CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS.

ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

Targeting cancer-associated fibroblasts in the bone marrow prevents resistance to CART-cell therapy in multiple myeloma.

Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Age Estimation Based on Computed Tomography Analysis of the Scapula.

Background and Objectives: Age estimation from skeletal remains and in living individuals is an important issue for human identification, and also plays a critical role in judicial proceedings for migrants. Forensic analysis of ossification centers is the main evaluation method for age estimation, and ossification degree can be determined using computed tomography analysis. The purpose of this study is to investigate the applicability of CT (computed tomography) in the analysis of left scapula ossification centers, for forensic age estimation in Turkish society. Materials and Methods: We analyzed six ossification centers of the left scapula and these ossification centers are the coracoid, subcoracoid, coracoid apex, acromial, glenoid, and inferior angle ossification centers. A pediatric radiologist analyzed these six ossification centers of the scapula by using a staging method defined by Schmeling et al. in 2004. Two months after the first assessment, 20 randomly selected cases was reanalyzed by the first observer and by another pediatric radiologist. Correlation between the age and ossification stage was assessed using Spearman's nonparametric correlation test. Linear regression analysis was performed using a backwards model. Cohen's kappa coefficient was used for evaluating interobserver and intraobserver variability. Results: In this retrospective study, 397 (248 male and 149 female) cases were evaluated. Ages ranged between 7.1 and 30.9. The mean age was 19.83 ± 6.49. We determined a positive significant correlation between the age and the ossification stages of ossification centers analyzed in both sexes. In each ossification center, except inferior angle, all of the stage 1 and 2 cases in both sexes were under 18 years old. Intraobserver and interobserver evaluations showed that reproducibility and consistency of the method was relatively good. Conclusions: The present study indicated that CT analysis of scapula ossification centers might be helpful in forensic age assessment of living individuals and dry bones.

Leukemic extracellular vesicles induce chimeric antigen receptor T cell dysfunction in chronic lymphocytic leukemia.

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1+ CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients.

Applicability of T1-weighted MRI in the assessment of forensic age based on the epiphyseal closure of the humeral head.

This work investigates the value of magnetic resonance imaging analysis of proximal epiphyseal fusion in research examining the growth and development of the humerus and its potential utility in establishing forensic age estimation. In this study, 428 proximal humeral epiphyses (patient age, 12-30 years) were evaluated with T1-weighted turbo spin echo (T1 TSE) sequences in coronal oblique orientation on shoulder MRI images. A scoring system was created following a combination of the Schmeling and Kellinghaus methods. Spearman's rank correlation analysis revealed a significant positive relationship between age and ossification stage of the proximal humeral epiphysis (all subjects: rho = 0.664, p < 0.001; males: 0.631, p < 0.001; females: rho = 0.651, p < 0.001). The intra- and inter-observer reliability assessed using Cohen's kappa statistic was κ = 0.898 and κ = 0.828, respectively. The earliest age of epiphysis closure was 17 years for females and 18 years for males. MRI of the proximal humeral epiphysis can be considered advantageous for forensic age estimation of living individuals in a variety of situations, ranging from monitoring public health to estimating the age of illegal immigrants/asylum seekers, minors engaged in criminal activities, and illegal participants in competitive sports, without the danger of radiation exposure.

Forensic age diagnostics by magnetic resonance imaging of the proximal humeral epiphysis.

The most commonly used radiological method for age estimation of living individuals is X-ray. Computed tomography is not commonly used due to high radiation exposure, which raises ethical concerns. This problem can be solved with the use of magnetic resonance imaging (MRI), which avoids the use of ionizing radiation. The purpose of the present study was to evaluate the utility of MRI analysis of the proximal humeral epiphyses for forensic age estimations of living individuals. In this study, 395 left proximal humeral epiphyses (patient age 12-30 years) were evaluated with fast-spin-echo proton density-weighted image (FSE PD) sequences in a coronal oblique orientation on shoulder MRI images. A five-stage scoring system was used following the method of Dedouit et al. The intra- and interobserver reliabilities assessed using Cohen's kappa statistic were κ = 0.818 and κ = 0.798, respectively. According to this study, stage five first appeared at 20 and 21 years of age in males and females, respectively. These results are not directly comparable to any other published study due to the lack of MRI data on proximal humeral head development. These findings may provide valuable information for legally important age thresholds using shoulder MRI. The current study demonstrates that MRI of the proximal humerus can support forensic age estimation. Further research is needed to establish a standardized protocol that can be applied worldwide.

Novel interactions between erythroblast macrophage protein and cell migration.

Erythroblast macrophage protein is a novel protein known to mediate attachment of erythroid cells to macrophages to form erythroblastic islands in bone marrow during erythropoiesis. Emp-null macrophages are small with round morphologies, and lack cytoplasmic projections which imply immature structure. The role of Emp in macrophage development and function is not fully elucidated. Macrophages perform varied functions (e.g. homeostasis, erythropoiesis), and are implicated in numerous pathophysiological conditions such as cellular malignancy. The objective of the current study is to investigate the interaction of Emp with cytoskeletal- and cell migration-associated proteins involved in macrophage functions. A short hairpin RNA lentiviral system was use to down-regulate the expression of Emp in macrophage cells. A cell migration assay revealed that the relocation of macrophages was significantly inhibited when Emp expression was decreased. To further analyze changes in gene expression related to cell motility, PCR array was performed by down-regulating Emp expression. The results indicated that expression of mitogen-activated protein kinase 1 and thymoma viral proto-oncogene 1 were significantly higher when Emp was down-regulated. The results implicate Emp in abnormal cell motility, thus, warrants to assess its role in cancer where tumor cell motility is required for invasion and metastasis.

Computed tomography evaluation of the iliac crest apophysis: age estimation in living individuals.

Determination of the ossification properties of the iliac apophysis is important not only in the clinical evaluation of patients undergoing orthopedic surgery but also in age estimation studies for forensic purposes. The literature includes both anthropological and radiological (conventional radiography, ultrasonography, and magnetic resonance imaging modalities) investigations of the different staging systems used for these purposes. In this study, we assessed the utility of computed tomography (CT) of the iliac crest apophysis in estimating forensic age. CT scans of the iliac crest apophysis of 380 patients (187 females, 193 males, and 10-29 years of age) were evaluated according to the four-stage system. Further subclassification did not give data properly due to the reference length measurement of the iliac wing with CT. Thus, in our series, stage 2 was first seen in 12 years of age and stage 3 in those 14 years of age in both sexes and on both sides of the pelvis. Stage 4 was first seen in 17 years of both sexes but only on the right side; on the left side, it appeared in females 18 years of age and in males 17 years of age. Present data was found consistent with previous pelvic radiographic findings. First seen ages for stage 2 and 3 are 12 and 14 years respectively which presented valuable information for legally important age thresholds. However, disadvantages of CT, including high-dose radiation exposure to gonads, the difficulty of evaluating the iliac crest, and the age boundary of 17 years, could make this method infeasible, as compared with hand wrist and pelvic radiographic methods. CT of the iliac crest has probably a greater utility where preexisting CT scans of the pelvic region are available, and it may be considered as a supportive method for age-estimation purposes.

Protective effect of Naringin on experimental hindlimb ischemia/reperfusion injury in rats.

This study was designed to investigate the antioxidant effects of Naringin, in ischemia/reperfusion (I/R)-induced skeletal muscle injury in rats. The rats were randomly allocated into three groups including control, I/R and I/R + Naringin groups. Muscle tissues of I/R groups revealed significantly higher antioxidant enzyme activities, and increased levels of malondialdehyde, as specific a marker of the lipid peroxidation and tissue damage, compared to the control group (p < 0.05). Levels of these parameters in muscle revealed significant reductions in the I/R + Naringin group compared to the I/R group (p < 0.05). Histopathological examination of ischemia muscles in the I/R group showed significant degeneration and inflammation compared to the control group, whereas ischemic muscles of Naringin-administered group showed significant reduction in degeneration and inflammation compared to the I/R group (p < 0.05). We suggest that the protective effect of Naringin may reduce the I/R injury in cases of extremity injuries with acute vascular complications, extremity surgery with prolonged tourniquet application.

Virtual Assessment of Sex: Linear and Angular Traits of the Mandibular Ramus Using Three-Dimensional Computed Tomography.

Morphometric analysis of the mandibular ramus (MR) provides highly accurate data to discriminate sex. The objective of this study was to demonstrate the utility and accuracy of MR morphometric analysis for sex identification in a Turkish population.Four hundred fifteen Turkish patients (18-60 y; 201 male and 214 female) who had previously had multidetector computed tomography scans of the cranium were included in the study. Multidetector computed tomography images were obtained using three-dimensional reconstructions and a volume-rendering technique, and 8 linear and 3 angular values were measured. Univariate, bivariate, and multivariate discriminant analyses were performed, and the accuracy rates for determining sex were calculated.Mandibular ramus values produced high accuracy rates of 51% to 95.6%. Upper ramus vertical height had the highest rate at 95.6%, and bivariate analysis showed 89.7% to 98.6% accuracy rates with the highest ratios of mandibular flexure upper border and maximum ramus breadth. Stepwise discrimination analysis gave a 99% accuracy rate for all MR variables.Our study showed that the MR, in particular morphometric measures of the upper part of the ramus, can provide valuable data to determine sex in a Turkish population. The method combines both anthropological and radiologic studies.

Estimation of forensic age using substages of ossification of the medial clavicle in living individuals.

Forensic age estimation based on staging of ossification of the medial clavicular bone is one of the methods recommended by the Study Group on Forensic Age Diagnostics of the German Association of Forensic Medicine. In the present study, we analyzed the stages of ossification of the medial clavicular epiphyses on thin-sliced (1 mm) computed tomography (CT) images using the substages defined within stages 2 and 3. The retrospective CT analysis involved 193 subjects (129 males, 64 females) ranging in age from 13 to 28 years. Spearman's correlation analysis revealed a positive correlation between age and ossification stage in both male and female subjects. Stage 3c was first observed at 19 years of age in both sexes and may thus serve as a valuable forensic marker for determining an age of 18 years. Although further research is needed on the ossification stages of the medial clavicular epiphyses, the present findings could contribute to existing reports on observers' experiences using CT analysis of ossification combined with analysis of substages.

Forensic age estimation by the Schmeling method: computed tomography analysis of the medial clavicular epiphysis.

The variability of anthropometric measures, such as the degree of ossification, among societies should be taken into account when estimating age. The degree of ossification of the medial clavicle can be determined with thin-section computed tomography (CT), which is one of the methods recommended by the Study Group on Forensic Age Diagnostics of the German Association of Forensic Medicine. The purpose of this retrospective study was to investigate the applicability of thin-section CT analysis of the degree of ossification of the medial clavicle in a Turkish population. We evaluated the CT images (1-mm slice thickness) of 503 patients (362 male, 141 female; age, 10-35 years) using the Schmeling five-stage method. The Spearman's correlation analysis revealed a positive correlation between age and ossification stage in both male and female patients (total group: rho = 0.838, p < 0.001; male: rho = 0.831, p < 0.001; female: rho = 0.856, p < 0.001). The linear regression analysis results indicated that the ossification stage of the medial clavicle is a good predictor when estimating age (r (2) = 0.735 for all patients, 0.734 for male patients, 0.741 for female patients). Sex differences in ossification stages were observed only for stage 1 and 4 ossification. We believe that future research could expand the database on this topic and contribute to improvements in this measurement method.

Magnetic resonance imaging of distal tibia and calcaneus for forensic age estimation in living individuals.

In recent years, methods by which to decrease radiation exposure during age estimation have gained importance and become a main research area in the forensic sciences. Imaging tools such as X-ray and computed tomography (CT) are accepted as the main diagnostic methods for evaluation of the epiphysis in living individuals; however, radiation exposure and superimposition are the main disadvantages of these techniques. Magnetic resonance (MR) imaging provides an advantage in terms of preventing radiation exposure. In this study, we performed an MR analysis of the degree of fusion of the distal tibia and calcaneal epiphysis and investigated the utility of this technique in the Turkish population. Using the three-stage method described by Saint-Martin et al., we retrospectively evaluated 167 MR images (97 males, 70 females; mean age, 17.7 ± 4.8 years for males and 17.6 ± 4.9 years for females; age range of all subjects, 8-25 years). Intraobserver and interobserver evaluation showed good repeatability and consistency of this method. Stages 2 and 3 ossification of the distal tibial epiphysis first occurred at age 14 and 15 years in males and 12 and 14 years in females, respectively. Stages 2 and 3 ossification of the calcaneal epiphysis first occurred at age 14 and 16 years in males and 10 and 12 years in females, respectively. When performed alone, MR analysis of the distal tibial and calcaneal epiphysis offers limited information for forensic age estimation. However, we suggest that MR analysis can be used as a supportive method when it is necessary to avoid repeated radiation exposure.

The apoptotic thanatotranscriptome associated with the liver of cadavers.

Gene expression investigations are well-established components of ante mortem studies with broad applications ranging from elucidating basic mechanisms responsible for normal physiological processes to discovering therapeutic targets in pathophysiological conditions. However, gene expression studies and their application in the medico-legal field are still in their infancy. Therefore, the present study focuses on RNA using PCR array in the analysis of gene expression associated with tissues taken from actual criminal cases. RNA was extracted from the liver tissues of bodies with PMIs between 6 and 48 h. The results demonstrated that mRNA was stable up to 48 h postmortem. Further, as cell death is an indispensable and necessary part of the biological life cycle, apoptotic gene expression profiles were investigated. The gene expression related to the programmed cell death found in body tissues after death is defined as the apoptotic thanatotranscriptome (thanatos-, Greek for death). On comparison of control and decaying tissues, the results show that with time, pro-apoptotic genes such as caspases are up-regulated and the expression of genes responsible for anti-apoptosis such as BCL2 and BAG3 were down-regulated. Thus, this current work gives a unique perspective of the apoptotic thanatotranscriptome that is affected after death. Up to the present time, gene expression in bodies from criminal cases has not been reported in literature using PCR array techniques. Thus, this thanatotranscriptome study provides insight into postmortem gene activity with potential applications in medico-legal investigations.

The use of maxillary sinus dimensions in gender determination: a thin-slice multidetector computed tomography assisted morphometric study.

Gender determination is an important step in identification. For gender determination, anthropometric evaluation is one of the main forensic evaluations. In the present study, morphometric analysis of maxillary sinuses was performed to determine gender. For morphometric analysis, coronal and axial paranasal sinus computed tomography (CT) scan with 1-mm slice thickness was used. For this study, 140 subjects (70 women and 70 men) were enrolled (age ranged between 18 and 63). The size of each subject's maxillary sinuses was measured in anteroposterior, transverse, cephalocaudal, and volume directions. In each measurement, the size of the maxillary sinus is significantly small in female gender (P < 0.001). When discrimination analysis was performed, the accuracy rate was detected as 80% for women and 74.3% for men with an overall rate of 77.15%. With the use of 1-mm slice thickness CT, morphometric analysis of maxillary sinuses will be helpful for gender determination.

Forensic age estimation by spheno-occipital synchondrosis fusion degree: computed tomography analysis.

The analysis of ossification points plays a considerable role in forensic age estimation. Although traditional methods are still in use, researchers are working on different age estimation procedures especially within the development of radiologic methods. One of these methods is to define spheno-occipital synchondrosis fusion degree. Spheno-occipital synchondrosis, an important growth point on cranial base, provides noteworthy information about age estimation through its late stage ossification nature. This study aimed to investigate spheno-occipital synchondrosis fusion degree for age estimation in the Turkish population. In our study, 1-mm-sectioned computed tomography images of 638 (399 men and 139 women) subjects within the age of 10 to 25 years were retrospectively examined. It is stated in our study that spheno-occipital syncondrosis fusion begins superiorly and progresses inferiorly until it is completed. Spheno-occipital syncondrosis is known to be totally open at the mean (SD) age of 11.5 (1.5) years in men and 10.7 (0.8) years in women. In addition, fusion degree is known to be increased with age. Fusion starts approximately 2 years earlier in women than in men, and the process of fusion completes at the age of 17 years in both sexes. An analysis of fusion degree between sex groups showed significance at the age of 11 to 15 years, and Spearman rank correlations indicate a significant positive relationship between age and degree of spheno-occipital fusion (P < 0.001; men, ρ = 0.714; women, ρ = 0.698). Consequently, 5-staged analysis of spheno-occipital synchondrosis fusion degree in use with 1-mm computed tomography images will be helpful for age estimation between 11 and 17 years.

Assessment of hearing sequelae in individuals tested post-trauma.

BACKGROUND: Over 5% of the global population (430 million people) require rehabilitation for hearing loss. Individuals with hearing impairments face significant challenges in business, daily life, and social participation. Hearing loss (HL) and other permanent physical and sensory disabilities escalate dramatically in cases with brain damage and temporal bone trauma associated with head injuries. This study aims to identify the significant risk factors for hearing loss following head trauma, utilizing current data, and discuss the findings in the context of the literature. This could contribute to the development of standard approaches for assessing such cases. METHODS: This retrospective study reviewed files and reports from individuals assessed for hearing loss at Dokuz Eylül University Faculty of Medicine, Department of Forensic Medicine. The study included cases that applied at least 12 months post-trauma, between January 1, 2016, and December 31, 2022, after their recovery process was completed. Sociodemographic data, types of temporal bone fractures, initial otoscopic examination findings, presence or absence of intracranial injury, type of hearing loss, and audiometry test results for air and bone conduction pure tone threshold averages were evaluated. Data analysis was conducted using SPSS 26.0 (Statistical Package for the Social Sciences). RESULTS: Out of 244 cases, 177 (72.5%) were male and 67 (27.5%) were female. It was observed that the majority of trauma cases occurred in the 19-40 age group (49.2%; n=120). In the initial otoscopic examinations post-trauma, otorrhagia/otorrhea was the most common finding, both as an isolated symptom (n=59, 24.2%) and when accompanied by other symptoms. No temporal bone fractures were detected in 43 cases (17.6%). Longitudinal fractures were found in 141 cases (57.8%), transverse fractures in 48 (19.7%), and mixed-type fractures in 12 (4.9%). The statistical difference in air conduction and bone conduction pure tone threshold averages between groups with and without intracranial injury was significant (p<0.001). CONCLUSION: Post-traumatic examinations should employ a multidisciplinary approach, adhering to standard medical improvement and assessment timelines. It is essential to verify whether each patient's medical improvement process has reached its maximum potential. We believe that adhering to these recommendations and utilizing standardized classifications for hearing loss will prevent the loss of rights.

Mesenchymal stromal cells with chimaeric antigen receptors for enhanced immunosuppression.

Allogeneic mesenchymal stromal cells (MSCs) are a safe treatment option for many disorders of the immune system. However, clinical trials using MSCs have shown inconsistent therapeutic efficacy, mostly owing to MSCs providing insufficient immunosuppression in target tissues. Here we show that antigen-specific immunosuppression can be enhanced by genetically modifying MSCs with chimaeric antigen receptors (CARs), as we show for E-cadherin-targeted CAR-MSCs for the treatment of graft-versus-host disease in mice. CAR-MSCs led to superior T-cell suppression and localization to E-cadherin+ colonic cells, ameliorating the animals' symptoms and survival rates. On antigen-specific stimulation, CAR-MSCs upregulated the expression of immunosuppressive genes and receptors for T-cell inhibition as well as the production of immunosuppressive cytokines while maintaining their stem cell phenotype and safety profile in the animal models. CAR-MSCs may represent a widely applicable therapeutic technology for enhancing immunosuppression.

Hyperphosphorylated PTEN exerts oncogenic properties.

PTEN is a multifaceted tumor suppressor that is highly sensitive to alterations in expression or function. The PTEN C-tail domain, which is rich in phosphorylation sites, has been implicated in PTEN stability, localization, catalytic activity, and protein interactions, but its role in tumorigenesis remains unclear. To address this, we utilized several mouse strains with nonlethal C-tail mutations. Mice homozygous for a deletion that includes S370, S380, T382 and T383 contain low PTEN levels and hyperactive AKT but are not tumor prone. Analysis of mice containing nonphosphorylatable or phosphomimetic versions of S380, a residue hyperphosphorylated in human gastric cancers, reveal that PTEN stability and ability to inhibit PI3K-AKT depends on dynamic phosphorylation-dephosphorylation of this residue. While phosphomimetic S380 drives neoplastic growth in prostate by promoting nuclear accumulation of ß-catenin, nonphosphorylatable S380 is not tumorigenic. These data suggest that C-tail hyperphosphorylation creates oncogenic PTEN and is a potential target for anti-cancer therapy.

AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.