In vitro CAR-T cell killing: validation of the potency assay.

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

Flow Index: a novel, non-invasive, continuous, quantitative method to evaluate patient inspiratory effort during pressure support ventilation.

BACKGROUND: The evaluation of patient effort is pivotal during pressure support ventilation, but a non-invasive, continuous, quantitative method to assess patient inspiratory effort is still lacking. We hypothesized that the concavity of the inspiratory flow-time waveform could be useful to estimate patient's inspiratory effort. The purpose of this study was to assess whether the shape of the inspiratory flow, as quantified by a numeric indicator, could be associated with inspiratory effort during pressure support ventilation. METHODS: Twenty-four patients in pressure support ventilation were enrolled. A mathematical relationship describing the decay pattern of the inspiratory flow profile was developed. The parameter hypothesized to estimate effort was named Flow Index. Esophageal pressure, airway pressure, airflow, and volume waveforms were recorded at three support levels (maximum, minimum and baseline). The association between Flow Index and reference measures of patient effort (pressure time product and pressure generated by respiratory muscles) was evaluated using linear mixed effects models adjusted for tidal volume, respiratory rate and respiratory rate/tidal volume. RESULTS: Flow Index was different at the three pressure support levels and all group comparisons were statistically significant. In all tested models, Flow Index was independently associated with patient effort (p < 0.001). Flow Index prediction of inspiratory effort agreed with esophageal pressure-based methods. CONCLUSIONS: Flow Index is associated with patient inspiratory effort during pressure support ventilation, and may provide potentially useful information for setting inspiratory support and monitoring patient-ventilator interactions.

Non-invasive assessment of respiratory muscle activity during pressure support ventilation: accuracy of end-inspiration occlusion and least square fitting methods.

Pressure support ventilation (PSV) should be titrated considering the pressure developed by the respiratory muscles (Pmusc) to prevent under- and over-assistance. The esophageal pressure (Pes) is the clinical gold standard for Pmusc assessment, but its use is limited by alleged invasiveness and complexity. The least square fitting method and the end-inspiratory occlusion method have been proposed as non-invasive alternatives for Pmusc assessment. The aims of this study were: (1) to compare the accuracy of Pmusc estimation using the end-inspiration occlusion (Pmusc,index) and the least square fitting (Pmusc,lsf) against the reference method based on Pes; (2) to test the accuracy of Pmusc,lsf and of Pmusc,index to detect overassistance, defined as Pmusc ≤ 1 cmH2O. We studied 18 patients at three different PSV levels. At each PSV level, Pmusc, Pmusc,lsf, Pmusc,index were calculated on the same breaths. Differences among Pmusc, Pmusc,lsf, Pmusc,index were analyzed with linear mixed effects models. Bias and agreement were assessed by Bland-Altman analysis for repeated measures. The ability of Pmusc,lsf and Pmusc,index to detect overassistance was assessed by the area under the receiver operating characteristics curve. Positive and negative predictive values were calculated using cutoff values that maximized the sum of sensitivity and specificity. At each PSV level, Pmusc,lsf was not different from Pmusc (p = 0.96), whereas Pmusc,index was significantly lower than Pmusc. The bias between Pmusc and Pmusc,lsf was zero, whereas Pmusc,index systematically underestimated Pmusc of 6 cmH2O. The limits of agreement between Pmusc and Pmusc,lsf and between Pmusc and Pmusc,index were ± 12 cmH2O across bias. Both Pmusc,lsf ≤ 4 cmH2O and Pmusc,index ≤ 1 cmH2O had excellent negative predictive value [0.98 (95% CI 0.94-1) and 0.96 (95% CI 0.91-0.99), respectively)] to identify over-assistance. The inspiratory effort during PSV could not be accurately estimated by the least square fitting or end-inspiratory occlusion method because the limits of agreement were far above the signal size. These non-invasive approaches, however, could be used to screen patients at risk for absent or minimal respiratory muscles activation to prevent the ventilator-induced diaphragmatic dysfunction.

Potency Assessment of Dendritic Cell Anticancer Vaccine: Validation of the Co-Flow DC Assay.

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC-T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.

Randomized trial comparing fork-tip and side-fenestrated needles for EUS-guided fine-needle biopsy of solid pancreatic lesions.

BACKGROUND AND AIMS: The aim of this study was to compare the performance of EUS-guided fine-needle biopsy using fork-tip or side-fenestrated needles in patients with solid pancreatic lesions. METHODS: A randomized controlled study was conducted in a single academic center on patients who underwent sampling with fork-tip or side-fenestrated 22-gauge or 25-gauge needles. Three passes were performed, each independently evaluated by a blinded pathologist and by endosonographers for macroscopic on-site evaluation (MOSE). The primary outcome was histologic yield; secondary aims were safety, diagnostic yield, sample quality, number of needle passes required to establish a diagnosis, and reliability of MOSE. RESULTS: One hundred ninety-two patients were enrolled. Both 22-gauge and 25-gauge fork-tip needles retrieved significantly higher rates of histologic samples than side-fenestrated needles (P < .013). Safety and diagnostic accuracy were comparable in the 2 arms, whereas sample quality (tissue integrity and blood contamination) was significantly better in the fork-tip group (P < .0001). The median number of diagnostic passes was lower using fork-tip needles (P = .054). The agreement between MOSE and pathologic evaluation was almost perfect in the fork-tip group and fair in the side-fenestrated group. CONCLUSIONS: Both needles showed equivalent safety and diagnostic accuracy. However, fork-tip needles provided a higher rate of extremely good-quality histologic samples and required fewer needle passes to reach a diagnosis. MOSE is a highly reliable tool when fork-tip needles are used compared with side-fenestrated needles. (Clinical trial registration number: NCT03622229.).

Genetic variability following selection for scrapie resistance in six autochthonous sheep breeds in the province of Bolzano (northern Italy).

Scrapie is an ovine transmissible spongiform encephalopathy, and its susceptibility is associated with polymorphisms in the prion protein gene (PRNP). Genetic selection is currently the most effective mean for eradication of the susceptible VRQ allele in favour of resistant ARR allele. Maintenance of genetic diversity should be one of the major objectives in breeding programmes, especially in endangered breeds, and genetic information are an excellent alternative to pedigree data where these information are missing. The aim of our study was to determine changes of genetic variability in six native sheep breeds from autonomous province of Bolzano, northern Italy, following simulation of scrapie selection scenarios. A total of 684 rams were investigated for PRNP polymorphisms and for 10 microsatellite loci to estimate genetic variability. Across all loci, a total of 163 alleles were detected with a mean of 10.4 alleles per locus. Average observed (Ho) and unbiased expected (uHe) heterozygosity overall loci were 0.74 and 0.78, respectively, showing a statistically significant deviation from Hardy-Weinberg equilibrium (HWE) in all breeds. This heterozygosity deficit was confirmed by a positive fixation index (Fis), determining a moderate inbreeding in each breed. Simulating a soft selection, where only rams having at least a VRQ allele should be excluded from reproduction, Ho, uHe and Fis values remained almost unchanged, indicating that genetic variability should not be affected by the removal of these individuals. With a mild selection scenario, considering only rams with at least one ARR allele, we observed a decrease in the mean alleles per breed (8.9) and the maintenance of heterozygosity deficiency, except for two breeds, where it was any longer significant. These results showed that selection strategies allowing use of heterozygous as well homozygous ARR rams might be the right compromise to improve resistance to scrapie and to do not dramatically affect genetic variability of these breeds.

Role of SIRT-3, p-mTOR and HIF-1α in Hepatocellular Carcinoma Patients Affected by Metabolic Dysfunctions and in Chronic Treatment with Metformin.

The incidence of hepatocellular carcinoma deriving from metabolic dysfunctions has increased in the last years. Sirtuin- (SIRT-3), phospho-mammalian target of rapamycin (p-mTOR) and hypoxia-inducible factor- (HIF-1α) are involved in metabolism and cancer. However, their role in hepatocellular carcinoma (HCC) metabolism, drug resistance and progression remains unclear. This study aimed to better clarify the biological and clinical function of these markers in HCC patients, in relation to the presence of metabolic alterations, metformin therapy and clinical outcome. A total of 70 HCC patients were enrolled: 48 and 22 of whom were in early stage and advanced stage, respectively. The expression levels of the three markers were assessed by immunohistochemistry and summarized using descriptive statistics. SIRT-3 expression was higher in diabetic than non-diabetic patients, and in metformin-treated than insulin-treated patients. Interestingly, p-mTOR was higher in patients with metabolic syndrome than those with different etiology, and, similar to SIRT-3, in metformin-treated than insulin-treated patients. Moreover, our results describe a slight, albeit not significant, benefit of high SIRT-3 and a significant benefit of high nuclear HIF-1α expression in early-stage patients, whereas high levels of p-mTOR correlated with worse prognosis in advanced-stage patients. Our study highlighted the involvement of SIRT-3 and p-mTOR in metabolic dysfunctions that occur in HCC patients, and suggested SIRT-3 and HIF-1α as predictors of prognosis in early-stage HCC patients, and p-mTOR as target for the treatment of advanced-stage HCC.

Skewing effect of sulprostone on dendritic cell maturation compared with dinoprostone.

BACKGROUND: Dendritic cells (DCs) are the most efficient antigen-presenting cells and act at the center of the immune system owing to their ability to control both immune tolerance and immunity. In cancer immunotherapy, DCs play a key role in the regulation of the immune response against tumors and can be generated ex vivo with different cytokine cocktails. METHODS: We evaluated the feasibility of dinoprostone (PGE2) replacement with the molecular analog sulprostone, in our good manufacturing practice (GMP) protocol for the generation of DC-based cancer vaccine. We characterized the phenotype and the function of DCs matured in the presence of sulprostone as a potential substitute of dinoprostone in the pro-inflammatory maturation cocktail consisting of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and IL-6. RESULTS: We found that sulprostone invariably reduces the recovery, but does not significantly modify the viability and the purity of DCs. The presence of sulprostone in the maturation cocktail increases the adhesion of single cells and of clusters of DCs to the flask, making them more similar to their immature counterpart in terms of adhesion and spreading proprieties. Moreover, we observed that sulprostone impairs the expression of co-stimulatory molecules and the spontaneous as well as the directed migration capacity of DCs. DISCUSSION: These findings underscore that the synthetic analog sulprostone strongly reduces the functional quality of DCs, thus cannot replace dinoprostone in the maturation cocktail of monocyte-derived DCs.

Interplay Between SIRT-3, Metabolism and Its Tumor Suppressor Role in Hepatocellular Carcinoma.

Sirtuins (SIRT), first described as nicotinamide adenine dinucleotide (NAD+)-dependent type III histone deacetylases, are produced by cells to support in the defense against chronic stress conditions such as metabolic syndromes, neurodegeneration, and cancer. SIRT-3 is one of the most studied members of the mitochondrial sirtuins family. In particular, its involvement in metabolic diseases and its dual role in cancer have been described. In the present review, based on the evidence of SIRT-3 involvement in metabolic dysfunctions, we aimed to provide an insight into the multifaceted role of SIRT-3 in many solid and hematological tumors with a particular focus on hepatocellular carcinoma (HCC). SIRT-3 regulatory effect and involvement in metabolism dysfunctions may have strong implications in HCC development and treatment. Research literature widely reports the relationship between metabolic disorders and HCC development. This evidence suggests a putative bridge role of SIRT-3 between metabolic diseases and HCC. However, further studies are necessary to demonstrate such interconnection.

Vaccination with autologous dendritic cells loaded with autologous tumor lysate or homogenate combined with immunomodulating radiotherapy and/or preleukapheresis IFN-α in patients with metastatic melanoma: a randomised "proof-of-principle" phase II study.

BACKGROUND: Vaccination with dendritic cells (DC) loaded with tumor antigens elicits tumor-specific immune responses capable of killing cancer cells without inducing meaningful side-effects. Patients with advanced melanoma enrolled onto our phase II clinical studies have been treated with autologous DC loaded with autologous tumor lysate/homogenate matured with a cytokine cocktail, showing a clinical benefit (PR + SD) in 55.5% of evaluable cases to date. The beneficial effects of the vaccine were mainly restricted to patients who developed vaccine-specific immune response after treatment. However, immunological responses were only induced in about two-thirds of patients, and treatments aimed at improving immunological responsiveness to the vaccine are needed. METHODS/DESIGN: This is a phase II, "proof-of-principle", randomized, open-label trial of vaccination with autologous DC loaded with tumor lysate or homogenate in metastatic melanoma patients combined with immunomodulating RT and/or preleukapheresis IFN-α. All patients will receive four bi-weekly doses of the vaccine during the induction phase and monthly doses thereafter for up to a maximum of 14 vaccinations or until confirmed progression. Patients will be randomized to receive:(1.) three daily doses of 8 Gy up to 12 Gy radiotherapy delivered to one non-index metastatic field between vaccine doses 1 and 2 and, optionally, between doses 7 and 8, using IMRT-IMAT techniques;(2.) daily 3 MU subcutaneous IFN-α for 7 days before leukapheresis;(3.) both 1 and 2;(4.) neither 1 nor 2.At least six patients eligible for treatment will be enrolled per arm. Daily 3 MU IL-2 will be administered subcutaneously for 5 days starting from the second day after each vaccine dose. Serial DTH testing and blood sampling to evaluate treatment-induced immune response will be performed. Objective response will be evaluated according to immune-related response criteria (irRC). DISCUSSION: Based upon the emerging role of radiotherapy as an immunologic modifier, we designed a randomized phase II trial adding radiotherapy and/or preleukapheresis IFN-α to our DC vaccine in metastatic melanoma patients. Our aim was to find the best combination of complementary interventions to enhance anti-tumor response induced by DC vaccination, which could ultimately lead to better survival and milder toxicity.

Molecular identity and prevalence of Cryptococcus spp. nasal carriage in asymptomatic feral cats in Italy.

Cryptococcosis is a life-threatening fungal disease that infects humans and animals worldwide. Inhalation of fungal particles from an environmental source can cause primary infection of the respiratory system. As animals can be considered a sentinel for human diseases, the aim of this study was to determine the prevalence and molecular identity of Cryptococcus spp. in the nasal cavity of feral cats. Cats from 162 urban and rural feral cat colonies were sampled over 3 years. Of 766 cats from which nasal swabs were obtained, Cryptococcus spp. were recovered from 95 (12.6%), including 37 C. magnus (4.8%), 16 C. albidus (2.0%), 15 C. carnescens (1.9%), 12 C. neoformans (1.6%), as well as C. oeirensis (n = 3), C. victoriae (n = 3), C. albidosimilis (n = 2), Filobasidium globisporum (n = 2), C. adeliensis (n = 1), C. flavescens (n = 1), C. dimnae (n = 1), C. saitoi (n = 1), and C. wieringae (n = 1) with prevalence <1%. Thirteen Cryptococcus species were identified by polymerase chain reaction and sequencing of internal transcribed spacer amplicons. Statistical analysis did not identify any predisposing factors that contributed to nasal colonization (eg, sex, age, season, or habitat). Results suggest that asymptomatic feral cats may carry C. neoformans and other Cryptococcus species in their sinonasal cavity. Genotyping of the specific cryptococcal isolates provides a better understanding of the epidemiology of these yeasts.

Effects of Disinfectants on Bacterium Paenibacillus larvae in Laboratory Conditions.

American foulbrood is an infectious disease of the honeybee brood that causes multiple types of damage to beekeeping. The causative agent of the disease is the bacterium Paenibacillus larvae, which forms resistant infective spores and is viable for decades. After the eradication measures have been implemented, in cases of clinically visible disease, it is necessary to conduct effective final disinfections of equipment and tools. This study aimed to determine the effect of ten commercially available and commonly used disinfectants on certified strains of P. larvae under laboratory conditions, as well as to compare the obtained results among individual genotypes of P. larvae. Selected products were tested by determining the zone of inhibition using an agar diffusion test, a suspension test for viable bacteria, a surface disinfectant test, and a sporicidal effect in the suspension test. Incidin OxyFoam S and Sekusept Aktiv are both effective against all examined genotypes of P. larvae. Despadac and Despadac Secure have a bactericidal effect, but their sporocidal effect is not as satisfactory as that of Genox. Genoll does not exhibit a sporicidal effect, and Ecocide S at 1%, Bee protect H forte, and Bee protect F did not exhibit a satisfactory sporocidal effect. Additionally, EM® PROBIOTIC FOR BEES did not exhibit any bactericidal effect. The effective application of control measures and proper application of final disinfection can reduce the reoccurrence of visible clinical signs of disease, whereas methods of early diagnosis can significantly reduce the incidence of the disease.

The oriental hornet (Vespa orientalis) as a potential vector of honey bee's pathogens and a threat for public health in North-East Italy.

BACKGROUND: Oriental hornets are large predatory hymenoptera that occur in the southern part of Asia and the southeastern Mediterranean. Among many pests of bee colonies, Vespa orientalis was recorded to be one of the most destructive. OBJECTIVES: The aim of this study was to: (1) monitor the presence of pathogens carried by V. orientalis that could potentially threaten honey bees and public health; (2) describe the hornet's predatory behavior on honey bee colonies and (3) collect the medical history of a V. orientalis sting suffered by a 36-year-old woman. METHODS: Observations of V. orientalis predatory behavior and the catches of hornets for parasitological and microbiological examination, using molecular and bacteriological analyses, were carried out in three experimental apiaries, both in spring in order to capture the foundress queens and during the summer to capture the workers. Furthermore, the medical history and photographic documentation of a V. orientalis sting suffered by a 36-year-old woman have been collected. RESULTS: The results obtained highlight that V. orientalis is capable of causing serious damage to beekeeping by killing bees, putting under stress the honey bee colonies and by potentially spreading honey bee pathogens among apiaries. These hornets may also become a public health concern, since they are capable of inflicting multiple, painful stings on humans. CONCLUSIONS: Only the development of an Integrated Management Control Program will be able to contain the negative effects of anomalous population growth and the potentially negative impact on honey bees and public health of V. orientalis.

Low-dose temozolomide before dendritic-cell vaccination reduces (specifically) CD4+CD25++Foxp3+ regulatory T-cells in advanced melanoma patients.

BACKGROUND: In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. METHODS: Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. RESULTS: Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. CONCLUSIONS: Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

Insects and Public Health: An Overview.

Insects are, by far, the most common animals on our planet. The ubiquity and plethora of ecological niches occupied by insects, along with the strict and sometimes forced coexistence between insects and humans, make insects a target of public health interest. This article reports the negative aspects historically linked to insects as pests and vectors of diseases, and describes their potential as bioindicators of environmental pollution, and their use as food and feed. Both negative and positive impacts of insects on human and animal health need to be addressed by public health professionals who should aim to strike a balance within the wide range of sometimes conflicting goals in insect management, such as regulating their production, exploiting their potential, protecting their health and limiting their negative impact on animals and humans. This requires increased insect knowledge and strategies to preserve human health and welfare. The aim of this paper is to provide an overview of traditional and emerging topics bridging insects and public health to highlight the need for professionals, to address these topics during their work. The present and future role and activities of public health authorities regarding insects are analyzed.

Stability Program in Dendritic Cell Vaccines: A "Real-World" Experience in the Immuno-Gene Therapy Factory of Romagna Cancer Center.

Advanced therapy medical products (ATMPs) are rapidly growing as innovative medicines for the treatment of several diseases. Hence, the role of quality analytical tests to ensure consistent product safety and quality has become highly relevant. Several clinical trials involving dendritic cell (DC)-based vaccines for cancer treatment are ongoing at our institute. The DC-based vaccine is prepared via CD14+ monocyte differentiation. A fresh dose of 10 million DCs is administered to the patient, while the remaining DCs are aliquoted, frozen, and stored in nitrogen vapor for subsequent treatment doses. To evaluate the maintenance of quality parameters and to establish a shelf life of frozen vaccine aliquots, a stability program was developed. Several parameters of the DC final product at 0, 6, 12, 18, and 24 months were evaluated. Our results reveal that after 24 months of storage in nitrogen vapor, the cell viability is in a range between 82% and 99%, the expression of maturation markers remains inside the criteria for batch release, the sterility tests are compliant, and the cell costimulatory capacity unchanged. Thus, the data collected demonstrate that freezing and thawing do not perturb the DC vaccine product maintaining over time its functional and quality characteristics.

Matrix metalloproteinases and their inhibitors in canine mammary tumors.

BACKGROUND: Malignant canine mammary tumors represent 50% of all neoplasms in female dogs. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression, and they are also associated with the reactive stroma, which provides structural and vascular support for tumor growth. RESULTS: MMP-2, MMP-9 and MT1-MMP were expressed at both the mRNA and protein levels in tumor samples. MMP-2 and MMP-9 immunohistochemical reactions were evident both in the epithelial tumor cells and in the stromal compartment to varying degrees; in particular, the intensity of the MMP-2 staining was stronger in the stromal fibroblasts close to epithelial tumor cells in simple carcinomas than in adenomas. These data were supported by gelatin-zymography; bands for the active form of MMP-2 were found in 94% of carcinoma samples, compared with 17% of benign tumor samples. The gene expression and immunohistochemical results for MT1-MMP were comparable to those for MMP-2. The immunoreactivity for MMP-13 and TIMP-2 was lower in carcinomas than in adenomas, confirming the mRNA data for MMP-13 and the other MMP inhibitors that were evaluated. The active form of MMP-9, but not the active form of MMP-2, was identified in the plasma of all of the tested dogs. CONCLUSIONS: Our findings suggest that MMP-9, MMP-2 and MT1-MMP, which are synthesized by epithelial cancer cells and cancer-associated fibroblasts, play an important role in malignant canine mammary tumors. The reduction of MMP-13 and TIMP-2 could also be a significant step in malignant transformation. MMP-2 and MT1-MMP could be further evaluated as future biomarkers for predicting the progression and prognosis of canine mammary tumors.

Radiotherapy and High-Dose Interleukin-2: Clinical and Immunological Results of a Proof of Principle Study in Metastatic Melanoma and Renal Cell Carcinoma.

High-dose interleukin-2 (HD IL-2) has curative potential in metastatic melanoma (MM) and renal cell carcinoma (RCC). Radiotherapy (RT) kills cancer cells and induces immunomodulatory effects. Prospective trials exploring clinical and immunological properties of combined RT/HD IL-2 are still needed. We designed a phase II, single-arm clinical trial for patients with MM and RCC. The treatment schedule consisted of 3 daily doses of 6-12 Gy of RT to 1-5 non-index metastatic fields, before IL-2 at the first and third treatment cycle. HD IL-2 was administered by continuous infusion for 72 hours and repeated every 3 weeks for up to 4 cycles, thereafter every 4 weeks for a maximum of 2 cycles. The primary endpoint was the immunological efficacy of the combined RT/HD IL-2 treatment (assessed by IFN-γ ELISPOT). Nineteen out of 22 patients were evaluable for immunological and clinical response. Partial response occurred in 3 (15.7%) patients and stable disease was observed in 7 (36.8%). The disease control rate was 52.6% after a median follow up of 39.2 months. According to Common Terminology Criteria for Adverse Events 4.0 (CTCAE 4.0), the majority of toxicities were grade 1-2. Immunological responses were frequent and detected in 16 (84.2%) patients. Increased levels of IL-8 and IL-10 in melanoma, circulating effector memory CD4+ and intratumoral CD8+ T cells in both tumor types were detected after therapy. Overall the treatment was well tolerated and immunologically active. Immunomonitoring and correlative data on tumor and peripheral blood cell subsets suggest that this combination treatment could be a promising strategy for patients progressing after standard treatments.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens.

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.

Tumor endothelial marker 8 expression levels in dendritic cell-based cancer vaccines are related to clinical outcome.

Previous studies have shown that tumor endothelial markers (TEMs 1-9) are up modulated in immunosuppressive, pro-angiogenic dendritic cells (DCs) found in tumor microenvironments. We recently reported that monocyte-derived DCs used for vaccination trials may accumulate high levels of TEM8 gene transcripts. Here, we investigate whether TEM8 expression in DC preparations represents a specific tumor-associated change of potential clinical relevance. TEM8 expression at the mRNA and protein level was evaluated by quantitative real-time RT-PCR and cytofluorimetric analysis in human clinical grade DCs utilized for the therapeutic vaccination of 17 advanced cancer patients (13 melanoma and 4 renal cell carcinoma). The analyses revealed that DCs from patients markedly differ in their ability to up-modulate TEM8. Indeed, mDCs from eight non-progressing patients [median overall survival (OS) = 32 months, all positive to the delayed-type hypersensitivity test (DTH)], had similar TEM8 mRNA expression levels [mDCs vs. immature iDCs; mean fold increase (mfi) = 1.97] to those found in healthy donors (mfi = 2.7). Conversely, mDCs from nine progressing patients (OS < 5 months, all but one with negative DTH) showed an increase in TEM8 mRNA levels (mfi = 12.88, p = 0.0018). The present observations suggest that TEM8 expression levels in DC-based therapeutic vaccines would allow the selection of a subgroup of patients who are most likely to benefit from therapeutic vaccination.