Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL), are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin-ligase (CRL) with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiological KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the BTB domain of KLHL6 disrupted its localization and heterodimerization, and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL.

Zinc oxide nanoparticles and Klebsiella sp. SBP-8 alleviates chromium toxicity in Brassica juncea by regulation of antioxidant capacity, osmolyte production, nutritional content and reduction in chromium adsorption.

Heavy metals are one of the most damaging environmental toxins that hamper growth of plants. These noxious chemicals include lead (Pb), arsenic (As), nickel (Ni), cadmium (Cd) and chromium (Cr). Chromium is one of the toxic metal which induces various oxidative processes in plants. The emerging role of nanoparticles as pesticides, fertilizers and growth regulators have attracted the attention of various scientists. Current study was conducted to explore the potential of zinc oxide nanoparticles (ZnONPs) alone and in combination with plant growth promoting rhizobacteria (PGPR) Klebsiella sp. SBP-8 in Cr stress alleviation in Brassica juncea (L.). Chromium stress reduced shoot fresh weight (40%), root fresh weight (28%), shoot dry weight (28%) and root dry weight (34%) in B. juncea seedlings. Chromium stressed B. juncea plants showed enhanced levels of malondialdehyde (MDA), electrolyte leakage (EL), hydrogen peroxide (H2O2) and superoxide ion (O2• -). However, co-supplementation of ZnONPs and Klebsiella sp. SBP-8 escalated the activity of antioxidant enzymes i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) in B. juncea grown in normal and Cr-toxic soil. It is further proposed that combined treatment of ZnONPs and Klebsiella sp. SBP-8 may be useful for alleviation of other abiotic stresses in plants.

GNAS mutation inhibits growth and induces phosphodiesterase 4D expression in colorectal cancer cell lines.

Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.

The Programmed Cell Death Ligand 1 and Lipocalin 2 Expressions in Primary Breast Cancer and Their Associations with Molecular Subtypes and Prognostic Factors.

Purpose: Breast cancers exhibit molecular heterogeneity, leading to diverse clinical outcomes and therapeutic responses. Immune checkpoint inhibitors targeting PD-L1 have shown promise in various malignancies, including breast cancer. Lipocalin 2 (LCN2) has also been associated with tumor aggressiveness and prognostic potential in breast cancers. However, the expression of PD-L1 and LCN2 in breast cancer subtypes and their prognostic implications remains poorly investigated. Methods: A retrospective analysis of 89 primary breast cancer cases was conducted to assess PD-L1 and LCN2 expressions using immunohistochemistry. Cases were classified into four different molecular subtypes based on ER, PR, HER2, and Ki-67 status. Associations between PD-L1 and LCN2 expressions and various prognostic factors were examined. Results: Although low expression of LCN2 (Allred score of <3) was observed even in normal breast tissue, LCN2 expression with increasing Allred score (≥3) positively correlated with the histological grade, high Ki-67 proliferation index, and ER/PR negativity. Significant elevations of LCN2 and PD-L1 expressions were observed in triple-negative and HER2-positive breast cancers. Conclusion: The results of the study highlight the association of LCN2 with known prognostic factors and molecular subtypes. To identify potential immunotherapy recipients, it would be useful to evaluate LCN2 as well as PD-L1 immune targets in different subgroups of breast cancer patients. Further studies with larger patient numbers are warranted to validate these observations and establish standardized scoring criteria for LCN2 expression assessment.

Calcium oxide nanoparticles mitigate lead stress in Abelmoschus esculentus though improving the key antioxidative enzymes, nutritional content and modulation of stress markers.

Lead (Pb) is thought to be one of most injurious metals on the earth. Lead stress in plants enhances synthesis of highly toxic reactive oxygen species (ROS). During present research, impact of calcium-oxide nanoparticles (CaO-NPs) was observed on antioxidative defense mechanism in Abelmoschus esculentus plants prone to Pb stress. A CRD experiment was employed with 5 replicates having four treatments (T0 = Control, T1 = Pb stress (200 ppm), T2 = CaO-NPs and T3 = Pb + CaO-NPs). Pb-stressed seedlings exhibited decreased root growth, shoot growth, chlorophyll concentration and biomass accumulation. Moreover, higher synthesis of hydrogen-peroxide (H2O2), malondialdehyde (MDA) and electrolyte leakage (EL) resulting in cellular injuries were noted in plants growing in Pb spiked conditions. Similarly, stressed plants showed higher accumulation of total soluble sugar and proline content besides elevated activity of antioxidative enzymes counting catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX). On the contrary side, CaO-NPs alleviated the Pb induced phytotoxicity through improving activity of antioxidative enzymes. The elevated activity of antioxidant enzymes reduced biosynthesis of H2O2 and MDA which was revealed through the increased growth parameters. In addition, CaO-NPs persuaded enhancement in plant defence machinery by decreased chlorophyll deprivation and augmented the uptake of plant nutrients including K and Ca content. Hence, CaO-NPs can be potent regulators of the antioxidative enzymes and stress markers to ameliorate abiotic stresses.

Proliferation and migration of ML1 follicular thyroid cancer cells are inhibited by IU1 targeting USP14: role of proteasome and autophagy flux.

USP14 is a deubiquitinating enzyme involved in protein degradation by interacting with the proteasome and removal of poly-ubiquitin chains on target proteins. USP14 can influence cellular processes such as cell survival, DNA repair, ER stress, endocytosis, and the inflammatory response. USP14 further plays a role in tumor growth, and the inhibition of USP14 by compounds such as IU1 may affect cancer cell migration and invasion. Here we have studied the mechanisms for the action of IU1 in ML1 follicular thyroid cancer cells, comparing them with control, primary thyroid cells. Treatment with IU1 reduced proliferation of ML1 cells in a concentration-dependent manner, and more prominently than in control cells. IU1 decreased basal migration of ML1 cells, and after stimulation of cells with the bioactive compound, sphingosine-1-phosphate. The sphingosine-1-phosphate receptor 3 was increased in ML1 cells as compared with control thyroid cells, but this was not influenced by IU1. Further studies on the mechanism, revealed that IU1 enhanced the proteasome activity as well as LC3B-dependent autophagy flux in ML1 cells with an opposite effect on control thyroid cells. This indicates that IU1 elicits a cell-type dependent autophagy response, increasing it in ML1 cancer cells. The IU1-mediated stimulation of autophagy and proteasomes can likely contribute to the reduced cell proliferation and migration observed in ML1 cells. The precise set of proteins affected by IU1 in ML1 thyroid and other cancer cells warrant further investigations.

N-Glycomic Profiling of Microsatellite Unstable Colorectal Cancer.

Aberrant glycosylation affects cancer progression and immune evasion. Approximately 15% of colorectal cancers (CRCs) demonstrate microsatellite instability (MSI) and display major differences in outcomes and therapeutic responses, as compared to corresponding microsatellite stable (MSS) tumors. We compared the N-glycan profiles of stage II and IV MSI CRC tumors, further subdivided into BRAFV600E wild-type and mutated subgroups (n = 10 in each subgroup), with each other and with those of paired non-neoplastic mucosal samples using mass spectrometry. Further, the N-glycans of BRAFV600E wild-type stage II MSI tumors were compared to corresponding MSS tumors (n = 9). Multiple differences in N-glycan profiles were identified between the MSI CRCs and control tissues, as well as between the stage II MSI and MSS samples. The MSI CRC tumors showed a lower relative abundance of high-mannose N-glycans than did the control tissues or the MSS CRCs. Among MSI CRC subgroups, acidic N-glycans showed tumor stage and BRAF mutation status-dependent variation. Specifically, the large, sulfated/phosphorylated, and putative terminal N-acetylhexosamine-containing acidic N-glycans differed between the MSI CRC subgroups, showing opposite changes in stages II and IV, when comparing BRAF mutated and wild-type tumors. Our results show that molecular subgroups of CRC exhibit characteristic glycan profiles that may explain certain carcinogenic properties of MSI tumors.

Azo dyes degradation by microorganisms - An efficient and sustainable approach.

Synthetic aromatic compounds consisting of various functional groups are known as dyes. These colored compounds are often discharged in effluents, and they are very dangerous to aquatic life. Basically, the dye industry started by using natural plant and insect sources, and then suddenly turned into artificial manufacturing. Natural equilibrium of our environment gets changed by the reduction in photosynthetic activity due to the dyes. In China 900,000 tons of all kinds of dyes are usually produced, which are used in many industries like food, textile, food, paper and leather. Untreated wastewater contaminates aquatic bodies by causing eutrophication, change in water color, oxygen depletion which affect aquatic organisms to a great extent. Dye wastewater is now the key environmental pollution form. In recent eras an extensive study line has been developed to explore the dye decolorization and biodegradation under both aerobic as well as anaerobic conditions. In this review, the chemistry, toxicity and microbial biodegradation/decolorization are presented. Some recent studies along with the new techniques and methodologies of remediating the dye pollution are also discussed to provide the bases of their handling. Overall, efficient and high biodegradation potential make microbes an impending foundation for green chemistry to eradicate toxic dyes from industrial wastewater.

Morpho-palynological and anatomical studies in desert cacti (Opuntia dillenii and Opuntia monacantha) using light and scanning electron microscopy.

Opuntia is the most diverse and widely distributed drought resistant promising genus of family Cactaceae. The cladodes were utilized to quantify the chemical composition of these plants helpful in lignocellulose conversion and their application towards biofuel production. The present study was aimed to evaluate and compare the taxonomic relationship based on morphology, stem anatomy and palynology of important desert cacti including Opuntia dillenii and Opuntia monacantha. This study also evaluates the potential usefulness of morphological, anatomical and pollen traits using light and scanning electron microscopy. The obtained microcharacters of stem and flowers are considered diagnostic at the generic and specific levels. Some distinguishing morphological features observed were elliptical to obovate cladodes, 1-7 spines per areole and presence of glochidia in O. dillenii. Pollen and stem anatomical characters of the studied taxa are considered highly diagnostic at the generic and species levels. The epidermis has irregular and wavy cells, with straight to sinuate wall pattern and paracytic stomata. Pollen grains appear as pantoporate and prolate spheroidal having reticulate to perforate-reticulate sculpturing while exine semi-tectate to tectate. The taxonomic features studied could be valuable to elaborate and helpful in correctly identification of Opuntia species. The methods of diverse microscopic examination also providing sufficient evidence about the taxonomy of the Opuntia species. RESEARCH HIGHLIGHTS: Description and illustration of desert cacti Opuntia. Morpho-anatomy and palynology were studied with LM and SEM. Highly variation in taxonomic qualitative and quantitative features. Systematic significance based on taxonomic characters was presented.

Pollen morphology of selected melliferous plants and its taxonomic implications using microscopy.

This research aimed to explore the melliferous bee flora pollen from arid zones of District Attock, Islamabad Capital and Rawalpindi. Morpho-palynological features has provided key information for the classification and help to explain the taxonomy of several genera and species. Therefore, in this work, we examined the morphological characters of pollen potentially to be used for the species delimitation of bee flora. A total of 18 bee floral species, classified into 12 different families were collected, pressed, identified and then acetolyized to observe the grains under microscopy. Bee pollen can be described by small, medium to large sized morphotypes presenting five types of shapes, prolate spheroidal in (seven species), sub-prolate (six species), oblate spheroidal (three species), oblate and prolate (one species each) was examined. The observed polar and equatorial diameter were found maximum in Zea mays 73.5 µm and Pelargonium inquinans 66.75 µm respectively. Pollen of six different types was found namely: tricolporate, 3-zonocolporate, monoportae, tetracolporate, tetraporate and mononsulcate. Whereas highest value of colpi length was measured in Grewia tenax (24.55 µm). Exine surface nature of pollen was examined echinate psilate and scabrate. Exine thickness noted to be maximum in Verbena tenuisecta (8.40 µm) and minimum in Citrus macrocarpa (0.4 µm). Bee floral species considered difficult to identify based on other morphological traits were successfully distinguished using pollen quantitative and qualitative traits, confirming the importance of pollen morphology to diagnose characters to correctly identify honeybee flora. RESEARCH HIGHLIGHTS: Pollen diversity among honeybee flora. Highly variations observed via microscopy in pollen micromorphology. Palyno-morph has taxonomic value for the identification of honeybee floral species.

Light and scanning electron microscopic observation of palynological characteristics in spineless Astragalus L. (Fabaceae) and its taxonomic significance.

Palynological characterization is considered to be one of the significant taxonomic tools for the delimitation and identification of morphologically complicated taxa. Hence, the pollen morphology of 12 species of spineless Astragalus L. was examined using light and scanning electron microscopy. Studied pollen were small to medium, monad, prolate to per-prolate and tricolporate type in all studied taxa. The exine sculpturing varied from reticulate to microreticulate whereas colpus ornamentation ranged from scabrate to granulate. Furthermore, maximum polar and equatorial diameter was recorded in Astragalus leucocephalus Bunge. (45.00 µm) and A. pyrrhotrichus Boiss. (22.91 µm) while minimum in A. amherstianus Benth. ex Royle (28.75 µm) and A. amherstianus Benth. ex Royle (15.00 µm), respectively. Similarly, the ratio of polar to equatorial diameter was recorded maximum in A. ophiocarpus Boiss. (2.05). The width of colpi was larger in A. hamosus L. (1.29 µm) and smaller in A. ophiocarpus Boiss. (0.62 µm). We have also found the maximum value of mesocolpium in A. retamocarpus Boiss. (2.08 µm) while minimum in A. oxyglottis Steven ex M.Bieb. (1.87 µm). The quantitative pollen attributes helped in the development of pollen keys for the accurate and quick identification of the studied species. Furthermore, ordination and cluster analysis were performed for the differentiation of the investigated taxa at species level. Based on our results, we conclude that pollen features can be used for the delimitation and identification of the studied taxa. RESEARCH HIGHLIGHTS: Pollen micromorphology is a useful tool for classifying complicated taxa. The pollen micromorphology of 12 spineless species of Astragalus L. was studied using LM and SEM. The observed pollen characteristics aided in Astragalus L. serve for the identification and classification of taxa at specific level.

Spatial variations in the biochemical potential of okra [Abelmoschus esculentus L. (Moench)] leaf and fruit under field conditions.

Okra (Abelmoschus esculentus L. (Moench) plays a significant role in humans nutrition because its fresh leaves, stems, flowers, pods and seeds, are used for multiple purposes. The present study attempted to determine the spatial variations in biochemical attributes of osmoprotectants and the oxidative defense system of okra plants. Samples of soil and okra plants (leaves and fruits) were collected from three different locations: Faisalabad region-1 (7 JB-I), Faisalabad region-2 (7 JB-II) and Pindi Bhattian. Chlorophyll contents, glycine betaine (GB), ascorbic acid (AsA), total phenolics, hydrogen peroxide (H2O2), proline, and malondialdehyde (MDA) contents were analyzed in the leaves and fruits of okra plants. Soil analyses showed that pH, electrical conductivity (EC), phosphorus (P), potassium (K), iron (Fe), and saturation of soil were higher in Faisalabad region 2, while organic matter, sand, Zn, and Cu were higher in the Pindi Bhattian region. The results from okra leaves showed that Pindi Bhattian had higher chlorophyll a, GB and H2O2 contents, while Faisalabad region 1 had a higher ratio of chlorophyll a/b compared to the other regions. However, Faisalabad regions 2 and 1 had higher leaf phenolic contents, Faisalabad regions 1 and 2 showed higher leaf proline contents, and Faisalabad region 2 possessed higher AsA and MDA contents. Analyses of okra fruits showed that Faisalabad region 2 had higher chlorophyll a and total chlorophyll contents, while Faisalabad region 1 had higher chlorophyll b contents. Faisalabad region 2 and Pindi Bhattian had higher ratios of chlorophyll a/b, and Faisalabad region 1 showed higher phenolic, AsA, H2O2, and MDA contents of okra fruit, whereas the Faisalabad regions exhibited higher proline and GB contents than the Pindi Bhattian region. Overall, okra leaves and fruits showed better responses in the Faisalabad regions, and these results may be used to screen for okra cultivars with better tolerance under different environmental conditions.

Palyno-morphological diversity of Asteraceous and Poaceous allergenic plant using microscopic techniques in lesser Himalaya-Pakistan.

In classification and identification of plant species, palyno morphological character has remain helpful for plant taxonomist. The aim to investigation this study was to identify the characteristic features of Asteraceae and Poaceae family member collected from lower Himalayas of Pakistan. Light microscope and Scanning electron microscope were used for feature study of allergenic pollen. Our study includes 10 species of Asteraceae and Poaceae family. The most dominant shape of pollen was Oblate-spheroidal, Prolate-Spheroidal, Prolate and Suboblate shape of pollen. Quantitative character of pollen includes maximum pollen diameter found in Dichanthium annulatum (46.0 µm) and minimum polar axis pollen diameter found in Parthenium hysterophorous (16.0 µm). Similarly highest value of P/E ratio was noted in Cenchrus echinatus (1.05) and smallest value of P/E ratio was observed in Erigeron bonariensis (0.88). Poaceae family member were monoporate. Character feature of exine sculpturing include Microechinate, granulate, echinate, conical base, lacunae Nanogammate, Aerolate-Scabratre, Verrucate, like variation found in exine of both family member. Highest value of pollen fertility was observed in P. hysterophorous 93%, Similarly maximum value of pollen sterility was noted in E. bonariensis and Brachiaria reptans 24%. This investigation provides the characteristic feature of allergenic pollen species and help to arrange them in define class. RESEARCH HIGHLIGHTS: Pollen has affected many people around the world. Using microscopic techniques, morpho-palynological analysis of Asteraceae and Poaceae families were recognized. LM and SEM study show both qualitative and quantitative character of pollen. Identification of allergenic pollen and microscopic identified character play important role for proper guideline of identification of pollen having allergenic potential.

Frequency and assessment of Nutritional Status of school going children in rural areas of Islamabad.

BACKGROUND AND OBJECTIVES: Nutritional status is considered a significant and positive health indicator. It determines anthropometric measurements of preschool children, the height of children at the time of school entry and prevalence of low birth weight. The objective of the study was to determine the frequency of nutritional status and socio-demographic factors influencing under nutrition among school children of rural Islamabad. METHODS: A cross-sectional study was conducted among school children of the age (4-16 years) from January 2017 to September 2019. The Sample size was 1710. Schools were selected through convenient sampling technique. Frequency and percentages were calculated and inferential statistics were computed to analyze the association of health status with categorical variables by using chi-square by keeping the level of significance <0.05 through SPSS version 20. RESULTS: The mean age of the sample was 9.38 ± 4.14 with the maximum number of children (49.1%) in the age bracket of 5-9. Out of 1710 children, 54.4% had normal weight for age, 25.3% were underweight, 7.5% overweight and 12.8% were found to be obese. Stunting was found to be 26%. Prevalence of being underweight was higher than overweight /obesity particularly in younger and higher age groups as indicated by p-value of 0.000. Comparing with females, male students had significantly higher frequency of being underweight and stunted as reflected by p-value of 0.004 and 0.000 respectively. Univariate analysis also showed a strong association between age and nutritional status as mean weight increased from 39.22 ±5.21 to 63.50± 4.66 and height from 35.67±5.76 to 113.73± 29.22 with advancing age. CONCLUSIONS: Undernutrition remains an ongoing health problem in school going children of rural Islamabad; particularly in male students of younger age groups. School health programs and nutritional interventions need to be strengthened particularly in rural areas of Islamabad.

Interactive effects of gibberellic acid and NPK on morpho-physio-biochemical traits and organic acid exudation pattern in coriander (Coriandrum sativum L.) grown in soil artificially spiked with boron.

It was aimed to examine the role of gibberellic acid (GA3) and NPK fertilizer in alleviating boron (B) toxicity in coriander (Coriandrum sativum L.) plants. Two weeks old C. sativum seedlings were subjected to different NPK fertilizers [low NPK (30 kg ha-1) and normal NPK (60 kg ha-1)], which were also supplied by GA3 (50 mg L-1), under varying levels of B i.e., 0, 200 and 400 mg kg-1 in the soil. Results revealed that B toxicity led to a substantial decreased in the plant growth and biomass, photosynthetic pigments, gas exchange characteristics, sugars and essential nutrients in the roots and shoots of C. sativum seedlings. However, B toxicity boosted the production of reactive oxygen species (ROS) by increasing the contents of malondialdehyde (MDA), which is the indication of oxidative stress in C. sativum seedlings and was also manifested by hydrogen peroxide (H2O2) contents and electrolyte leakage (EL) to the membrane bounded organelles. Although, activities of various antioxidative enzymes like superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), non-enzymatic antioxidants like phenolic, flavonoid, ascorbic acid and anthocyanin contents and organic acids from the roots such as oxalic acid, malic acid, formic acid, citric acid, acetic acid and fumaric acid contents were increased with the increasing levels of B in the soil. The application if NPK and GA3 mitigated B toxicity by stimulated plant growth and biomass, photosynthetic efficiency, nutritional status and antioxidant machinery of the plant by decreasing MDA contents, H2O2 initiation and EL (%) in the roots and leaves of C. sativum seedlings. In addition, the application of NPK and GA3 further decreased the organic acids exudation contents in the roots C. sativum seedlings. Research findings, therefore, suggested that NPK and GA3 application can ameliorate B toxicity in C. sativum seedlings and resulted in improved plant growth and composition under B stress as depicted by balanced contents of organic acids.

Adenovirus Armed With TNFa and IL2 Added to aPD-1 Regimen Mediates Antitumor Efficacy in Tumors Refractory to aPD-1.

Immune checkpoint inhibitors such as anti-PD-1 have revolutionized the field of oncology over the past decade. Nevertheless, the majority of patients do not benefit from them. Virotherapy is a flexible tool that can be used to stimulate and/or recruit different immune populations. T-cell enabling virotherapy could enhance the efficacy of immune checkpoint inhibitors, even in tumors resistant to these inhibitors. The T-cell potentiating virotherapy used here consisted of adenoviruses engineered to express tumor necrosis factor alpha and interleukin-2 in the tumor microenvironment. To study virus efficacy in checkpoint-inhibitor resistant tumors, we developed an anti-PD-1 resistant melanoma model in vivo. In resistant tumors, adding virotherapy to an anti-PD-1 regimen resulted in increased survival (p=0.0009), when compared to anti-PD-1 monotherapy. Some of the animals receiving virotherapy displayed complete responses, which did not occur in the immune checkpoint-inhibitor monotherapy group. When adenoviruses were delivered into resistant tumors, there were signs of increased CD8 T-cell infiltration and activation, which - together with a reduced presence of M2 macrophages and myeloid-derived suppressor cells - could explain those results. T-cell enabling virotherapy appeared as a valuable tool to counter resistance to immune checkpoint inhibitors. The clinical translation of this approach could increase the number of cancer patients benefiting from immunotherapies.

Oncolytic Adenovirus Coding for a Variant Interleukin 2 (vIL-2) Cytokine Re-Programs the Tumor Microenvironment and Confers Enhanced Tumor Control.

The notion of developing variants of the classic interleukin 2 (IL-2) cytokine has emerged from the limitations observed with the systemic use of human IL-2 in the clinic: severe adverse events accompanied by low therapeutic response rate in treated patients. Modifications made in the IL-2 receptor-binding structure leads to preferential binding of IL-2 variant cytokine to receptors on effector anti-tumor lymphocytes over T regulatory (TReg) cells. Because of their inherent immunogenicity, oncolytic adenoviruses are useful for expression of immunomodulatory molecules in tumors, for induction of a pro-inflammatory state in the tumor microenvironment. In the present study, we constructed an adenovirus coding for an IL-2 variant (vIL-2) protein, Ad5/3-E2F-d24-vIL2. Functionality of the new virus was tested in vitro, and anti-tumor efficacy and mechanism of action studies were performed in immunocompetent hamsters bearing pancreatic tumors. Ad5/3-E2F-d24-vIL2 treatment elicited efficient anti-tumor response, with 62.5% monotherapy complete response. Moreover, it promoted substantial repression of genes associated with myeloid cells mediated immunosuppression (CD11b, ARG1, CD206). This was seen in conjunction with upregulation of genes associated with tumor-infiltrating lymphocyte (TIL) cytotoxicity (CD3G, SAP, PRF1, GZMM and GZMK). In summary, Ad5/3-E2F-d24-vIL2 demonstrates therapeutic potential by counteracting immunosuppression and in efficiently coordinating lymphocytes mediated anti-tumor response in immunosuppressive tumors. Thus, Ad5/3-E2F-d24-vIL2 is a promising candidate for translation into clinical trials in human immunosuppressive solid tumors.

Systemic Delivery of Oncolytic Adenovirus to Tumors Using Tumor-Infiltrating Lymphocytes as Carriers.

Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.

Kidney Transplantation and COVID-19: Two Case Reports.

Immunocompromised populations are at great risk of the current 2020 global emergency of coronavirus disease 2019 (COVID-19), and treatment of kidney transplant recipients with COVID-19 is currently not declared. Hence, the purpose of the study is to set a clear treatment regimen. We report here a therapeutic course of 2 patients who underwent transplant surgery in March 2020 and got infected soon after. Since the transplant, these 2 patients have received triple maintenance immunosuppressive therapy with oral tacrolimus, mycophenolate mofetil (MMF), and prednisone, and they have been regularly followed up at our hospital. The tacrolimus trough level was between 10 and 12 ng/mL. After the diagnosis of COVID-19, MMF was stopped and the tacrolimus dose was reduced so that blood level was between 4 and 6 ng/mL. The first patient was a 30-year-old man who, despite being treated with hydroxychloroquine, favipiravir, oseltamivir, and azithromycin therapy, died because of the presence of other comorbidities. The second case was a 58-year-old man who fully recovered from COVID-19 pneumonia with treatment with methylprednisolone, MMF, azithromycin, favipiravir, hydroxychloroquine, and reduction in immunosuppression dosage. This reflects the importance of using glucocorticoids in the treatment of COVID-19 along with other medications and the decreased mortality rate associated with their use.

Ad5/3 is able to avoid neutralization by binding to erythrocytes and lymphocytes.

Oncolytic adenoviruses are promising cancer therapeutic agents. Clinical data have shown adenoviruses' ability to transduce tumors after systemic delivery in human cancer patients, despite antibodies. In the present work, we have focused on the interaction of a chimeric adenovirus Ad5/3 with human lymphocytes and human erythrocytes. Ad5/3 binding with human lymphocytes and erythrocytes was observed to occur in a reversible manner, which allowed viral transduction of tumors, and oncolytic potency of Ad5/3 in vitro and in vivo, with or without neutralizing antibodies. Immunodeficient mice bearing xenograft tumors showed enhanced tumor transduction following systemic administration, when Ad5/3 virus was bound to lymphocytes or erythrocytes (P < 0.05). In conclusion, our findings reveal that chimeric Ad5/3 adenovirus reaches non-injected tumors in the presence of neutralizing antibodies: it occurs through reversible binding to lymphocytes and erythrocytes.