects. CONCLUSIONS: These results confirm the feasibility of a simple therapeutic immuno-treatment against L. infantum infected dogs, which is a promising tool for future developments.

The role of cytology in patients undergoing pressurized intraperitoneal aerosol chemotherapy (PIPAC) treatment for peritoneal carcinomatosis.

Objectives: Cytology of ascites or peritoneal washing is a routine part of staging of peritoneal metastases (PM). We aim to determine value of cytology in patients undergoing pressurized intraperitoneal aerosol chemotherapy (PIPAC). Methods: Single-center retrospective cohort study included consecutive patients having PIPAC for PM of different primary between January 2015 and January 2020. Results: A total of 75 patients (median 63 years (IQR 51-70), 67 % female) underwent a total of 144 PIPAC. At PIPAC 1 59 % patients had positive and 41 % patients had negative cytology. Patients with negative and positive cytology only differed in terms of symptoms of ascites (16% vs. 39 % respectively, p=0.04), median ascites volume (100 vs. 0 mL, p=0.01) and median PCI (9 vs. 19, p<0.01). Among 20 patients who completed 3 PIPACs (per protocol), cytology changed in one from positive to negative, and in two from negative to positive. Median overall survival was 30.9 months in the per protocol group and 12.9 months in patients having <3 PIPACs (=0.519). Conclusions: Positive cytology under PIPAC treatment is more frequently encountered in patients with higher PCI and symptomatic ascites. Cytoversion was rarely observed and cytology status had no impact on treatment decisions in this cohort.

Nasal vaccination of six squirrel monkeys (Saimiri sciureus): Improved immunization protocol against Toxoplasma gondii with a nanoparticle-born vaccine.

Toxoplasma gondii is an intracellular protozoon found worldwide, which completes its life cycle between felids (its definitive host) and other warm-blooded animals. While the infection rarely leads to severe complications in humans, many animal species are very susceptible to this infection, for example the squirrel monkey (Saimiri sciureus) which is the subject of this study. Toxoplasmosis is lethal for 80% of cases in this species, and fatal outbreaks are frequently reported in zoological parks. No efficient treatment exists, but a new vaccine prepared with maltodextrin nanoparticles containing killed T. gondii antigens has been tested recently in French zoos. The animals were immunized through heterologous administrations, with two nasal doses at one-month interval, followed by nasal/subcutaneous boosts thereafter. No death has been reported since the beginning of this vaccination campaign, but we felt the protocol could be simplified. Here, an improved and less-invasive immunization protocol was evaluated on 6 Saimiri sciureus in the French zoo La Palmyre. It consisted of two nasal administrations at one-month interval, followed by a nasal boost at 6 months. A specific memory T-cell immunity was observed by ELISPOT after two administrations in all the animals, without humoral responses. The results suggest that 2 nasal administrations induce a protective immune response against T. gondii infection and might be sufficient to induce a strong Tcell memory, further improving immunity.

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species.

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.

Starch-based NP act as antigen delivery systems without immunomodulating effect.

The nasal route of immunization has become a real alternative to injections. It is indeed described as more efficient at inducing immune protection, since it initiates both mucosal and systemic immunity, thus protecting against both the infection itself and the transmission of pathogens by the host. However, the use of immunomodulators should be limited since they induce inflammation. Here we investigated in vitro the mechanisms underlying the enhancement of antigen immunogenicity by starch nanoparticles (NPL) delivery systems in H292 epithelial cells, as well as the NPL's immunomodulatory effect. We observed that NPL had no intrinsic immunomodulatory effect but enhanced the immunogenicity of an E. coli lysate (Ag) merely by increasing its intracellular delivery. Moreover, we demonstrated the importance of the NPL density on their efficiency by comparing reticulated (NPL) and non-reticulated particles (NPL·NR). These results show that an efficient delivery system is sufficient to induce a mucosal immune response without the use of immunomodulators.

Systematic comparison with autoimmune liver disease identifies specific histological features of immune checkpoint inhibitor-related adverse events.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have become a mainstay of cancer treatment. Their immune-boosting quality has one major drawback, their proclivity to induce a broad array of immune-related adverse events (irAEs) affecting, among others, the liver and sharing some similarities with classic autoimmune liver diseases (AILD).We aimed to compare clinical, laboratory and histological features of patients with liver-related irAEs and AILD. METHODS: We systematically compared liver irAEs with AILD, namely autoimmune hepatitis (AIH) and primary biliary cholangitis, regarding their clinical, laboratory, and histological features. RESULTS: Twenty-seven patients with liver irAEs (ICI group) and 14 patients with AILD were identified. We observed three distinct ICI-induced histological liver injury patterns: hepatitic (52%), cholangitic (19%), and mixed (29%). When comparing the ICI and AILD groups, centrilobular injury as well as granuloma formation were more prevalent in the former (p=0.067 and 0.002, respectively). CD4+/CD8+ T cell ratios were heterogeneous between the two groups, without statistically significant difference but with a trend toward increased CD8+ T cells among hepatitic irAEs as compared with AIH. Pattern of liver function test alteration was predictive for the type of irAEs but did not correlate with histological severity. CONCLUSIONS: Liver irAEs have broad clinical, laboratory and histological presentations. Histological features of irAEs and AILD are distinct, likely underpinning their different immunological mechanisms.

Multiple primary malignancies: synchronous lymphoma, pancreatic neuroendocrine tumour and colorectal cancer.

A 67-year-old woman underwent a medical check-up by her general practitioner after complaining of atypical pain in the shoulder girdle. Due to the important inflammatory syndrome noticed on blood testing, a polymyalgia rheumatica was suspected and she was started on corticosteroid treatment with good clinical response, but no impact on inflammation. She underwent extensive imaging with a thoraco-abdominal CT scanner that demonstrated a pancreatic mass, then later a PET-CT showed 3 different hyperactive lesions. Biopsies then revealed simultaneous diffuse large B-cell lymphoma (DLBCL), colorectal adenocarcinoma and pancreatic neuroendocrine tumour. She benefited from low rectal endoscopic excision of the colorectal tumour, R-CHOP chemotherapy for DLBCL and laparoscopic left pancreatectomy. Successful treatment required a good multidisciplinary collaboration between the different specialists. The patient made a good recovery and achieved complete remission at 1 year. This an unusual presentation of multiple primary malignancies.

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis.

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.

Case Report: Vasculitis Triggered by SIRT in a Patient With Previously Untreated Cholangiocarcinoma.

Arising from the biliary tract, cholangiocarcinoma is a rare and aggressive epithelial cancer. According to the primary site, it can be further classified into intrahepatic, perihilar and distal types. Due to the lack of symptoms early in the disease course, most patients are diagnosed at advanced stages. Being not candidates for curative surgical management, these patients are treated with palliative systemic chemotherapy, and their prognosis remains poor. Using radioisotopes like yttrium-90 -labeled microspheres (90Y), radioembolization represents a local approach to treat primary and secondary liver tumors. In the case of intrahepatic cholangiocarcinoma, radioembolization can be used as a primary treatment, as an adjunct to chemotherapy or after failing chemotherapy. An 88-year-old man underwent radioembolization for a previously untreated stage II intrahepatic cholangiocarcinoma. One week later, he presented to our clinic with a non-pruritic maculopapular rash of the lower extremities and abdomen, worsening fatigue and low-grade fever. Laboratory exams, including hepatitis screening, were within normal limits. Showing positive immunofluorescence staining for immunoglobulin M (IgM) and complement 3 (C3) in vessel walls without IgA involvement, the skin biopsy results were compatible with leukocytoclastic vasculitis. Apart from the anticancer intervention, there have been no recent medication changes which could explain this complication. Notably, we did not observe any side effects during or after the perfusion scan with technetium-99m macroaggregated albumin (MAA) performed prior to radioembolization. The symptoms resolved quickly after a short course of colchicine and did not reappear at cholangiocarcinoma progression. In the absence of other evident causes, we conclude that the onset of leukocytoclastic vasculitis in our patient was directly linked to the administration of yttrium-90 -labeled microspheres. Our report therefore demonstrates that this condition can be a rare but manageable complication of 90Y liver radioembolization.

Cav1/EREG/YAP Axis in the Treatment Resistance of Cav1-Expressing Head and Neck Squamous Cell Carcinoma.

The EGFR-targeting antibody cetuximab (CTX) combined with radiotherapy is the only targeted therapy that has been proven effective for the treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC). Recurrence arises in 50% of patients with HNSCC in the years following treatment. In clinicopathological practice, it is difficult to assign patients to classes of risk because no reliable biomarkers are available to predict the outcome of HPV-unrelated HNSCC. In the present study, we investigated the role of Caveolin-1 (Cav1) in the sensitivity of HNSCC cell lines to CTX-radiotherapy that might predict HNSCC relapse. Ctrl- and Cav-1-overexpressing HNSCC cell lines were exposed to solvent, CTX, or irradiation, or exposed to CTX before irradiation. Growth, clonogenicity, cell cycle progression, apoptosis, metabolism and signaling pathways were analyzed. Cav1 expression was analyzed in 173 tumor samples and correlated to locoregional recurrence and overall survival. We showed that Cav1-overexpressing cells demonstrate better survival capacities and remain proliferative and motile when exposed to CTX-radiotherapy. Resistance is mediated by the Cav1/EREG/YAP axis. Patients whose tumors overexpressed Cav1 experienced regional recurrence a few years after adjuvant radiotherapy ± chemotherapy. Together, our observations suggest that a high expression of Cav1 might be predictive of locoregional relapse of LA-HNSCC.

Importance of the Phospholipid Core for Mucin Hydrogel Penetration and Mucosal Cell Uptake of Maltodextrin Nanoparticles.

Nanoparticles (NPs) used as mucosal antigen delivery systems are a promising way to vaccinate. However, NPs have to cross the mucus gel and penetrate into mucosal cells to deliver antigens, and a mismatch exists between mucopenetrating NPs, rarely able to interact with cells, and NPs designed to deliver antigens into cells, but often described as mucoadhesives. Here, we compared the ability of cationic maltodextrin-based NPs, either without (NP+) or with an anionic phospholipid core (NPL), to interact with mucins and airway epithelial cells. We showed that their lipid core increased the NPL's mobility in mucin hydrogel by reducing interactions with mucins. Similarly, the uptake and protein delivery by NPLs into airway epithelial cells were not hindered by mucins. This highlights the importance of anionic lipids in the NPL, which are more efficient in crossing the mucin hydrogel while retaining the ability to interact with epithelial cells, an intermediate behavior between mucoadhesive and mucopenetrating NPs.

Prevention of influenza virus infection and transmission by intranasal administration of a porous maltodextrin nanoparticle-formulated vaccine.

Influenza vaccines administered intramuscularly exhibit poor mucosal immune responses in the respiratory tract which is the prime site of the infection. Intranasal vaccination is a potential route for vaccine delivery which has been demonstrated effective in inducing protective immune responses in both systemic and mucosal compartments. For this purpose, nanoparticles have been used as antigen delivery systems to improve antigen capture by immune cells. In this paper we demonstrate efficient delivery of viral antigens to airway epithelial cells, macrophages and dendritic cells, using polysaccharide nanoparticles (NPL), leading to a strong protection against influenza virus infection. A formulation combining split Udorn virus antigens with NPL and the mucosal protein adjuvant CTA1-DD was administered intranasally and resulted in an enhanced specific humoral immune response. Furthermore, NPL carrying split Udorn, with or without CTA1-DD, inhibited virus transmission from infected to uninfected naive mice. These results demonstrate that an intranasal delivery system combining NPL, mucosal adjuvant CTA1-DD and split virus antigens confers robust protection against influenza infection and inhibits virus transmission.

Protein delivery by porous cationic maltodextrin-based nanoparticles into nasal mucosal cells: Comparison with cationic or anionic nanoparticles.

Different types of biodegradable nanoparticles (NPs) have been studied as delivery systems for proteins into nasal mucosal cells, especially for vaccine applications. Such a nanocarrier must have the ability to be loaded with proteins and to transport this payload into mucosal cells. However, comparative data on nanoparticles' capacity for protein loading, efficiency of subsequent endocytosis and the quantity of nanocarriers used are either lacking or contradictory, making comparisons and the choice of a best candidate difficult. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: the NPL (cationic maltodextrin NP with an anionic lipid core), cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP, and cationic and anionic liposomes. We first quantified the protein association efficiency and NPL associated the largest amount of ovalbumin, used as a model protein. In vitro, the delivery of fluorescently-labeled ovalbumin into mucosal cells (airway epithelial cells, dendritic cells and macrophages) was assessed by flow cytometry and revealed that the NPL delivered protein to the greatest extent in all 3 different cell lines. Taken together, these data underlined the potential of the porous and cationic maltodextrin-based NPL as efficient protein delivery systems to mucosal cells.