Control Population Research Articles

Microstomia is associated with functional impairment and is a poor prognostic factor in systemic sclerosis - a single center observational study with survival analysis.

Objectives were to assess the mouth opening ability (MOA) among patients with systemic sclerosis (SSc) in comparison to a healthy control population. The impact of microstomia (decrease in any of the MOA parameters) on physical performance and long-term survival was also investigated. Interincisal (ID), interlabial (LD), the intercommissural distances at both opened mouth and closed mouth (OW, CW) and the oral area (OA) and circumference (OC) all were assessed in 131 SSc patients. Microstomia was defined based on the oral aperture values of a healthy control group (n = 63) with similar median age and sex distribution. Tests evaluating functional performance were performed. Survival analysis by univariate and multivariate Cox regression analysis was performed. Microstomia was present in 56.5% of the entire SSc cohort. Patients with microstomia had higher number of contractures compared to those with preserved oral aperture parameters (median 10 vs. 7, p < 0.01). LD, OA and OC correlated negatively with the number of joint contractures, both in the entire SSc cohort and separately in the diffuse (dcSSc) and limited cutaneous subsets (lcSSc), (rho: between - 0.267 and - 0.516, p < 0.05). Reduced ID was associated with worse Health Assessment Questionnaire Disability Index (HAQ-DI) only in dcSSc while reduced LD was linked to increased HAQ-DI scores in both SSc subsets. Decreased OA upon enrollment was associated with an increased risk in mortality by multivariate Cox regression analysis (HR: 2.74; 95% CI, 1.15-6.53). Microstomia was associated with joint damage, and higher overall disability based on HAQ-DI. Interlabial distance was a beneficial, convenient measurable parameter to characterize oral aperture. Oral area was an independent poor prognostic factor regarding long-term survival.

Delineating the psychiatric morbidity spectrum in congenital adrenal hyperplasia: a population-based registry study.

Clinical studies of psychiatric morbidity in patients with congenital adrenal hyperplasia (CAH) imply impaired mental health. To delineate psychiatric morbidity in a national CAH cohort by using complete data on psychiatric diagnoses from all Danish hospitals between 1977-2018 and on all psychiatric medication prescribed between 1995-2018. A registry-based cohort study. A public uniform healthcare system. Four-hundred-forty-eight patients (females: n=215) with CAH, hereof 410 with 21-hydroxylase deficiency (21-OHD) (females: n=255) and 44,527 age- and sex-matched general population controls. Diagnoses were analyzed by negative binomial regression yielding incidence rate ratios (IRR). Medication were analyzed by Cox regression yielding hazard ratios (HR). 21-OHD was associated with an increased risk of any psychiatric diagnosis; females: IRR=2.32 (confidence interval (CI) (1.48-3.64), males: IRR=2.74 (CI 1.31-5.71) as well as of medication related to psychiatric disorders; females: HR=1.74 (CI 1.42-2.13), males: HR=1.74 (CI1.30-2.33). Both females and males with 21-OHD had a significantly increased risk of alcohol use, stress- and adjustment disorders, and of suicidal behavior. For patients with more rare forms of CAH (n=24), the risk of any psychiatric diagnosis was significantly increased for males, IRR=12.85 (CI 1.78-92.87), but not for females, IRR=0.54 (CI 0.10-3.00). The risk of being prescribed psychiatric medication was not increased for neither females, HR=1.05 (CI 0.39-2.84), or males, HR=0.72 (CI 0.10-5.13), with rare forms of CAH. 21-OHD is associated with a significantly increased psychiatric morbidity. This study underlines a need for awareness of mental health in patients with 21-OHD.

Incidence of and risk of mortality after hip fractures in Rheumatoid Arthritis relative to the general population.

Osteoporosis, a known complication of rheumatoid arthritis (RA), increases the risk of hip fracture, which is associated with high morbidity and mortality. Fracture risk estimates in RA patients treated with contemporary treatment strategies are lacking. The objectives were 1) to estimate age- and sex-specific incidence rates and compare the risk of hip fractures in RA relative to age and sex-matched general population controls, and 2) to compare the risk of all-cause mortality in RA and general population controls after hip fracture. A longitudinal study of a population-based incident cohort of RA patients diagnosed between 1997 and 2009, followed until 2014, with age- and sex-matched controls from the general population of British Columbia, using administrative health data. Hip fracture outcomes (ICD9-CM codes 820.0 or 820.2; ICD10-CA code S72.0 to S72.2) and mortality at pre-defined intervals after fracture (in-hospital, 90 days, 1-year, 5-year) were identified. Hip fracture incidence rates for RA and controls, and incidence rate ratios (IRR) were calculated. Cox proportional hazards models compared hip fracture and mortality risk in RA vs. controls; logistic regression compared in-hospital mortality risk. Overall, 1314 hip fractures over 360,521 person-years were identified in 37,616 RA individuals and 2083 over 732,249 person-years in 75,213 controls, yielding a 28% greater fracture risk in RA (IRR 1.28 [95%CI, 1.20;1.37]). Mean age at time of fracture was slightly younger for RA than controls (79.6 + 10.8 vs. 81.6 + 9.3 yrs). Post-fracture mortality risk at 1- and 5-years did not differ between RA and general population controls. Results were similar in a sensitivity analysis including only RA individuals who received disease-modifying antirheumatic drugs (DMARDs). People with RA had a greater risk of hip fractures, but no greater risk of mortality post fracture, than the general population. The relative risk of hip fractures observed was not as high as previously reported, likely reflecting better treatment of inflammation and management of osteoporosis and its risk factors.

Phenotypic and transcriptomic profiling of induced pluripotent stem cell (iPSC)-derived NK cells and their cytotoxicity against cancers.

Adoptive immunotherapy using natural killer (NK) cells has attracted considerable interest in numerous clinical trials targeting both hematological and solid tumors. Traditionally, NK cells are primarily derived from either peripheral blood (PB) or umbilical cord blood (UCB). However, these methods can lead to variability and heterogeneity within the NK cell population. In contrast, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells provide a more controlled and uniform cellular population, suitable for large-scale clinical applications. This makes iNK cells a promising option for developing "off-the-shelf" immunotherapeutic products. Nevertheless, current NK cell differentiation protocols, which rely on embryoid body (EB) cultures, are labor-intensive and susceptible to unwanted heterogeneity during differentiation. Here, we developed a more efficient approach for generating iNK cells by employing a monolayer and feeder-free differentiation protocol, alongside optimized culture media. The iNK cells were generated using a two-step in vitro monolayer feeder-free system following NK cell development. To evaluate their maturity, phenotypic analysis was performed using flow cytometry, comparing with PB-NK cells and the NK-92 cell line. Additionally, single-cell RNA sequencing was performed to examine their transcriptomic profiles. The cytotoxic activity of the iNK cells was evaluated by co-culturing with cholangiocarcinoma (CCA) and breast cancer (BCA) cell lines in both monolayer (2D) and tumor spheroid (3D) co-culture systems. We successfully differentiated iPSCs into mesoderm (ME), hematopoietic stem/progenitor cells (HSPCs), and NK cells. The resulting iNK cells exhibited typical NK cell markers such as CD45, CD56, and CD16, and expressed key functional proteins, including both activating and inhibitory receptors. Single-cell RNA sequencing confirmed that the transcriptomic profile of our iNK cells closely resembles that of PB-NK cells. Importantly, our iNK cells demonstrated strong cytotoxic abilities against various CCA and BCA cell lines, surpassing the NK-92 cell line in both monolayer cultures and tumor spheroid cultures. This study highlights the potential of iPSCs as an effective alternative cell source for generating NK cells. Using a two-step in vitro monolayer feeder-free system, we successfully generated iNK cells that not only expressed key NK cell markers and their receptors but also displayed a transcriptomic profile closely resembling PB-NK cells. Furthermore, iNK cells exhibited cytotoxicity against CCA and BCA cell lines comparable to that of PB-NK cells. This approach could pave the way for off-the-shelf NK cell products, potentially enhancing the effectiveness of adoptive NK cell therapy.

Gut Bacterial Microbiome Profiles Associated with Colorectal Cancer Risk: A Narrative Review and Meta-Analysis

Objective: Recent studies have shown a potential link between the gut microbiome and colorectal cancer (CRC). A wide array of research into this topic was performed over the past decade, illustrating a keen interest in the potential causal relationship between the gut microbiome and CRC. However, the cancer research community is lacking a concise review of this kind, which aims to explore the evidence linking the human gut microbiome to the risk of developing CRC. Design: This narrative review was carried out by two independent reviewers who assessed the database outcomes from Medline and EMBASE during May 2020. A meta-analysis was undertaken to study the link between Helicobacter pylori and CRC. The meta-analysis was processed through Stata (StataCorp LLC, Lakeway Drive, College Station, Texas, USA). Results: Thirty one papers were included in this narrative review, of which 12 were included in the meta-analysis. From these papers, Fusobacterium and Bacteroides were reported most frequently as enriched in those with CRC versus the control populations. The meta-analysis showed an odds ratio of 1.49 (95% CI: 1.19–1.86), including a total of 20,001 events. This meta-analysis concluded that H. pylori infection significantly increases the risk of CRC, albeit with evidence of publication bias. Conclusion: Bacteria have been found to increase the risk of CRC; however, a definitive causal relationship cannot be concluded or excluded using case-control studies. To fully understand the potential link of the bacteria listed, alterations in research design and execution are required. The assessment found a need for a large-scale cohort study conducted over a significant period of time to thoroughly evaluate the potential relationship between gut microbiome and CRC risk.

Waste reduction decreases rat activity from peri-urban environment.

Globally, species in the genus Rattus (specifically Rattus rattus and Rattus norvegicus), are some of the most influential invasive taxa due to their high rates of competitive exclusion and large dietary breadth. However, the specific foraging strategies of urban-adjacent populations remain largely unknown. We examined Rattus spp. dependency on human food supplementation in a population on adjacent non-developed (or peri-urban) land. Via linear regression modeling, we measured rodent activity changes between native and invasive species before and after a decrease in human supplementation due to the COVID-19 lockdown in Santa Cruz, California, USA. We documented invasive rat activity via camera traps in normal (pre-COVID lockdown) conditions near dining halls and similar waste sources, and again under COVID lockdown conditions when sources of human supplementation were drastically decreased. After 120 trap nights we found a significant decrease (p < 0.001) in Rattus activity after the removal of human refuse, while native small mammal activity remained unchanged (p = 0.1). These results have strong conservation implications, as they support the hypothesis that proper waste management is an effective, less-invasive form of population control over conventional rodenticides.

Abstract 4141469: Left Ventricular Septum Displacement Implications in the Limited Cardiac Reserve, RV to Pc Uncoupling and Oxygen Uptake During Exercise in Heart Failure

Background: Dyspnea on exertion is cardinal symptom in heart failure (HF) and may be caused by an unfavorable dynamic of ventricular interaction. This concept has been poorly addressed over time and we hypothesized that a careful study of the interventricular septum adaptations during maximal exercise in HF might be implicated in the limited cardiac reserve and uncoupling of right ventricular (RV) to pulmonary circulation (Pc), both yielding to a limited O2 uptake. Aim: To study the pathophysiology behind biventricular interaction during exercise in HF, evaluating the role of septum displacement during exercise and how and whether it would affect peak exercise oxygen consumption (VO 2 peak) through a limited cardiac output (CO) increase and some degrees of RV to PC uncoupling assessed by TAPSE/PASP. Methods: 22 HF performed a combined cardiopulmonary exercise testing imaging (CPET imaging) with RV 3D-imaging analysis and were compared with a control population. 3D imaging of the RV chamber was examined off-line using the 4D RV TomTec software and obtained 3D mesh of the RV model using custom software to obtain the mean curvature value of IVS in 4 regions of: inflow tract (RVIT), outflow tract (RVOT), apical and body. We acquired measurements of curvature during end-diastole (ED) and at end systole (ES) phases and obtained a parametric curvature map. Results: HF patients (mean age 72±12, 27% female) typically showed an abnormal septal curve, with a more leftward configuration either at rest and under exercise (rest =−0.01±0.007 at ED, and −0.01±0.009 at ES; peak exercise= −0.01±0.006 at ED, and −0.01±0.007 at ES) compared to controls (rest=−0.02±0.002 at ED, and −0.02±0.006 at ES; peak exercise −0.02±0.0.006 at ED, and −0.02±0.01 at ES, Figure). Notably, the degree of the IVS curvature showed a linear correlation with an impaired gas exchange performance as indicated by a lower peak VO 2 , a limited CO and impairment of TAPSE/PASP in HF (respectively, r=0.64, p&lt;0.001, r=0.5, p&lt;0.001, r=0.53, p&lt;0.001 at ED during exercise; r=0.61, p&lt;0.001 at ES during exercise; Figure). Conclusions: In HF, the evidence of right to left IVS displacement appears worth of investigation, by intervening strictly to a reduced CO response, and RV-PA uncoupling during exercise. These findings clearly point to the utility of assessing how new and current therapeutic strategies may modulate the negative septum displacement and overall cardiac mechanics.

Cancer Risk Among Patients With Multiple Sclerosis: A 10-Year Nationwide Retrospective Cohort Study.

Previous literature has been diverging on cancer risk in people with multiple sclerosis (PwMS). Therefore, this study compared the risk of cancer in PwMS and a matched sample from the French general population. This 10-year nationwide retrospective matched cohort study (2012-2021) used data from the national French administrative health care database (99% coverage of the French population) to determine the time to the first incident cancer. PwMS were identified using their long-term disease (LTD) status, hospitalizations, and multiple sclerosis (MS)-specific drug reimbursements. The control population was matched 4:1 on age, sex, residence, insurance scheme, and cohort entry date. Participants were included if they had no history of cancer in the 3 years before inclusion. Patients with cancer were identified through LTD status, hospitalizations, chemotherapy, radiotherapy, or prostate cancer-specific drug reimbursements. Overall and cancer location-specific hazard ratios (HRs) for the first incident cancer were obtained from Fine and Gray models, and age- and sex-stratified estimates were reported. Participation in cancer screening through the 3 national programs (breast, colorectal, and cervical) were compared between groups. Cancer incidence was 799 per 100,000 person-years (PYs) (n = 8,368) among the 140,649 PwMS and 736 per 100,000 PYs (n = 31,796) among the 562,596 matched controls (70.8% of women; follow-up: 7.6 ± 3.2 years). A small overall risk increase was observed for PwMS (HR 1.06, 95% CI 1.03-1.08), mostly in women (HR 1.08, 95% CI 1.05-1.11). Risk varied by cancer types and was lower for prostate (HR 0.80, 95% CI 0.73-0.88), breast (HR 0.91, 95% CI 0.86-0.95), and colorectal (HR 0.90, 95% CI 0.84-0.97) cancer and higher for bladder (HR 1.71, 95% CI 1.54-1.89), brain (HR 1.68, 95% CI 1.42-1.98), and cervical (HR 1.24, 95% CI 1.12-1.38) cancer in PwMS. Cancer risk was higher in PwMS younger than 55 years (HR 1.20, 95% CI 1.15-1.24) but decreased in PwMS aged 65 years and older (HR 0.89, 95% CI 0.85-0.94). This trend was found in all cancer locations. There were fewer PwMS getting screened than controls (all programs), with a particularly pronounced difference among those aged 65 years and older. Cancer risk was slightly increased in PwMS, particularly for urogenital cancers, possibly due to surveillance bias. Risk fluctuated depending on age, perhaps due to varying generational screening practices (i.e., diagnosis neglect in the older PwMS) and risk factors.

Abstract 4113263: Serum Metabolomics Adds Predictive Value to Clinical and Polygenic Risk Scores for Atrial Fibrillation: A UK Biobank study of &gt;240,000 Patients

Background: Atrial fibrillation (AF) poses substantial morbidity and mortality burdens globally. It is critical to identify individuals at high-risk for AF to implement both lifestyle modifications and close monitoring for initiation of oral anticoagulants. Currently, risk prediction in AF relies on clinical scores such as CHARGE-AF. Small molecule metabolites are thought to play a role in AF pathogenesis, but the additional predictive value of metabolomics over clinical and genetic risk factors is unknown. Using the UK Biobank (UKBB), we sought to perform this evaluation. Methods: We included all UKBB participants with complete 1 H-NMR metabolite measurements. We excluded participants with prior history of AF or with incomplete AF Polygenic Risk Score (AF-PRS) or CHARGE-AF components. The final cohort included 240,628 patients. In-patient records, primary care notes, and death registries were queried to identify patients who had developed incident AF within 5 years of enrollment. The cohort was divided into a 80/20:Train/Test split. We compared the performance of an Elastic-Net regularized Cox Proportional Hazards (EN-CPH) model trained on CHARGE-AF, AF-PRS, and the full 170 metabolite panel to a CPH model trained only on CHARGE-AF and AF-PRS. Results: Within 5 years, 4,174 (1.7%) patients developed AF. Compared to the control population, the incident AF cases were more likely to be older (62 vs. 56, p&lt;0.001), male (65% vs 45%, p&lt;0.001), and have higher CHARGE-AF and AF-PRS scores (p&lt;0.001). After training the EN-CPH model on CHARGE-AF, AF-PRS, and 170 metabolites, the final model included 8 metabolites on top of CHARGE-AF (HR: 1.28) and AF-PRS (HR: 1.11). After adjusting for CHARGE-AF and AF-PRS, creatinine level was associated with increased risk of AF (HR: 1.01) while linoleic acid level (HR: 0.985) was associated with decreased risk. Furthermore, total cholesterol, esterified cholesterol, free cholesterol, cholesteryl esters in IDL, omega-6 fatty acids, and cholesterol in large LDL were associated with HR 0.993-0.999. The EN-CPH model out-performed the CPH model without metabolomics on the test set (AUC 0.786 vs 0.753, p &lt; 0.001). Conclusions: The addition of metabolomics to clinical and genomic risk scores improves prediction of 5-year incident AF within a general population. This study highlights the significance of the metabolome as an independent prognosticator of AF risk. Further study of the mechanisms by which selected metabolites alter AF risk is needed.

Abstract Su603: Out-of-hospital cardiac arrest presenting with ST-segment elevation myocardial infarction: Real world assessment and treatment

Background: Coronary artery disease is a common cause of out-of-hospital cardiac arrest (OHCA). Early recognition myocardial infarction (MI) and initiating appropriate therapy has been shown to improve outcomes in OHCA. While early coronary intervention is part of guidelines-based care, the implementation of these recommendation in actual US practice remains poorly described. Objectives: This study aims to characterize patients presenting with OHCA and MI in a cohort from 4 hospitals, to assess care of these patients in actual practice. Methods: We employed a database of OHCA patients admitted to four hospitals within the University of Pennsylvania Hospital System between 01/2019-12/2023. For this study, we included all patients that were diagnosed with STEMI during emergency department evaluation, and collected demographic, arrest, coronary angiographic, and ECG data from electronic health records. SAS was used for statistical analysis. Results: A total of 43 MI cases were included; mean age was 60.6±11.8 y, 65% male, mean BMI 30.0±6.3, 58.1% African American, 27.9% White or Caucasian, and 16.3% had a medical history of myocardial infarction. 74.4% were witnessed, and 44.2% received bystander CPR[BA1]. Initial shockable rhythm was present in 34.9%. 38 survived to hospital admission, and 9 were discharged alive. The first obtained 12-lead ECG (median time to ECG: 13min, (IQR: 3.5-26.5)) found that ST-segment elevations were present in 88.4%, ST-segment depressions in 44.2%, T-wave inversions in 4.7%, QT prolongation in 9.3%, and wide QRS in 18.6%. Sinus rhythm was most common (41.9%), followed by junctional rhythm (9.3%). Left and right bundle branch blocks (4.3% and 7%, respectively) and AV block (7%) were recorded. Coronary angiography (median time to angiography: 1.95hr (IQR: 1.3-4.2)) was performed in 29 patients and revealed that left anterior descending artery (28.6%) was the most common location for a culprit lesion, followed by the right coronary artery (21.4%), and left circumflex artery (10.7%). Notably, in 21.4% of cases, no culprit lesion was identified. 13 patients underwent angioplasty with stent, 4 received balloon angioplasty, and 1 underwent aspiration thrombectomy. Conclusion: This study highlights important attributes of OHCA patients presenting with MI. This paves the way for larger studies comparing ECG and coronary angiography findings between MI and control populations to help identify and improve acute care for OHCA MI patients.

Clinical and demographic factors linked to low-value emergency department visits in pediatric patients with spina bifida.

Identifying factors associated with emergency visits that could be delivered at lower-cost sites may help guide population health strategies for pediatric patients with spina bifida. Emergency department encounters (2016-2023) by patients with spina bifida (< 18-years-old) in Pediatric Health Information System were identified. Absence of clinical and imaging charges was defined as low-value emergency visit. We utilized a control population of patients (<18-years-old) with obstructive/reflux uropathy who presented for emergency department encounters (2016-2023). Mixed effects (with repeated individual measurements as random effect) logistic regression was fitted to model odds of low-value emergency visit. In total, we included 22,672 emergency visits by patients with spina bifida. 20.7% of these emergency visits were low-value versus 17.7% in controls (p<0.001). Costs related to low-value emergency visits account for 3.8% of all costs for emergency visit-related encounters in patients with spina bifida. Low-value emergency visits were associated with younger age [OR 1.05 (1.04 - 1.06) per year younger], Hispanic/Latino [OR 1.21 (1.06 - 1.39) compared to non-Hispanics], black [OR 1.35 (1.16 - 1.58) compared to white], public insurance [OR 1.14 (1.01 - 1.29) compared to private insurance], and genitourinary encounter diagnosis [OR 1.16 (1.04 - 1.30)]. Using a standard patient, we found that the odds of low-value emergency visit across hospitals ranged from 0.31 to 5.36. Younger age, Hispanic/Latino ethnicity, black and other race, public insurance, and genitourinary encounter diagnosis were associated with higher odds for low-value emergency visits in pediatric patients with spina bifida. There was wide variation across hospitals that warrants further investigation to elucidate best practices.

Survival After Shunt Therapy in Normal-Pressure Hydrocephalus: A Meta-Analysis of 1614 Patients

Background: Ventriculoperitoneal (VP) shunt therapy is a crucial intervention for normal-pressure hydrocephalus (NPH). This meta-analysis delves into survival time and the impact of baseline symptom burden on survival after VP shunt therapy for NPH, employing reconstructed pooled survival curves and a one-stage meta-analysis. Methods: IPD regarding overall survival (OS) were acquired from published Kaplan–Meier charts, utilizing the R package IPDfromKM in R (Version 4.3.1, the R Foundation for Statistical Computing). Reconstructed Kaplan–Meier charts were then generated from the pooled IPD data. Both one-stage and two-stage meta-analyses were executed, with hazard ratios (HRs) employed as metrics to evaluate effectiveness. Results: From the initial screening of 216 records, five articles encompassing 1614 patients met the eligibility criteria for inclusion. In two of the five included studies, overall survival was stratified by gait score (1–4 vs. ≥4) in 1043 patients, continence score (1–3 vs. ≥4) in 1022 patients, and mRS (0–2 vs. ≥3) in 956 patients. Patients with good gait demonstrated a mean survival of 8.24 years, while those with poor gait had a mean survival of 6.19 years (log-rank test: p &lt; 0.001). The HR for gait was 2.25 (95% CI: 1.81–2.81, p &lt; 0.001). Continence score stratification revealed a significant difference in survival time (log-rank test: p &lt; 0.001), with an HR of 1.66 (95% CI: 1.33–2.06, p &lt; 0.001). Similarly, mRS stratification demonstrated a significant survival difference (log-rank test: p &lt; 0.001), with an HR of 2.21 (95% CI: 1. 74–2.80, p &lt; 0.001). The reconstructed survival curves for all NPH patients treated with VP shunt therapy, pooling data from five studies, revealed a median survival time of 8.82 years (95% CI: 8.23–9.40). Survival rates at 1, 3, 5, 7, 9, 11, and 13 years were 95.7%, 83.8%, 70.5%, 59.5%, 48.7%, 35.8%, and 25.4%, respectively. Comparison with a general control population showed an HR of 1.79 (95% CI: 1.62–1.98, p &lt; 0.001). Conclusions: This comprehensive meta-analysis underscores the influence of baseline symptom burden on survival after VP shunt therapy in NPH. Therapy in the early stages for those without significant comorbidities may enhance survival.

DDDR-45. INVESTIGATING TEMOZOLOMIDE RESISTANCE IN GLIOBLASTOMA USING A CLINICALLY RELEVANT DOSING REGIMEN

Abstract The standard of care of management for glioblastoma, the most common primary malignant brain tumor in adults, involves maximal safe surgical resection followed by adjuvant chemoradiotherapy and six cycles of adjuvant temozolomide (TMZ). Approximately half of all treated patients develop chemoresistance to TMZ. The evolution of chemoresistance to temozolomide in vitro has historically involved continuous exposure to drug, as opposed to clinical dosing schedules, which involve five days of treatment followed by 23 days of recovery. Thus, in this study, we examined the emergence of chemoresistance to TMZ in vitro, using a clinical dosing regimen. Temozolomide dose response to RN1, an MGMT promoter unmethylated, IDH-wildtype, patient-derived glioblastoma cell line, was determined using cell titer glo. Cells were treated with TMZ for five days, followed by 23 days of recovery, in cycles. In each subsequent cycle, the dose of TMZ used was adjusted to previous cycle’s IC50, the concentration at which 50% of cell growth is inhibited. This protocol was repeated over three rounds. Cells treated with TMZ, as compared to the DMSO-treated control population, gradually acquired chemoresistance with a continuous increase in IC50. Our protocol therefore highlights how a clinically relevant dosing regimen may help capture the trajectory by which chemoresistance emerges, also allowing for the exploration of the molecular mechanisms underlying this phenomenon.

CTIM-33. IMMUNE BIOMARKERS OF TREATMENT RELATED IMAGING CHANGES IN GLIOBLASTOMA PATIENTS TREATED WITH STANDARD OF CARE PLUS PEMBROLIZUMAB

Abstract Pseudoprogression is a post-treatment imaging phenomenon in glioblastoma (GBM) that mimics true progression. Immune checkpoint inhibition with pembrolizumab has shown clinical benefits in multiple cancers and is a burgeoning treatment in GBM. Discerning pseudoprogression, or treatment-related imaging changes, from true progression is essential for appropriate treatment recommendations. However, there are no reliable biomarkers that can differentiate between pseudoprogression and true progression. Our prospective single-arm study (NCT 03347617) evaluated immune cell sub-populations (B-cells, T-cells, NK cells, monocytes, and myeloid cells) in newly diagnosed GBM patients who received standard-of-care chemoradiation plus concurrent pembrolizumab after maximal safe resection. Flow cytometry was performed on blood collected at predetermined timepoints (pre-treatment, post-radiation, suspected progression, and confirmed progression) and immune cell sub-populations were evaluated as covariates in univariable and multivariable logistic regression analysis. Of the 56 patients enrolled in this trial, 51 completed blood collection. Median survival was 13.84 months. Fifteen (29%) patients had pseudoprogression by modified RANO criteria and 36 (70.5%) had suspected or confirmed progression. Univariable analysis identified the following serum derived immune populations as being significantly associated with pseudoprogression: cytotoxic T-cell percentage (CD3+CD8+; HR: 1.10; p=0.045); total NK Cells percentage (CD3+CD56+; HR:0.912; p=0.0.053); IGD+CD27-naïve B-cells of B-cell percentage (HR: 0.976; p=0.014); IGD+CD27+non-switched B of B-cell percentage (HR:1.07; p=0.036). Backward selection multivariable logistic regression analysis confirmed the significant association of cytotoxic T-cell percentage (CD3+CD8+; HR:1.02; p=0.049) with pseudoprogression while controlling for IGD+CD27+non-switched B of B-cell percentage (HR:1.01, p=0.116). Increasing levels of serum cytotoxic T-cells in GBM patients treated with standard-of-care plus pembrolizumab is associated with pseudoprogression and may be used to differentiate between true progression. Validation of results is needed in a control population treated with standard-of-care prior to clinical implementation. Future studies will utilize serum immune biomarkers coupled with radiomic features to better characterize the pseudoprogression signature in GBM.

IMMU-04. G-AMINOBUTYRIC ACID DRIVES GLIOBLASTOMA IN FEMALES IN AN IMMUNE-DEPENDENT MANNER

Abstract Communication with a neuronal network and accumulation of myeloid-derived suppressive cells (MDSCs) support GBM growth and therapeutic resistance. GBM incidence and outcomes differ between males and females. We previously demonstrated that granulocytic (gMDSCs) drive GBM in females. However, a lack of understanding of the host factors regulating distinct MDSC subset function remains to be a major obstacle for therapeutically targeting these cells in GBM. In this study, we identified γ-aminobutyric acid (GABA) pathway as a modulator of gMDSC activity. gMDSCs from both healthy adults and glioma patients, expressed significantly higher levels of GABA receptor B1 (GABBR1) and GABA receptor B2 (GABBR2) compared to monocytic MDSCs (mMDSCs). On contrary, GABA receptor A (GABAAR) subunits were not expressed in either MDSC subtype. Given these differences, we investigated the effect of GABA on MDSC metabolism and found an increase in L-arginine metabolism specifically in female gMDSCs following GABA treatment while no significant metabolic changes were observed in any other groups. In MDSCs, L-arginine can be metabolized via nitric oxide synthase (NOS2) or Arginase-1 (Arg-1). While treatment with GABA or the GABBR agonist baclofen increased NOS2 production in specifically in female gMDSCs, Arg-1 was not affected. Mechanistically, baclofen enhanced gMDSC-mediated T cell suppression in females. In preclinical models of GBM, baclofen treatment resulted in a truncated survival only in immunocompetent female mice, which was reversed by pharmaceutical inhibition of NOS2. Clinically, female patients on GABA analogs (pregabalin, gabapentin) had significantly lower 12-month survival probabilities compared to control population. This effect was not observed in male patients. Conversely, inhibition of GABBR with CGP35348 prolonged survival in female GBM models and reprogramed the immunosuppressive tumor microenvironment. Collectively, these results highlight that GABA alters anti-tumor immune response in a sex-specific manner. These results support the future assessment of GABA pathway inhibitors as potential immunotherapy agents in GBM.

Establishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi

BackgroundGenetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions.MethodsTo address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi, we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti, exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR’s applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex (AngDsx) splicing module in An. stephensi. Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope.ResultsOur results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species.ConclusionsSEPARATOR’s independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes.Graphical

Synthetic translational coupling element for multiplexed signal processing and cellular control.

Repurposing natural systems to develop customized functions in biological systems is one of the main thrusts of synthetic biology. Translational coupling is a common phenomenon in diverse polycistronic operons for efficient allocation of limited genetic space and cellular resources. These beneficial features of translation coupling can provide exciting opportunities for creating novel synthetic biological devices. Here, we introduce a modular synthetic translational coupling element (synTCE) and integrate this design with de novo designed riboregulators,toehold switches. A systematic exploration of sequence domain variants for synTCEs led to the identification of critical design considerations for improving the system performance. Next, this design approach was seamlessly integrated into logic computations and applied to construct multi-output transcripts with well-defined stoichiometric control. This module was further applied to signaling cascades for combined signal transduction and multi-input/multi-output synthetic devices. Further, the synTCEs can precisely manipulate the N-terminal ends of output proteins, facilitating effective protein localization and cellular population control. Therefore, the synTCEs could enhance computational capability and applicability of riboregulators for reprogramming biological systems, leading to future applications in synthetic biology, metabolic engineering and biotechnology.

Creating an Environment Free from Sexual Violence Against Women and Children Within the Cultural Values of Mitawe in West Sulawesi

This research aims to create an environment free from sexual violence against women and children in West Sulawesi Province through the application of Mitawe cultural values. The Mitawe culture, which emphasizes politeness, respect, and dignity, is seen as an important tool in preventing sexual violence within the Mandar community. The research method used is empirical law, involving observation, in-depth interviews with traditional leaders and community leaders, as well as document analysis from the Office of Women's Empowerment, Child Protection, Population Control, and Family Planning (DP3AP2KB) of West Sulawesi Province. The research findings show that Mitawe values can be effectively integrated into sexual violence prevention programs through various interventions carried out in local communities. The implementation of these values has successfully raised public awareness about the importance of respecting the rights of women and children, as well as reducing the incidence of sexual violence in several districts. Moreover, collaboration with DP3AP2KB has enabled the implementation of more structured and sustainable prevention strategies, including training for victim advocates and public awareness campaigns. This research also found that strengthening local cultural values, such as Mitawe, can be an effective approach in creating a safer society free from sexual violence. These findings provide a foundation for developing more inclusive and culturally sensitive policies in the effort to protect women and children in West Sulawesi.

Gestational breast cancer: distinctive molecular and clinico-epidemiological features

Gestational breast cancer (GBC), defined as breast cancer (BC) diagnosed during pregnancy or the first-year post-partum, accounts for 6–15% of BC cases in women aged 20–44 years. GBC has worse prognosis than non-GBC, but reasons behind are not clear. The GEICAM/2012–03 Study (Molecular Characterization of Gestational Breast Cancer) is a multicenter prospective/retrospective observational registry of patients diagnosed with GBC. From November 2014 to June 2015 seventy patients diagnosed with GBC were included in the study, 30 diagnosed during pregnancy and 40 after delivery. Our current study was aimed to explore differences in epidemiological, clinico-pathological and gene expression features of GBC tumors, from the GEICAM/2012–03 Study, compared to non-GBC tumors from patients of similar age (< 43 years) from six different GEICAM studies, used as non- GBC control population. As per the main objective, the study found multiple differences showing GBC tumors as a different biological entity. GBC showed a more aggressive biology, with higher Ki67 levels, higher incidence of breast and/or ovarian cancer family history, and germline deleterious BRCA1/2 mutations, and are enriched in basal-like intrinsic subtype. GBC patients showed a lower number of tumor infiltrating lymphocytes, while specific genetic signatures highlight differences in GBC´s distinctive transcriptome. Our study shows that GBC is potentially a clinically and molecularly different entity, with specific epidemiological, clinical, and histological features, as well as a distinctive altered immune state and genetic signature. Nevertheless, further studies are needed to better understand the biology of GBC and to identify new targets against which develop new, more effective, targeted therapies.

Pathological changes on the skin due to the combined effects of polycyclic aromatic hydrocarbons and ultraviolet radiation

Introduction. In the National Healthcare Project, one of the key areas is the federal project "Fight against oncological diseases for 2019-2024". The influence of the nature of work activities of workers in contact with industrial carcinogens — polycyclic aromatic hydrocarbons (PAHs) significantly increases the risk of skin neoplasms, as evidenced by differences in the frequency of their occurrence among representatives of different labor categories. The leading factor in the development of skin neoplasms is ultraviolet radiation (UV) of the A and B spectrum. There is literature evidence of a possible mutual enhancement of the combined effects of PAHs and UV in the occurrence of skin neoplasms and their malignancy. The study aims to evaluate the role of the combined effects of polycyclic aromatic hydrocarbons (PAHs) and ultraviolet radiation (UV) in the occurrence of pigmented skin tumors in workers engaged in the construction of highways and highways and in housing and communal services. Materials and methods. During periodic medical examinations (PMEs) in 2022–2024, specialists have examined 140 people who formed 4 study groups. The researchers have conducted a physical examination of skin tumors; examination in the rays of a Wood lamp; dermatoscopy of skin tumors using a Heine Delta 20 dermatoscope. Hyperpigmentation was assessed according to the hyperpigmentation area and severity index (NASI). Results. During the clinical examination, the authors assessed the presence of pigmentation and skin neoplasms (lentigo, ephelides, seborrheic keratomas). The most frequent localizations of pigmentation and skin neoplasms were open areas of the body: face, hands, upper third of the back. At the same time, in asphalt concrete workers — group I, working under the influence of PAHs and solar insolation (spectrum A and B, in the range of 320–400 nm and 280–320 nm), specialists revealed pigmented neoplasms in a larger number of individuals compared with the population control for all skin localizations. The HASI index (in the surveyed groups) showed that the highest rates (24–48) of HASI were observed in a larger number of individuals — 57.1% — from group I (UV+PAHs), compared with groups of individuals (II, III, IV) who do not work under conditions of exposure to solar insolation (spectra A and B, in the range of 320–400 nm and 280–320 nm). The authors noted that the intensity of pigmentation is higher and the lesion area is larger when assessing the foci of hyperpigmentation in the rays of the Wood lamp compared with the clinical examination in all the examined groups. Limitations. The study is limited by the number of subjects examined, corresponding to the design of the study and the time interval of the study. Conclusion. To obtain objective results of the study of the role of the combined effects of polycyclic aromatic hydrocarbons (PAHs) and ultraviolet irradiation (UV) in the occurrence of pigmented skin tumors, scientists used a method for determining the hyperpigmentation area index and severity index (HASI) in the rays of a Wood's lamp. When comparing the results obtained, experts revealed an increase in the area of hyperpigmentation and its intensity with localization in open areas of the body in workers engaged in the construction of highways and housing and communal services, in contact with the combined effects of carcinogens (PAHs) and ultraviolet irradiation (UV A and B). Further in-depth studies are needed to study the etiopathogenesis of photogenotoxicity depending on the dose-effect ratio. Ethics. The work was performed in accordance with the ethical standard set out in the Helsinki Declaration of the World Medical Association of 1964 (as amended and supplemented in 2013). The authors conducted all the studies after receiving informed consent and a decision of the Local Ethics Committee (extract from Protocol No. 7 dated 10/21/2020).