The safety and potential efficacy of exosomes overexpressing CD24 (EXO-CD24) in mild-moderate COVID-19 related ARDS.

INTRODUCTION: EXO-CD24 are exosomes genetically manipulated to over-express Cluster of Differentiation (CD) 24. It consists of two breakthrough technologies: CD24, the drug, as a novel immunomodulator that is smarter than steroids without any side effects, and exosomes as the ideal natural drug carrier. METHODS: A randomized, single blind, dose-finding phase IIb trial in hospitalized patients with mild to moderate Coronavirus disease 2019 (COVID-19) related Acute Respiratory Distress Syndrome (ARDS) was carried out in two medical centers in Athens. Patients received either 109 or 1010 exosome particles of EXO-CD24, daily, for five consecutive days and monitored for 28 days. Efficacy was assessed at day 7 among 91 patients who underwent randomization. The outcome was also compared in a post-hoc analysis with an income control group (n = 202) that fit the inclusion and exclusion criteria. RESULTS: The mean age was 49.4 (± 13.2) years and 74.4% were male. By day 7, 83.7% showed improved respiratory signs and 64% had better oxygen saturation (SpO2) (p < 0.05). There were significant reductions in all inflammatory markers, most notably in C-reactive protein (CRP), lactate dehydrogenase (LDH), ferritin, fibrinogen and an array of cytokines. Conversely, levels of the anti-inflammatory cytokine Interleukin-10 (IL-10) were increased (p < 0.05). Of all the documented adverse events, none were considered treatment related. No drug-drug interactions were noted. Two patients succumbed to COVID-19. Post-hoc analysis revealed that EXO-CD24 patients exhibited greater improvements in clinical and laboratory outcomes compared to an observational income control group. CONCLUSIONS: EXO-CD24 presents a promising therapeutic approach for hyper-inflammatory state and in particular ARDS. Its unique combination of exosomes, as a drug carrier, and CD24, as an immunomodulator, coupled with inhalation administration, warrants further investigation in a larger, international, randomized, quadri-blind trial against a placebo.

Inhaled CD24-Enriched Exosomes (EXO-CD24) as a Novel Immune Modulator in Respiratory Disease.

Acute Respiratory Distress Syndrome (ARDS) is a major health concern with urgent unmet need for treatment options. There are three million new ARDS cases annually, and the disease's mortality rate is high (35-46%). Cluster of differentiation 24 (CD24), a long-known protein with multifaceted functions, is a small, heavily glycosylated, membrane-anchored protein which functions as an immune checkpoint control. CD24 allows for immune discrimination between Damage-Associated Molecular Patterns and Pathogen-Associated Molecular Patterns derived from pathogens. Exosomes are intraluminal vesicles which play an important role in intercellular communication. Exosomes offer the advantage of targeted delivery, which improves safety and efficacy. The safety and efficacy of EXO-CD24 is promising, as was shown in >180 ARDS patients in phase 1b/2a, phase 2b, and compassionate use. CD24 binds Damage-associated molecular patterns (DAMPs) and inhibits the activation of the NF-ĸB pathway, a pivotal mediator of inflammatory responses. In contrast to anti-inflammatory therapies that are cytokine-specific or steroids that shut down the entire immune system, EXO-CD24 acts upstream, reverting the immune system back to normal activity. Herein, the safety and efficacy of mEXO-CD24 is shown in murine models of several pulmonary diseases (sepsis, allergic asthma, Chronic Obstructive Pulmonary Disease(COPD), fibrosis). EXO CD24 can suppress the hyperinflammatory response in the lungs in several pulmonary diseases with a significant unmet need for treatment options.

The APC I1307K allele conveys a significant increased risk for cancer.

This study is the first attempt to evaluate the association between the APC I1307K variant and overall cancer risk. It is unique in both its large sample size and in the reliability of data in the control group. The findings described in this article have major implications in terms of identifying asymptomatic individuals who are at increased risk to harbor cancer and therefore targeted to be enrolled in specific early detection and prevention programs. The prevalence of the APC I1307K missense mutation among Ashkenazi Jews is ∼ 6%. Carriers are at an increased risk for colorectal neoplasia. In this study, we examined the association of this variant with non-colorectal cancers. Consecutive 13,013 healthy subjects who underwent screening at the Integrated Cancer Prevention Center between 2006 and 2014 were enrolled. This population was supplemented with 1,611 cancer patients from the same institution. Demographics, medical history, and pathological data were recorded. Mortality data were obtained from the Ministry of Health's registry. The prevalence of APC I1307K in cancer patients and healthy subjects was compared. The APC I1307K variant was detected in 189 (11.8%) cancer patients compared to 614 (4.7%) healthy subjects, reflecting an adjusted age and sex odds ratio (OR) of 2.53 (p < 0.0001). History of two or more cancer types was associated with a positive carrier prevalence (OR = 4.38 p < 0.0001). Males had significantly increased carrier prevalence in lung, urologic, pancreatic, and skin cancers. The carrier prevalence among females was significantly higher only in breast and skin cancers. Female carriers developed cancer at a significantly older age compared to non-carriers (average 62.7 years vs. 57.8, respectively, p = 0.027), had better survival rates (HR = 0.58, p = 0.022) and overall increased longevity (average age of death 78.8 vs. 70.4 years, respectively, p = 0.003). In conclusion, the APC I1307K variant is a reliable marker for overall cancer risk (OR 2.53). Further studies are needed to evaluate its use for specific cancer types-particularly in males. Female carriers have better prognosis and increased lifespan.

CD24 knockout prevents colorectal cancer in chemically induced colon carcinogenesis and in APC(Min)/CD24 double knockout transgenic mice.

Increased expression of CD24 is seen in a large variety of solid tumors, including up to 90% of gastrointestinal (GI) tumors. Stable derivatives of SW480 colorectal cancer (CRC) cells that overexpress CD24 proliferate faster, and increase cell motility, saturation density, plating efficiency, and growth in soft agar. They also produce larger tumors in nude mice as compared to the parental SW480 cells. Most significantly, even depletion of one copy of the CD24 allele in the APC(Min/+) mice of a transgenic mouse model led to a dramatic reduction in tumor burden in all sections of the small intestine. Homozygous deletion of both CD24 alleles resulted in complete abolishment of tumor formation. Moreover, CD24 knockout mice exhibited resistance to chemically induced inflammation-associated CRC. Finally, a new signal transduction pathway is suggested: namely, CD24 expression downstream to COX2 and PGE2 synthesis, which is directly regulated by ß-catenin. CD24 is shown in vitro and in vivo as being an important oncogene in the gut, and one that plays a critical role in the initiation and progression of carcinogenesis.

Aspirin but not meloxicam attenuates early atherosclerosis in apolipoprotein E knockout mice.

BACKGROUND: Atherosclerosis is a complex vascular inflammatory disease. In the last decade it was suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) and in particular inhibition of cyclooxygenase (COX)-2 are associated with an increase in cardiovascular morbidity and mortality. Aspirin is known to reduce the incidence and mortality from ischemic heart disease and is a mainstay in the prevention of vascular complications of atherosclerosis. OBJECTIVES: To examine the effect of meloxicam, a selective COX-2 inhibitor, or low dose aspirin on the development of experimental atherosclerosis in apoE knockout (KO) compared to wild-type (WT) mice. We aimed to test the hypothesis that meloxicam, a potential vasculitis inducer, would exacerbate atherosclerotic lesions while aspirin, which is known to reduce the incidence of thrombosis occlusive events, would increase protection in this model. METHODS: We randomly divided 36 male apoE KO and 36 WT mice, 8 weeks old. Mice were treated for 10 weeks with 0.1 mg/ml aspirin, or 0.05 mg/ml meloxicam, dissolved in their drinking water. Control groups received regular drinking water. At sacrifice, the hearts were removed for histochemical staining and plaque size and composition were examined. RESULTS: Aspirin-treated animals displayed a decreased atherosclerotic lesion area compared to the untreated control mice, while meloxicam had a null effect on the extent of atherosclerosis in Apo E KO mice. CONCLUSIONS: These results suggest that low dose aspirin reduces early atherosclerosis, while inhibition of COX-2 by meloxicam is not associated with an increase in atherosclerotic plaque size in this mouse model.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer.

BACKGROUND: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. RESULTS: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our "gene therapy" approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~35% tumor progression in vivo in already established tumors. CONCLUSIONS: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

CD24 in Head and Neck Malignancies-An Uprising Biomarker?

INTRODUCTION: CD24 is often overexpressed in human tumors as a regulator of cell migration, invasion and proliferation. It has been associated with poor prognosis and chemoresistance in laryngeal cancer. In oral cavity tumors, it was correlated with better overall survival. In this study, we aimed to evaluate the role of CD24 in peripheral blood leukocytes (PBLs) as a potential marker for head and neck malignancies. MATERIALS AND METHODS: CD24/CD11b expression in peripheral blood leukocytes (PBLs) of head and neck cancer patients and matched healthy controls was analyzed via flow cytometry. Tumors and healthy tissues were immune-stained for CD24 expression and the intensity of stain was ranked. Clinical data including tumor site, size, locoregional or metastatic spread, histopathological characteristics and recurrence events were analyzed. RESULTS: CD24 expression in PBLs was significantly higher in a cohort of 101 head and neck cancer patients compared with 101 matched healthy controls (26.9 ± 12.9 vs. 22.4 ± 13.8; p = 0.02). No significant differences in CD24 levels in PBLs were found between different head and neck subsites involved with malignancy. Higher CD24 levels did not correlate with any adverse feature, i.e., perineural invasion or lymphovascular invasion, advanced T stage or regional spread. Immunohistochemistry analysis demonstrated that CD24 was highly expressed in tumor tissue in comparison to healthy surrounding tissue. CONCLUSIONS: CD24 is a possible uprising marker for tumor identification, overexpressed in PBLs and is intensely stained in tumor tissue and pre-malignant lesions. Tumor-PBLs should be further studied.

Renal Tubular CD24 Upregulation Aggravates Folic Acid Induced Acute Kidney Injury: A Possible Role for T Regulatory Cells Inhibition in Mice.

Acute kidney injury (AKI) is characterized by cell death and inflammation. CD24 is a protein induced during tissue damage and is not expressed in mature renal tissue. We explored the role of CD24 in the pathogenesis of folic acid-induced AKI (FA-AKI) in mice. A single Intraperitoneal (IP) injection of folic acid induced AKI in WT and CD24-/- mice. Renal function tests, histological analysis, immunohistochemistry, Western blot analysis, and ELISA were performed to assess the severity of renal damage and the intensity of the inflammatory response. FA-AKI induced CD24 in the distal tubular epithelial cells. Compared to WT mice, FA-AKI CD24-/- mice exhibited an attenuated reduction in renal function and histological injury, lower serum IL-10 and interferon γ, and decreased expression of renal TNFα. In contrast, renal and systemic IL-33 upregulation were augmented. CD24-/- FA-AKI animals exhibited increased splenic margination and renal infiltration of regulatory T cells (Tregs). At day 7, FA-AKI CD24-/- mice exhibited increased expression of tubular pro-apoptotic and decreased anti-apoptotic proteins compared to WT animals. Anti-CD24 antibody administration to FA-AKI mice attenuated the decrease in renal function as well as the histological injury. Renal biopsies from patients with ATN stained strongly for CD24 in the distal tubules. In conclusion, during AKI, upregulation of CD24 promotes renal inflammation through inhibition of Treg infiltration and diversion of cell death towards necrosis rather than apoptosis. Neutralization of CD24 may prove a target for future therapies in AKI.

Inhaled Exosomes Genetically Manipulated to Overexpress CD24 (EXO-CD24) as a Compassionate Use in Severe ARDS Patients.

RATIONALE: Acute respiratory distress syndrome (ARDS) is a major global health concern with a significant unmet need. EXO-CD24 is delivered via inhalation-reduced cytokines and chemokine secretion and lung injury in ARDS and improved survival in mice models of ARDS, influenza, and sepsis. OBJECTIVES: This clinical paper aims to evaluate the potential of EXO-CD24, a novel immunomodulatory treatment, in the compassionate care of critically ill, intubated patients with post-infection-induced acute respiratory distress syndrome (ARDS). METHODS: Eleven critically ill patients diagnosed with post-infection ARDS (10 with COVID-19 and one with an adenovirus-associated infection) were administered EXO-CD24 in four medical centers across Israel. The patients had multiple co-morbidities, including cancer, hypertension, diabetes, and ischemic heart disease, and met the criteria for severe ARDS according to the Berlin classification. EXO-CD24 was administered via inhalation, and adverse events related to its use were carefully monitored. MEASUREMENTS AND MAIN RESULTS: The administration of EXO-CD24 did not result in any recorded adverse events. The median hospitalization duration was 11.5 days, and the overall mortality rate was 36%. Notably, patients treated at the Tel Aviv Medical Center (TASMC) showed a lower mortality rate of 12.5%. The WBC and CRP levels decreased in comparison to baseline levels at hospitalization, and rapid responses occurred even in patients with kidney transplants who were off the ventilator within a few days and discharged shortly thereafter. The production of cytokines and chemokines was significantly suppressed in all patients, including those who died. Among the patients at TASMC, four had kidney transplants and were on immunosuppressive drugs, and all of them fully recovered and were discharged from the hospital. CONCLUSIONS: EXO-CD24 holds promise as a potential therapeutic agent for all stages of ARDS, even in severe intubated cases. Importantly, EXO-CD24 demonstrated a favorable safety profile without any apparent side effects with promising efficacy. Furthermore, the potential of EXO-CD24 as a platform for addressing hyper-inflammatory states warrants exploration. Further research and larger-scale clinical trials are warranted to validate these preliminary findings.

Data From a One-Stop-Shop Comprehensive Cancer Screening Center.

PURPOSE: Cancer is the second leading cause of death globally. However, by implementing evidence-based prevention strategies, 30%-50% of cancers can be detected early with improved outcomes. At the integrated cancer prevention center (ICPC), we aimed to increase early detection by screening for multiple cancers during one visit. METHODS: Self-referred asymptomatic individuals, age 20-80 years, were included prospectively. Clinical, laboratory, and epidemiological data were obtained by multiple specialists, and further testing was obtained based on symptoms, family history, individual risk factors, and abnormalities identified during the visit. Follow-up recommendations and diagnoses were given as appropriate. RESULTS: Between January 1, 2006, and December 31, 2019, 8,618 men and 8,486 women, average age 47.11 ± 11.71 years, were screened. Of 259 cancers detected through the ICPC, 49 (19.8%) were stage 0, 113 (45.6%) stage I, 30 (12.1%) stage II, 25 (10.1%) stage III, and 31(12.5%) stage IV. Seventeen cancers were missed, six of which were within the scope of the ICPC. Compared with the Israeli registry, at the ICPC, less cancers were diagnosed at a metastatic stage for breast (none v 3.7%), lung (6.7% v 11.4%), colon (20.0% v 46.2%), prostate (5.6% v 10.5%), and cervical/uterine (none v 8.5%) cancers. When compared with the average stage of detection in the United States, detection was earlier for breast, lung, prostate, and female reproductive cancers. Patient satisfaction rate was 8.35 ± 1.85 (scale 1-10). CONCLUSION: We present a proof of concept study for a one-stop-shop approach to cancer screening in a multidisciplinary outpatient clinic. We successfully detected cancers at an early stage, which has the potential to reduce morbidity and mortality as well as offer substantial cost savings.[Media: see text].

The CD24 protein inducible expression system is an ideal tool to explore the potential of CD24 as an oncogene and a target for immunotherapy in vitro and in vivo.

CD24 is a cell surface, heavily glycosylated glycosylphosphatidylinositol-anchored mucin-like protein that is overexpressed in various human malignancies. To accurately analyze CD24 function and dissect its biological role in a defined genetic background, it is critical to tightly regulate its expression and be able to turn it on/off in a restricted environment and at a specific time. The tetracycline-induced expression system is most promising as it exhibits such regulation, lack of pleiotropic effects, and high and rapid induction levels. To evaluate the oncogenic and immunotherapeutic potential of CD24 by applying the Tet-On system, the human CD24 gene was cloned downstream to two tetracycline operator sequences, resulting in pCDNA4/TO-CD24, which was then transfected into tetracycline (Tet) repressor-expressing cells (293T-REx), allowing tight on/off regulation, thereby resulting in a very low background or leaky CD24 expression. Selected clones were chosen for further studies and characterized in vitro and in vivo, and several treatment modalities were examined. In addition, the role of CD24 in promoting cell proliferation and tumor growth was studied. The tetracycline-dependent system was successfully implemented. Tetracycline treatment induced CD24 expression in a dose- and time-dependent fashion, which was abrogated following treatment with anti-CD24 monoclonal antibodies (mAbs). CD24-induced expression led to an increased proliferation rate that was inhibited by mAb treatment. In vivo, significantly larger tumors were developed in tetracycline-fed mice. The CD24 Tet-On system is a good model to unravel the role and underlying CD24 pathogenesis in vivo. This valuable tool allows the successful study of novel treatment options, whose effectiveness depends on the CD24 expression level. This set of experiments supports CD24 oncogenic properties.

An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice.

BACKGROUND & AIMS: Effective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells. METHODS: We conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice. RESULTS: The immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice. CONCLUSIONS: Anti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

The Big Potential of Small Particles: Lipid-Based Nanoparticles and Exosomes in Vaccination.

Some of the most significant medical achievements in recent history are the development of distinct and effective vaccines, and the improvement of the efficacy of previously existing ones, which have contributed to the eradication of many dangerous and life-threatening diseases. Immunization depends on the generation of a physiological memory response and protection against infection. It is therefore crucial that antigens are delivered in an efficient manner, to elicit a robust immune response. The recent approval of COVID-19 vaccines containing lipid nanoparticles encapsulating mRNA demonstrates the broad potential of lipid-based delivery systems. In light of this, the present review article summarizes currently synthesized lipid-based nanoparticles such as liposomes, lipid-nano particles, or cell-derived exosomes.

Insights into CD24 and Exosome Physiology and Potential Role in View of Recent Advances in COVID-19 Therapeutics: A Narrative Review.

Cluster of differentiation (CD) 24, a long-known protein with multifaceted functions, has gained attention as a possible treatment for Coronavirus Disease 19 (COVID-19) due to its known anti-inflammatory action. Extracellular vesicles (EVs), such as exosomes and microvesicles, may serve as candidate drug delivery platforms for novel therapeutic approaches in COVID-19 and various other diseases due to their unique characteristics. In the current review, we describe the physiology of CD24 and EVs and try to elucidate their role, both independently and as a combination, in COVID-19 therapeutics. CD24 may act as an important immune regulator in diseases with complex physiologies characterized by excessive inflammation. Very recent data outline a possible therapeutic role not only in COVID-19 but also in other similar disease states, e.g., acute respiratory distress syndrome (ARDS) and sepsis where immune dysregulation plays a key pathophysiologic role. On the other hand, CD24, as well as other therapeutic molecules, can be administered with the use of exosomes, exploiting their unique characteristics to create a novel drug delivery platform as outlined in recent clinical efforts. The implications for human therapeutics in general are huge with regard to pharmacodynamics, pharmacokinetics, safety, and efficacy that will be further elucidated in future randomized controlled trials (RCTs).

A novel platform for attenuating immune hyperactivity using EXO-CD24 in COVID-19 and beyond.

A small but significant proportion of COVID-19 patients develop life-threatening cytokine storm. We have developed a new anti-inflammatory drug, EXO-CD24, a combination of an immune checkpoint (CD24) and a delivery platform (exosomes). CD24 inhibits the NF-kB pathway and the production of cytokines/chemokines. EXO-CD24 discriminates damage-from pathogen-associated molecular patterns (DAMPs and PAMPs) therefore does not interfere with viral clearance. EXO-CD24 was produced and purified from CD24-expressing 293-TREx™ cells. Exosomes displaying murine CD24 (mCD24) were also created. EXO-CD24/mCD24 were characterized and examined, for safety and efficacy, in vitro and in vivo. In a phase Ib/IIa study, 35 patients with moderate-high severity COVID-19 were recruited and given escalating doses, 108 -1010 , of EXO-CD24 by inhalation, QD, for 5 days. No adverse events related to the drug were observed up to 443-575 days. EXO-CD24 effectively reduced inflammatory markers and cytokine/chemokine, although randomized studies are required. EXO-CD24 may be a treatment strategy to suppress the hyper-inflammatory response in the lungs of COVID-19 patients and further serve as a therapeutic platform for other pulmonary and systemic diseases characterized by cytokine storm.

An improved molecular inversion probe based targeted sequencing approach for low variant allele frequency.

Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10-56.25% (P < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.

Gene expression following exposure to celecoxib in humans: pathways of inflammation and carcinogenesis are activated in tumors but not normal tissues.

BACKGROUND: The Cox-2 inhibitor, celecoxib (Pfizer Inc., N.Y., USA), is a promising chemopreventive agent [Arber et al.: N Engl J Med 2006;355:885-895; Bertagnolli et al.: N Engl J Med 2006;355:873-884]. This study aims to explore its mechanism by defining changes in gene expression between neoplastic and normal tissue samples before and after treatment. METHODS: Patients with documented colorectal neoplasia in screening colonoscopy, destined to undergo surgical colectomy, were randomized for treatment with celecoxib (n = 11; 400 mg/day) or placebo (n = 3) for 30 days. Tissue samples were taken from the tumor and from normal adjacent mucosa during both colonoscopy and surgery. RNA was extracted and analyzed using Affymetrix Genechip®. RESULTS: 687 genes differentiated tumor samples before and after treatment, among which 310 genes did not show the same differential expression in the placebo group or normal samples. These genes were significantly related to pathways of cell cycle regulation and inflammation, and of note was the TGF-ß pathway, which held a strong association with the list of genes formerly found to be associated with the colorectal cancer expression profile in microarray analyses, as summarized in a meta-analysis by Cardoso et al. [Biochim Biophys Acta 2007;1775:103-137]. CONCLUSIONS: Celecoxib selectively affects genes and pathways involved in inflammation and malignant transformation in tumor but not normal tissues, this may assist in the development of safer and more effective chemopreventive agents.

Behavioral Characterizing of CD24 Knockout Mouse-Cognitive and Emotional Alternations.

CD24 is a small, glycophosphatidylinositol-anchored cell surface protein, mostly investigated with respect to cancer, inflammation, and autoimmune diseases. CD24 knockdown or inhibition has been used to test various biochemical mechanisms and neurological conditions; however, the association between CD24 and behavioral phenotypes has not yet been examined. This study aims to characterize cognitive and emotional functions of CD24 knockout mice (CD24-/-) compared with CD24 wild-type mice at three time-points: adolescence, young adulthood, and adulthood. Our results show that CD24-/- mice exhibited better cognitive performance and less anxiety-like behavior compared with WT mice, with no effect on depression-like behavior. This phenotype was constant from childhood (2 months old) to adulthood (6 months old). The results from our study suggest that CD24 may influence important behavioral aspects at the whole-organism level, which should be taken into consideration when using CD24 knockout models.

High Expression Level of PPARγ in CD24 Knockout Mice and Gender-Specific Metabolic Changes: A Model of Insulin-Sensitive Obesity.

BACKGROUND: The heat-stable HSA/CD24 gene encodes a protein that shows high expression levels in adipocyte precursor cells but low levels in terminally differentiated adipocytes. Its high expression in many types of human cancer suggests an association between cancer, diabetes, and obesity, which is currently unclear. In addition, peroxisome proliferator-activated receptor gamma (PPARγ) is a regulator of adipogenesis that plays a role in insulin sensitivity, lipid metabolism, and adipokine expression in adipocytes. AIM: To assess gender-dependent changes in CD24 KO and its association with PPARγ expression. EXPERIMENTAL APPROACH: WT and CD24 KO mice were monitored from birth up to 12 months, and various physiological and molecular characteristics were analysed. Mean body weight and adipose mass were higher in KO mice than in WT mice. Male, but not female, KO mice showed increased insulin sensitivity, glucose uptake, adipocyte size, and PPARγ expression than WT mice. In addition, enteric bacterial populations, assessed through high-throughput sequencing of stool 16S rRNA genes, were significantly different between male KO and WT mice. CONCLUSIONS: CD24 may negatively regulate PPARγ expression in male mice. Furthermore, the association between the CD24 and insulin sensitivity suggests a possible mechanism for diabetes as a cancer risk factor. Finally, CD24 KO male mice may serve as a model of obesity and insulin hyper-sensitivity.

Innovative dual system approach for selective eradication of cancer cells using viral-based delivery of natural bacterial toxin-antitoxin system.

The inactivation of p53, a tumor suppressor, and the activation of the RAS oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin-antitoxin (TA) system can be used for selective and effective eradication of RAS-mutated cancer cells. This out of the box strategy holds great promise for effective cancer treatment and management. We provide proof of concept for a novel platform to selectively eradicate cancer cells using an adenoviral delivery system based on the adjusted natural bacterial system. We generated adenoviral vectors carrying the mazF toxin (pAdEasy-Py4-SV40mP-mCherry-MazF) and the antitoxin mazE (pAdEasy-RGC-SV40mP-MazE-IRES-GFP) under the regulation of RAS and p53, resp. The control vector carries the toxin without the RAS-responsive element (pAdEasy-ΔPy4-SV40mP-mCherry-MazF). In vitro, the mazF-mazE TA system (Py4-SV40mP-mCherry-MazF+RGC-SV40mP-MazE-IRES-GFP) induced massive, dose-dependent cell death, at 69% compared to 19% for the control vector, in a co-infected HCT116 cell line. In vivo, the system caused significant tumor growth inhibition of HCT116 (KRASmut/p53mut) tumors at 73 and 65% compared to PBS and ΔPY4 control groups, resp. In addition, we demonstrate 65% tumor growth inhibition in HCT116 (KRASmut/p53wt) cells, compared to the other two control groups, indicating a contribution of the antitoxin in blocking system leakage in WT RAS cells. These data provide evidence of the feasibility of using mutations in the p53 and RAS pathway to efficiently kill cancer cells. The platform, through its combination of the antitoxin (mazE) with the toxin (mazF), provides effective protection of normal cells from basal low activity or leakage of mazF.