Exploring the therapeutic potential of CAR-engineered T-cells targeting endothelial markers on tumour and inflamed vasculature

T-cells engineered to target tumour antigens through surface-expressed chimeric antigen receptors (CARs) are highly effective in treating some leukaemias. The challenge is to extend this success to solid tumours. Tumour endothelial marker 8 (TEM8) is a conserved transmembrane protein overexpressed on the vasculature of many solid tumours but low or undetectable on healthy tissues, making it a potential CAR T -cell target. This thesis explores the safety and therapeutic efficacy of this approach by generating five human TEM8-specific CARs, expressing them in T-lymphocytes, and characterising their functional responses to TEM8 in vitro. Four of the five CARs showed unexpected reactivity to control cells, and in mouse studies some of these proved toxic while most were selectively lost from the circulation, an effect that was TEM8-dependent. Only one CAR selectively responded to target cells overexpressing human TEM8 in vitro but was unable to recognise mouse TEM8, so further in vivo studies were not possible. These results highlight the sensitivity and potency of CAR -engineered T -cells and demonstrate the need for additional safety measures if targeting TEM8. The thesis also demonstrates that another TEM, CLEC14A, is overexpressed in some inflammatory liver diseases, and identifies a suitable mouse model for exploring the therapeutic potential ofCLEC14A-specific CAR-expressing regulatory T-cells

Hepatitis C transmission using lymphocytes as vectors: mechanism and therapeutic interventions and Neutrophil recruitment to microvascular endothelium: the impact of mesenchymal stem cells

Project 1: Hepatitis C virus (HCV) can be transmitted from B-lymphocytes to hepatocytes – a process termed trans-infection. This project characterised trans-infection further by examining receptors involved in HCV transmission from B- and T-lymphocytes to hepatoma cells. Both lymphocyte classes were equally capable of trans-infection, which was significantly reduced by HCV entry receptor inhibitors or neutralising antibodies. The actin cytoskeleton of the hepatocyte was also involved. Further work is needed to fully characterise HCV trans-infection, which would provide us with crucial insights into the varied modes of HCV transmission. Project 2: Neutrophil recruitment to inflamed tissue is essential in physiological and pathological inflammation. Mesenchymal stem cells (MSC) reduce neutrophil adhesion to inflamed macrovascular endothelium, but may become immunostimulatory in chronically inflamed tissue. This project characterised neutrophil recruitment to microvascular endothelium, examined whether MSC could suppress it, and studied MSC phenotype changes on exposure to pro-inflammatory cytokines. Micro- and macrovascular endothelium supported neutrophil adhesion equally well, but MSC suppression of neutrophil recruitment was not observed. Prolonged stimulation with transforming growth factor β in low serum altered MSC surface expression. Further studies of MSC phenotype changes in inflammation and their effect on endothelial neutrophil recruitment may shed light on the complex cellular interactions within chronically inflamed tissue

Vrste roda Diaporthe/Phomopsis na soji u Srbiji

A complex of soybean diseases is caused by species from the genus Diaporthe /Phomopsis. Diaporthe phaseolorum (anamorph Phomopsis phaseoli) and Phomopsis longicolla (teleomorph unknown) are described as soybean pathogens. The first species includes three varieties: D. phaseolorum var. sojae, anamorph Phomopsis sojae, the causal agent of pod and stem blight, and D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis, agents of northern and southern stem canker. The species P. longicolla is the most common and most damaging agent of soybean seed decay. The diseases caused by parasites from this genus were first observed and described on soybean in the USA. Presently they are widespread in most soybean-growing regions around the world. Soybean in Serbia is attacked by all pathogens mentioned above, except for D. phaseolorum var. meridionalis, the causal agent of the southern stem canker. D. phaseolorum var. caulivora (northern stem canker) has the greatest economic importance because it causes wilt and drying of plants during pod development and grain filling. Most intensive outbreaks of the disease occurred in the 1980s in southern and southeastern Banat, southern Bačka and Srem. Prematurely wilted plants yielded 50% to 62% (depending on variety) less than healthy plants. Such heavy losses raised the question of the profitability of soybean growing. P. sojae and P. longicolla are less important in Serbia at the moment. Intensive infections of soybean seed break out occasionally. It was found that P. longicolla is the primary agent of seed decay and latent infections of seed in our country, although the other members of this genus may cause identical symptoms. Sexual cycle of development (teleomorph stage) of the fungi of the genus Diaporthe /Phomopsis form perithecia with asci and ascospores, while in the asexual cycle (anamorph stage) various types of conidia (α and β) are formed in pycnidia. The species P. longicolla has no teleomorph stage. Infected harvest residues and soybean seeds are main sources of the inoculum. D. phaseolorum var. caulivora forms perithecia with ascospores on overwintered harvest residues. During growing season, ascospores infect leaf laminas, petioles, or injured plant parts. P. sojae and P. longicolla form pycnidia (P. sojae sometimes forms perithecia), and they infect plants by means of conidia. Seed infection by conidia or ascospores unfolds via pods. Infected crop residues are the main source of the inoculum, while infected seeds help the parasites spread over large distances. Humidity and temperature (soil and air) are the mains factors that affect the dynamics of fruiting body formation, spore release, establishment of infection and the development of disease symptoms on soybean plants. Considerable attention is devoted to finding effective sources of resistance to the disease complex caused by species from the genus Diaporthe/Phomopsis. Resistance to the agents of seed decay was found in several introduced (PI) genotypes. This resistance is controlled by one or two pairs of dominant genes. Some cultivars are resistant to D. phaseolorum var. meridionalis (southern stem canker). Their resistance is controlled by two or four pairs of genes (Rdc). There is no physiological resistance to D. phaseolorum var. caulivora, the agent of northern stem canker, but significant differences in susceptibility level exist among soybean genotypes. Molecular techniques (RAPD, PCR-RFLP) showed that significant variability was present within the population of D. phaseolorum var. sojae. Other species of fungi were suspected to exist within that population, which could not be distinguished on the basis of their morphological characteristics. This was subsequently confirmed by the sequencing of different regions of ribosomal DNA.Kompleks oboljenja na soji izazivaju gljive iz roda Diaporthe/Phomopsis. Kao paraziti soje opisana su tri varijeteta vrste Diaporthe phaseolorum (anamorf Phomopsis phaseoli), i to: D. phaseolorum var. sojae (syn. P. sojae), prouzrokovač paleži mahuna i stabla (pod and stem blight), zatim D. phaseolorum var. caulivora i D. phaseolorum var. meridionalis prouzrokovači „severnog“ i „južnog“ tipa raka stabla (soybean stem canker). Phomopsis longicolla (teleomorf nepoznat) je najčešći prouzrokovač truleži semena (soybean seed decay), mada ovo oboljenje izazivaju i sva tri varijeteta gljive D. phaseolorum. Svi pomenuti pripadnici roda, izuzev D. phaseolorum var. meridionalis, prisutni su na soji u Srbiji, a najveći značaj ima D. phaseolorum var. caulivora, prouzrokovač raka stabla. Poslednjih nekoliko decenija sprovedena su brojna, sveobuhvatna proučavanja parazita roda Diaporthe/ Phomopsis na soji u našoj zemlji. Nedavno započeta istraživanja na molekularnom nivou ukazuju da su pored pomenutih na soji prisutne i druge vrste ovog roda. U ovom radu su analizirani rezultati višegodišnjih istraživanja u svetu i kod nas

TEM8/ANTXR1-specific CAR T cells mediate toxicity in vivo

Engineering T-cells to express receptors specific for antigens present on tumour tissue is proving a highly effective treatment for some leukaemias. However, extending this to solid tumours requires antigens that can be safely and effectively targeted. TEM8, a marker overexpressed on the vasculature of some solid tumours, has been proposed as one such target. A recent report stated that T-cells engineered to express a TEM8-specific chimeric antigen receptor (CAR), when injected into mouse models of triple negative breast cancer, are both safe and effective in controlling tumour growth. Here we report contrasting data with a panel of TEM8-specific CAR-T-cells including one generated from the same antibody used in the other study. We found that the CAR-T-cells demonstrated clear TEM8-specific cytotoxic and cytokine release responses in vitro, but when injected into healthy C57BL6 and NSG mice they rapidly and selectively disappeared from the circulation and in most cases caused rapid toxicity. Infusing CAR-T-cells into a TEM8-knockout mouse indicated that selective loss of cells from the circulation was due to targeting of TEM8 in healthy tissues. Histological analysis of mice treated with a TEM8-specific CAR revealed evidence of inflammation in the lung and spleen with large collections of infiltrating neutrophils. Therefore our data raise concerns over potential on-target off-tumour toxicity with CARs targeting TEM8 and these should be considered carefully before embarking upon clinical trials with such agents

Predictors of unfavourable outcome in aneurysmal subarachnoid haemorrhage

Background. Mortality rates following aneurysmal subarachnoid haemorrhage (aSAH) have decreased due to improvements in diagnoses and the management of complications, as well as early obliteration of the aneurysms. Neurogenic pulmonary oedema (NPO) is a clinical syndrome associated with an acute increase in intracranial pressure and a release of catecholamines into the circulation. This study investigated independent predictors of unfavourable outcomes (Glasgow Outcome Scores 1, 2 or 3) in patients with aSAH. Materials and methods. A total of 262 patients with aSAH (162 females) were included in this prospective study. Clinical characteristics were assessed, and electrocardiographic, serum cardiac and inflammatory biomarker measurements were recorded on admission. Outcomes were assessed three months after admission. Univariate and multivariate analyses of these data were used to predict unfavourable outcomes. Results. A total of 156 patients (59.54%) had unfavourable outcomes. Compared to those who had favourable outcomes, patients with unfavourable outcomes were significantly older (54.37 ± 10.56 vs. 49.13 ± 10.77 years; p < 0.001) and had more severe aSAHs (Hunt and Hess grades ≥ 3: 82.7% vs. 39.6%; p < 0.001). Patients with unfavourable outcomes were more likely to have NPO (10.3% vs. 2.8%; p = 0.023), hydrocephalus (34.0% vs. 20.8%; p = 0.02), and aneurysm reruptures (28.2% vs. 3.8%; p < 0.001).Independent predictors of an unfavourable outcome included Hunt and Hess grades ≥ 3 (odds ratio [OR], 4.291; 95% confidence interval [CI], 2.168–8.491; p < 0.001), increased systolic blood pressure on admission (OR, 1.020; 95% CI, 1.002–1.038; p = 0.03), increased heart rate (HR) on admission (OR, 1.024; 95% CI, 1.001–1.048; p = 0.04), and aneurysm rerupture (OR, 4.961; 95% CI, 1.461–16.845; p = 0.01).Conclusions. These findings suggest that aneurysm reruptures, as well as increased blood pressure and HR, are associated with unfavourable outcomes in patients with aSAH

Structured inquiry-based learning: Drosophila GAL4 enhancer trap characterization in an undergraduate laboratory course.

We have developed and tested two linked but separable structured inquiry exercises using a set of Drosophila melanogaster GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community

Narcissism and the strategic pursuit of short-term mating : universal links across 11 world regions of the International Sexuality Description Project-2.

Previous studies have documented links between sub-clinical narcissism and the active pursuit of short-term mating strategies (e.g., unrestricted sociosexuality, marital infidelity, mate poaching). Nearly all of these investigations have relied solely on samples from Western cultures. In the current study, responses from a cross-cultural survey of 30,470 people across 53 nations spanning 11 world regions (North America, Central/South America, Northern Europe, Western Europe, Eastern Europe, Southern Europe, Middle East, Africa, Oceania, Southeast Asia, and East Asia) were used to evaluate whether narcissism (as measured by the Narcissistic Personality Inventory; NPI) was universally associated with short-term mating. Results revealed narcissism scores (including two broad factors and seven traditional facets as measured by the NPI) were functionally equivalent across cultures, reliably associating with key sexual outcomes (e.g., more active pursuit of short-term mating, intimate partner violence, and sexual aggression) and sex-related personality traits (e.g., higher extraversion and openness to experience). Whereas some features of personality (e.g., subjective well-being) were universally associated with socially adaptive facets of Narcissism (e.g., self-sufficiency), most indicators of short-term mating (e.g., unrestricted sociosexuality and marital infidelity) were universally associated with the socially maladaptive facets of narcissism (e.g., exploitativeness). Discussion addresses limitations of these cross-culturally universal findings and presents suggestions for future research into revealing the precise psychological features of narcissism that facilitate the strategic pursuit of short-term mating

Care of patients with inborn errors of immunity in thirty J Project countries between 2004 and 2021

IntroductionThe J Project (JP) physician education and clinical research collaboration program was started in 2004 and includes by now 32 countries mostly in Eastern and Central Europe (ECE). Until the end of 2021, 344 inborn errors of immunity (IEI)-focused meetings were organized by the JP to raise awareness and facilitate the diagnosis and treatment of patients with IEI.ResultsIn this study, meeting profiles and major diagnostic and treatment parameters were studied. JP center leaders reported patients’ data from 30 countries representing a total population of 506 567 565. Two countries reported patients from JP centers (Konya, Turkey and Cairo University, Egypt). Diagnostic criteria were based on the 2020 update of classification by the IUIS Expert Committee on IEI. The number of JP meetings increased from 6 per year in 2004 and 2005 to 44 and 63 in 2020 and 2021, respectively. The cumulative number of meetings per country varied from 1 to 59 in various countries reflecting partly but not entirely the population of the respective countries. Altogether, 24,879 patients were reported giving an average prevalence of 4.9. Most of the patients had predominantly antibody deficiency (46,32%) followed by patients with combined immunodeficiencies (14.3%). The percentages of patients with bone marrow failure and phenocopies of IEI were less than 1 each. The number of patients was remarkably higher that those reported to the ESID Registry in 13 countries. Immunoglobulin (IgG) substitution was provided to 7,572 patients (5,693 intravenously) and 1,480 patients received hematopoietic stem cell therapy (HSCT). Searching for basic diagnostic parameters revealed the availability of immunochemistry and flow cytometry in 27 and 28 countries, respectively, and targeted gene sequencing and new generation sequencing was available in 21 and 18 countries. The number of IEI centers and experts in the field were 260 and 690, respectively. We found high correlation between the number of IEI centers and patients treated with intravenous IgG (IVIG) (correlation coefficient, cc, 0,916) and with those who were treated with HSCT (cc, 0,905). Similar correlation was found when the number of experts was compared with those treated with HSCT. However, the number of patients treated with subcutaneous Ig (SCIG) only slightly correlated with the number of experts (cc, 0,489) and no correlation was found between the number of centers and patients on SCIG (cc, 0,174).Conclusions1) this is the first study describing major diagnostic and treatment parameters of IEI care in countries of the JP; 2) the data suggest that the JP had tremendous impact on the development of IEI care in ECE; 3) our data help to define major future targets of JP activity in various countries; 4) we suggest that the number of IEI centers and IEI experts closely correlate to the most important treatment parameters; 5) we propose that specialist education among medical professionals plays pivotal role in increasing levels of diagnostics and adequate care of this vulnerable and still highly neglected patient population; 6) this study also provides the basis for further analysis of more specific aspects of IEI care including genetic diagnostics, disease specific prevalence, newborn screening and professional collaboration in JP countries

Narcisismo y búsqueda estratégica del emparejamiento a corto plazo a través de las culturas: Enlaces omnipresentes a través de 11 regiones mundiales del Proyecto de la descripción de la sexualidad internacional 2

Previous studies have documented links between sub-clinical narcissism and the active pursuit of short-term mating strategies (e.g., unrestricted sociosexuality, marital infidelity, mate poaching). Nearly all of these investigations have relied solely on samples from Western cultures. In the current study, responses from a cross-cultural survey of 30,470 people across 53 nations spanning 11 world regions (North America, Central/South America, Northern Europe, Western Europe, Eastern Europe, Southern Europe, Middle East, Africa, Oceania, Southeast Asia, and East Asia) were used to evaluate whether narcissism (as measured by the Narcissistic Personality Inventory; NPI) was universally associated with short-term mating. Results revealed narcissism scores (including two broad factors and seven traditional facets as measured by the NPI) were functionally equivalent across cultures, reliably associating with key sexual outcomes (e.g., more active pursuit of short-term mating, intimate partner violence, and sexual aggression) and sex-related personality traits (e.g., higher extraversion and openness to experience). Whereas some features of personality (e.g., subjective well-being) were universally associated with socially adaptive facets of Narcissism (e.g., self-sufficiency), most indicators of short-term mating (e.g., unrestricted sociosexuality and marital infidelity) were universally associated with the socially maladaptive facets of narcissism (e.g., exploitativeness). Discussion addresses limitations of these cross-culturally universal findings and presents suggestions for future research into revealing the precise psychological features of narcissism that facilitate the strategic pursuit of short-term mating.Estudios previos, en primer lugar a través de las muestras de culturas occidentales, han documentado asociaciones sistemáticas del narcisismo subclínico con múltiples indicadores de estrategias del emparejamiento a corto plazo (p. ej. sociosexualidad ilimitada, infidelidad, caza de pareja). En este estudio se han usado respuestas de la encuesta transcultural de 30.470 personas de 53 naciones de 11 regiones mundiales (América del Norte, América del Sur/América Central, Europa del Norte, Europa del Oeste, Europa del Este, Europa del Sur, Oriente Próximo, África, Asia del Sur/Sudoeste de Asia, Asia del Este y Oceanía) para evaluar si el narcisismo (medido por el Inventario de Personalidad Narcisista; NPI) se asocia panuniversalmente con los indicadores del emparejamiento a corto plazo, tanto en la dirección, como en la intensidad. Los resultados sugieren que el narcisismo (incluidos muchos aspectos suyos medidos por el NPI) tiene las mismas asociaciones básicas con los rasgos de personalidad relacionados con el sexo (p. ej. extraversión alta) y con los resultados sexuales claves (p. ej. búsqueda más activa de las estrategias del emparejamiento a corto plazo) a través de las 11 mayores regiones mundiales del PDSI 2. La discusión se enfoca en las implicaciones y limitaciones del estudio actual

Ag-doped As-S-Se chalcogenide glasses: a correlative study of structural and dielectrical properties

This work is focused on investigating the effect of Ag doping on the dielectric and structural properties of the As-S-Se glass system and their suitability for potential applications in optoelectronics, such as electronic and switching components. A series of glasses from the system Ag-x(As40S30Se30)(100-x) (x <= 5 at.% Ag) were prepared with melt-quenching technique. Morphological and compositional analysis was performed by scanning electron microscopy and energy-dispersive X-ray spectroscopy. Raman spectroscopy measurements have shown structural modifications of the glass network upon Ag doping, with creation of new Ag-(S,Se)-As structures, resulting in increased number of defect states. Variations in dielectric permittivity (epsilon ') and dielectric loss (epsilon '') were investigated in a wide frequency and temperature range, and explained by dominant dipolar and electronic polarization, as well as correlated with the observed structural changes. Improvement in dielectric properties is observed upon doping with Ag. Furthermore, the low dielectric tangent loss at high frequencies for all glassy samples points to a beneficial ability of the material to absorb energy of the external electric field. Good thermal stability of these glasses is confirmed by the calculated temperature coefficient of the dielectric permittivity. Density of localized states in all glasses was determined from AC conductivity and the dielectric tangent loss using a correlated barrier hopping model, and found to increase with Ag concentration. The overall results suggest that Ag doping enhances the electrical and optical quality of this type of glasses, making them suitable as nonlinear optical materials and their applications in devices