Prothymosin α and its C-Terminal Immunoreactive Decapeptide Show No Evidence of Acute Toxicity: A Preliminary In Silico, In Vitro and In Vivo Investigation.

BACKGROUND: Members of the α-thymosin family have long been studied for their immunostimulating properties. Among them, the danger-associated molecular patterns (DAMPs) prothymosin α (proTα) and its C-terminal decapeptide proTα(100-109) have been shown to act as immunomodulators in vitro, due to their ability to promote T helper type 1 (Th1) responses. Recently, we verified these findings in vivo, showing that both proTα and proTα(100-109) enhance antitumor-reactive T cell-mediated responses. METHODS: In view of the eventual use of proTα and proTα(100-109) in humans, we investigated their safety profile in silico, in human leukocytes and cancer cell lines in vitro, and in immunocompetent mice in vivo, in comparison to the proTα derivative thymosin alpha 1 (Τα1), a 28-mer peptide extensively studied for its safety in clinical trials. RESULTS: In silico prediction via computational tools showed that all three peptide sequences likely are non-toxic or do not induce allergic regions. In vitro, pro- Tα, proTα(100-109) and Tα1 did not affect the viability of human cancer cell lines and healthy donor-derived leukocytes, did not promote apoptosis or alter cell cycle distribution. Furthermore, mice injected with proTα, proTα(100-109) and Tα1 at doses equivalent to the suggested dose regimen of Tα1 in humans, did not show signs of acute toxicity, whereas proTα and proTα(100-109) increased the levels of proinflammatory and Th1- type cytokines in their peripheral blood. CONCLUSION: Our preliminary findings suggest that proTα and proTα(100-109), even at high concentrations, are non-toxic in vitro and in an acute toxicity model in vivo; moreover, we show that the two peptides retain their immunomodulatory properties in vivo and, eventually, could be considered for therapeutic use in humans.

4th Summer School in Immuno-Oncology, July 1st-3rd, 2021, Athens, Greece.

The 4th Summer School in Immuno-Oncology was held from July 1st-July 3rd as a web meeting. Many eminent researchers and leading oncologists from Europe and the USA working on basic, translational and clinical cancer research participated, presented, and discussed the most recent advances in cancer immunology and immunotherapy. Besides sharing the newest information in the field of cancer immunology and immunotherapy, the meeting also focused on the actual translation of new knowledge acquired in the lab to the clinical setting; particular emphasis was given to the mode of action of novel therapeutic modalities and to biomarkers helpful for treatment decision-making, as well as to means that may improve cancer immunotherapeutic protocols used for the treatment of a variety of malignancies. The main topics presented by the speakers included: (1) mechanisms of tumor immune evasion and resistance; (2) host-tumor interactions and means to regulate antitumor immunity; (3) exploitation of new biomarkers and tumor or immune signatures able to potentially guide therapeutic interventions; (4) emerging therapeutic modalities for cancer treatment and specific immunotherapeutics for thoracic, genito-urinary, gastrointestinal, skin and breast cancers; and (5) innovative treatment options and alternatives to minimize the toxic adverse events of cancer immunotherapy.

Aberrant Plasma Cell Contamination of Peripheral Blood Stem Cell Autografts, Assessed by Next-Generation Flow Cytometry, Is a Negative Predictor for Deep Response Post Autologous Transplantation in Multiple Myeloma; A Prospective Study in 199 Patients.

High-dose chemotherapy with autologous stem cell support (ASCT) is the standard of care for eligible newly diagnosed Multiple Myeloma (MM) patients. Stem cell graft contamination by aberrant plasma cells (APCs) has been considered a possible predictive marker of subsequent clinical outcome, but the limited reports to date present unclear conclusions. We prospectively estimated the frequency of graft contamination using highly sensitive next-generation flow cytometry and evaluated its clinical impact in 199 myeloma patients who underwent an ASCT. Contamination (con+) was detected in 79/199 patients at a median level 2 × 10-5. Its presence and levels were correlated with response to induction treatment, with 94%, 71% and 43% achieving CR, VGPR and PR, respectively. Importantly, con+ grafts conferred 2-fold and 2.8-fold higher patient-risk of not achieving or delaying reaching CR (4 vs. 11 months) and MRD negativity (5 vs. 18 months) post ASCT, respectively. Our data also provide evidence of a potentially skewed bone marrow (BM) reconstitution due to unpurged grafts, since con+ derived BM had significantly higher prevalence of memory B cells. These data, together with the absence of significant associations with baseline clinical features, highlight graft contamination as a potential biomarker with independent prognostic value for deeper responses, including MRD negativity. Longer follow-up will reveal if this corresponds to PFS or OS advantage.

Wnt and Src signals converge on YAP-TEAD to drive intestinal regeneration.

Wnt signalling induces a gradient of stem/progenitor cell proliferation along the crypt-villus axis of the intestine, which becomes expanded during intestinal regeneration or tumour formation. The YAP transcriptional co-activator is known to be required for intestinal regeneration, but its mode of regulation remains controversial. Here we show that the YAP-TEAD transcription factor is a key downstream effector of Wnt signalling in the intestine. Loss of YAP activity by Yap/Taz conditional knockout results in sensitivity of crypt stem cells to apoptosis and reduced cell proliferation during regeneration. Gain of YAP activity by Lats1/2 conditional knockout is sufficient to drive a crypt hyperproliferation response. In particular, Wnt signalling acts transcriptionally to induce YAP and TEAD1/2/4 expression. YAP normally localises to the nucleus only in crypt base stem cells, but becomes nuclear in most intestinal epithelial cells during intestinal regeneration after irradiation, or during organoid growth, in a Src family kinase-dependent manner. YAP-driven crypt expansion during regeneration involves an elongation and flattening of the Wnt signalling gradient. Thus, Wnt and Src-YAP signals cooperate to drive intestinal regeneration.

Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy.

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Engineering transplantable jejunal mucosal grafts using patient-derived organoids from children with intestinal failure.

Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.

Hallmarks of intestinal stem cells.

Intestinal stem cells (ISCs) are highly proliferative cells that fuel the continuous renewal of the intestinal epithelium. Understanding their regulatory mechanisms during tissue homeostasis is key to delineating their roles in development and regeneration, as well as diseases such as bowel cancer and inflammatory bowel disease. Previous studies of ISCs focused mainly on the position of these cells along the intestinal crypt and their capacity for multipotency. However, evidence increasingly suggests that ISCs also exist in distinct cellular states, which can be an acquired rather than a hardwired intrinsic property. In this Review, we summarise the recent findings into how ISC identity can be defined by proliferation state, signalling crosstalk, epigenetics and metabolism, and propose an update on the hallmarks of ISCs. We further discuss how these properties contribute to intestinal development and the dynamics of injury-induced regeneration.

The Transcription Co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation Into Enterocyte vs Secretory Lineages.

BACKGROUND & AIMS: Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established. METHODS: We sorted cells from Lgr5-GFP knockin intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription polymerase chain reaction and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing (RNA-seq). We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16. RESULTS: The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt-villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode Delta-like canonical Notch ligand and other secretory-specific transcription factors. CONCLUSIONS: In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1.

MYB - A regulatory factor in hematopoiesis.

MYB is a transcription factor which was identified in birds as a viral oncogene (v-MYB). Its cellular counterpart was subsequently isolated as c-MYB which has three functional domains - DNA binding domain, transactivation domain and negative regulatory domain. c-MYB is essential for survival, and deletion of both alleles of the gene results in embryonic death. It is highly expressed in hematopoietic cells, thymus and neural tissue, and required for T and B lymphocyte development and erythroid maturation. Additionally, aberrant MYB expression has been found in numerous solid cancer cells and human leukemia. Recent studies have also implicated c-MYB in the regulation of expression of fetal hemoglobin which is highly beneficial to the ß-hemoglobinopathies (beta thalassemia and sickle cell disease). These findings suggest that MYB could be a potential therapeutic target in leukemia, and possibly also a target for therapeutic increase of fetal hemoglobin in the ß-hemoglobinopathies.

g(HbF): a genetic model of fetal hemoglobin in sickle cell disease.

Fetal hemoglobin (HbF) is a strong modifier of sickle cell disease (SCD) severity and is associated with 3 common genetic loci. Quantifying the genetic effects of the 3 loci would specifically address the benefits of HbF increases in patients. Here, we have applied statistical methods using the most representative variants: rs1427407 and rs6545816 in BCL11A, rs66650371 (3-bp deletion) and rs9376090 in HMIP-2A, rs9494142 and rs9494145 in HMIP-2B, and rs7482144 (Xmn1-HBG2 in the ß-globin locus) to create g(HbF), a genetic quantitative variable for HbF in SCD. Only patients aged ≥5 years with complete genotype and HbF data were studied. Five hundred eighty-one patients with hemoglobin SS (HbSS) or HbSß0 thalassemia formed the "discovery" cohort. Multiple linear regression modeling rationalized the 7 variants down to 4 markers (rs6545816, rs1427407, rs66650371, and rs7482144) each independently contributing HbF-boosting alleles, together accounting for 21.8% of HbF variability (r2) in the HbSS or HbSß0 patients. The model was replicated with consistent r2 in 2 different cohorts: 27.5% in HbSC patients (N = 186) and 23% in 994 Tanzanian HbSS patients. g(HbF), our 4-variant model, provides a robust approach to account for the genetic component of HbF in SCD and is of potential utility in sickle genetic and clinical studies.

Weekly Personal Ozone Exposure and Respiratory Health in a Panel of Greek Schoolchildren.

BACKGROUND: The association of ozone exposure with respiratory outcomes has been investigated in epidemiologic studies mainly including asthmatic children. The findings reported had methodological gaps and inconsistencies. OBJECTIVES: We aimed to investigate effects of personal ozone exposure on various respiratory outcomes in school-age children generally representative of the population during their normal activities. METHODS: We conducted a panel study in a representative sample of school-age children in the two major cities of Greece, Athens and Thessaloniki. We followed 188, 10- to 11-y-old, elementary school students for 5 wk spread throughout the 2013­2014 academic year, during which ozone was measured using personal samplers. At the end of each study week, spirometry was performed by trained physicians, and the fractional concentration of nitric oxide in exhaled air (FeNO) was measured. Students kept a daily time­activity­symptom diary and measured PEF (peak expiratory flow) using peak flow meters. Mixed models accounting for repeated measurements were applied. RESULTS: An increase of 10 µg/m3 in weekly ozone concentration was associated with a decrease in FVC (forced vital capacity) and FEV1 (forced expiratory volume in 1 s) of 0.03 L [95% confidence interval (CI): −0.05, −0.01] and 0.01 L (95% CI: −0.03, 0.003) respectively. The same increase in exposure was associated with a 11.10% (95% CI: 4.23, 18.43) increase in FeNO and 19% (95% CI: −0.53, 42.75) increase in days with any symptom. The effect estimates were robust to PM10 adjustment. No inverse association was found between ozone exposure and PEF. CONCLUSIONS: The study provides evidence that airway inflammation and the frequency of respiratory symptoms increase, whereas lung function decreases with increased ozone exposure in schoolchildren. https://doi.org/10.1289/EHP635.

Effect of Ambient Ozone Exposure Assessed by Individual Monitors on Nasal Function and Exhaled NO Among School Children in the Area of Thessaloniki, Greece.

OBJECTIVES: The study of short-term effects of environmental ozone exposure on nasal airflow, lung function, and airway inflammation. METHODS: Ninety one children-47 underwent rhinomanometry-were included. The study was carried out during the 2013 to 2014 academic year. Activity questionnaires and personal O3 samplers were distributed and 1 week later, respiratory measurements were performed. Daily measurements of outdoor ozone were also considered. RESULTS: A 10 µg/m increase in weekly personal ozone exposure concentrations was associated with a non-statistically significant 12.7% decrease in nasal inspiratory airflow (29.4% during the high ozone period). When the outdoor exposure of the same and the previous day were taken into account the corresponding figures were 13.48% and 43.58% (P = 0.02). CONCLUSIONS: There is an indication for increased risk of nasal obstruction during exposure to high ozone.

Pneumothorax and asthma.

This review is focused on the relationship between asthma, pneumothorax and pneumomediastinum while presenting a number of case reports that include these conditions. The association between pneumothorax and asthma is not widely known. While asthma includes a common disorder and is prevalent worldwide, its morbidity and mortality is high when is associated with pneumothorax. Furthermore, the delayed diagnosis of pneumothorax while focusing on asthma includes the higher risk of coincidental pneumothorax in asthmatic patients. In addition, pneumomediastinum is considered benign and self-limiting condition that responds to conservative therapy. Although it is rare, the concurrence of pneumomediastinum with pneumothorax may prove fatal during a serious asthma attack. In conclusion, the symptoms of chest pain, dyspnea or focal chest findings when presented in asthmatic patients, must always create suspicion of pneumothorax or pneumomediastinum to the physician.

Preoperative evaluation for lung cancer resection.

During the last decades lung cancer is the leading cause of death worldwide for both sexes. Even though cigarette smoking has been proved to be the main causative factor, many other agents (e.g., occupational exposure to asbestos or heavy metals, indoor exposure to radon gas radiation, particulate air pollution) have been associated with its development. Recently screening programs proved to reduce mortality among heavy-smokers although establishment of such strategies in everyday clinical practice is much more difficult and unknown if it is cost effective compared to other neoplasms (e.g., breast or prostate cancer). Adding severe comorbidities (coronary heart disease, COPD) to the above reasons as cigarette smoking is a common causative factor, we could explain the low surgical resection rates (approximately 20-30%) for lung cancer patients. Three clinical guidelines reports of different associations have been published (American College of Chest Physisians, British Thoracic Society and European Respiratory Society/European Society of Thoracic Surgery) providing detailed algorithms for preoperative assessment. In the current mini review, we will comment on the preoperative evaluation of lung cancer patients.

Airway inflammation in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is an inflammatory airway disease whose incidence and mortality increases every year. It is associated with an abnormal inflammatory response of the lung to toxic particles or gases (usually cigarette smoke). A central role in the pathophysiology has been shown to play a chronic inflammation of the airways that is expressed primarily by hypersecretion of mucus, stenosis of the smaller airways and the establishment of pulmonary emphysema. There is an increasing trend for assessing the inflammatory pattern of inflammatory airway diseases through mediators measured by noninvasive techniques. Markers in biological fluids and exhaled air have been the subject of intense evaluation over the past few years, with some of them reaching their introduction into clinical practice, while others remain as research tools. Of particular interest for the scientific community is the discovery of clinically exploitable biomarkers associated with specific phenotypes of the disease. Studying the effects of therapeutic interventions in these biomarkers may lead to targeted therapy based on phenotype and this is perhaps the future of therapeutics in COPD.

Lady Windermere syndrome after cardiac surgery procedure: a case of Mycobacterium avium complex pneumonia.

We present a patient with Lady Windermere syndrome after coronary bypass operation. To avoid the sternal pain that occurred after every cough episode, this patient used to receive large doses of antitussive drugs. In a poorly drained lung, this usage leads to the development of regions of colonization with Mycobacterium avium complex. It is concerning that the lack of diagnosis for 18 months in this patient occurred because of the insidious symptoms that mimic bronchitis. This case emphasizes how important it is for patients after a median sternotomy to receive adequate analgesic and physiotherapeutic support in order to avoid chronic inflammation and infection of the lungs.