Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Smooth muscle cell-derived Cxcl12 directs macrophage accrual and sympathetic innervation to control thermogenic adipose tissue.

Sympathetic innervation of brown adipose tissue (BAT) controls mammalian adaptative thermogenesis. However, the cellular and molecular underpinnings contributing to BAT innervation remain poorly defined. Here, we show that smooth muscle cells (SMCs) support BAT growth, lipid utilization, and thermogenic plasticity. Moreover, we find that BAT SMCs express and control the bioavailability of Cxcl12. SMC deletion of Cxcl12 fosters brown adipocyte lipid accumulation, reduces energy expenditure, and increases susceptibility to diet-induced metabolic dysfunction. Mechanistically, we find that Cxcl12 stimulates CD301+ macrophage recruitment and supports sympathetic neuronal maintenance. Administering recombinant Cxcl12 to obese mice or leptin-deficient (Ob/Ob) mice is sufficient to boost macrophage presence and drive sympathetic innervation to restore BAT morphology and thermogenic responses. Altogether, our data reveal an SMC chemokine-dependent pathway linking immunological infiltration and sympathetic innervation as a rheostat for BAT maintenance and thermogenesis.

Augmented-reality-assisted intraoral scanning: A proof-of-concept study.

PURPOSE: The aims of the present study were (a) to compare the scanning time and image count to complete optical scans of a typodont between augmented-reality-assisted intraoral scanning (ARIOS) and intraoral scanning (IOS); (b) to compare the accuracy of the digital casts derived from ARIOS and IOS; (c) to compare participant-related outcomes between ARIOS and IOS. MATERIALS AND METHODS: A multi-session within-subject experiment was conducted to compare ARIOS and IOS. Thirty-one dental students participated in the study. Following a trial session, each participant obtained optical scans under ARIOS and IOS conditions. The time required to complete the scan, and the number of images taken were recorded. Participant feedback was collected using entry, exit, and NASA-Task Load Index (TLX) surveys. The accuracy of the digital casts derived from the optical scans was measured in root mean square error (RMSE). RESULTS: The present study found a 6.8% increase in preference for ARIOS from entry to exit survey. Slightly more participants favored the ARIOS setup compared to IOS; 54.8% of participants favored ARIOS, 9.7% were indifferent, and 35.5% favored IOS. NASA-TLX subscale ratings were higher for IOS in general apart from mental demand. The accuracy of the digital casts between ARIOS and IOS was comparable in RMSE. CONCLUSION: ARIOS was advantageous compared to IOS in ergonomics, improved scanner tracking, and ease of scanner orientation. However additional trials, increased field of view, and better superimposition of scanning status to the target site were improvements desired by the study participants.

Quantifying the Educational Value of a Student-Run Free Clinic.

BACKGROUND AND OBJECTIVES: Student-run free clinics (SRFCs) are settings in which students in health professions gain clinical experience, often while providing free or reduced-cost health care to the surrounding community. The current literature quantifies the many benefits these clinics provide to their patients and the impact they have on students' future careers; but few previous studies have assessed the financial impact of the education provided at an SRFC. We report on a net educational benefit, an educational benefit to educational cost ratio, and a net educational benefit to educational cost ratio of one SRFC from the perspective of the university. METHODS: We calculated the value of education by multiplying all student hours worked in the clinic by the associated value of 1 hour in the typical tuition-based curriculum. Clinic educational costs and student hours were obtained from clinic records from August 1, 2021 through July 31, 2022. RESULTS: We found the total educational value students received to be $73,571 over one academic year. The educational operating expenses of the clinic totaled $9,053, resulting in a benefit-cost ratio of 8.13. CONCLUSIONS: This analysis demonstrated a potential financial advantage of operating an SRFC when assessing clinic education expenses in relation to the value of university-generated education. Our research may serve as a starting point to showcase the economic benefit of SRFCs to their parent institutions and encourage further analysis of other benefits SRFCs may provide to institutions of higher education.

Platelet-derived growth factor receptor beta is required for embryonic specification and confinement of the adult white adipose lineage.

White adipose tissue (WAT) development and adult homeostasis rely on distinct adipocyte progenitor cells (APCs). While adult APCs are defined early during embryogenesis and generate adipocytes after WAT organogenesis, the mechanisms underlying adult adipose lineage determination and preservation remain undefined. Here, we uncover a critical role for platelet-derived growth factor receptor beta (Pdgfrß) in identifying the adult APC lineage. Without Pdgfrß, APCs lose their adipogenic competency to incite fibrotic tissue replacement and inflammation. Through lineage tracing analysis, we reveal that the adult APC lineage is lost and develops into macrophages when Pdgfrß is deleted embryonically. Moreover, to maintain the APC lineage, Pdgfrß activation stimulates p38/MAPK phosphorylation to promote APC proliferation and maintains the APC state by phosphorylating peroxisome proliferator activated receptor gamma (Pparγ) at serine 112. Together, our findings identify a role for Pdgfrß acting as a rheostat for adult adipose lineage confinement to prevent unintended lineage switches.

Accuracy of Estimated Fetal Weight by Ultrasound Versus Leopold Maneuver.

ABSTRACT: Estimated fetal weight (EFW) is frequently used for clinical decision-making in obstetrics. The goals of this study were to determine the accuracy of EFW assessments by Leopold and ultrasound and to investigate any associations with maternal characteristics. Postgraduate years 1 and 2 obstetrics and gynecology resident physicians from Harbor-UCLA Medical Center from 2014 to 2020 performed EFW assessments on 10 preterm (<37 weeks' gestational age) fetuses by ultrasound biometry and 10 full-term (≥37 weeks' gestational age) fetuses by ultrasound biometry and Leopold maneuver. Assessments were included if the patients delivered within 2 weeks of the assessments. One thousand six hundred ninety-seven EFW assessments on 1183 patients performed by 33 residents were analyzed; 72.6% of sonographic full-term EFWs, 69% of Leopold full-term EFWs, and 61.5% of sonographic preterm EFWs were within 10% of the neonatal birth weight (BW). The lowest estimation error in our study occurred when actual BW was 3600 to 3700 g. After adjusting for BW, residents were found to have lower accuracy when the mother had a higher body mass index (BMI) for full-term estimation methods (Leopold and ultrasound, ß = 0.13 and 0.12, P = 0.001 and 0.002, respectively). Maternal BMI was not related to estimation error for preterm fetuses ( ß = 0.01, P = 0.75). Clinical and sonographic EFW assessments performed by obstetrics and gynecology junior residents are within 10% of neonatal BW much of the time. In our cohort, they tended to overestimate EFWs of lower-BW infants and underestimate EFWs of higher-BW infants. Accuracy of full-term EFW assessments seems to decrease with increasing maternal BMI.

Humoral and Cellular Immunogenicity of 3 Doses of BNT162b2 in Children With Kidney Diseases.

Introduction: Patients with severe kidney diseases are at risk of complications from COVID-19; however, little is known about the effectiveness of COVID-19 vaccines in children and adolescents with kidney diseases. Methods: We investigated the immunogenicity and safety of an accelerated 3-dose primary series of COVID-19 vaccination among 59 pediatric patients with chronic kidney disease (CKD) (mean age 12.9 years; 30 male) with or without immunosuppression, dialysis, or kidney transplant. Dosage was 0.1 ml BNT162b2 to those aged 5 to 11 years, and 0.3 ml BNT162b2 to those aged 11 to 18 years. Results: Three doses of either vaccine type elicited significant antibody responses that included spike receptor-binding domain (S-RBD) IgG (90.5%-93.8% seropositive) and surrogate virus neutralization (geometric mean sVNT% level, 78.6%-79.3%). There were notable T cell responses. Weaker neutralization responses were observed among those on immunosuppression, especially those receiving higher number of immunosuppressants or on mycophenolate mofetil. Neutralization was reduced against Omicron BA.1 compared to wild type (WT, i.e., ancestral) (post-dose 3 sVNT% level; 82.7% vs. 27.4%; P < 0.0001). However, the T cell response against Omicron BA.1 was preserved, which likely confers protection against severe COVID-19. Infected patients exhibited hybrid immunity after vaccination, as evidenced by the higher Omicron BA.1 neutralization response among these infected patients who received 2 doses compared with those who were uninfected. Generally mild or moderate adverse reactions following vaccines were reported. Conclusion: An accelerated 3-dose primary series with BNT162b2 is immunogenic and safe in young children and adolescents with kidney diseases.

Unlocking the potential of allogeneic Vδ2 T cells for ovarian cancer therapy through CD16 biomarker selection and CAR/IL-15 engineering.

Allogeneic Vγ9Vδ2 (Vδ2) T cells have emerged as attractive candidates for developing cancer therapy due to their established safety in allogeneic contexts and inherent tumor-fighting capabilities. Nonetheless, the limited clinical success of Vδ2 T cell-based treatments may be attributed to donor variability, short-lived persistence, and tumor immune evasion. To address these constraints, we engineer Vδ2 T cells with enhanced attributes. By employing CD16 as a donor selection biomarker, we harness Vδ2 T cells characterized by heightened cytotoxicity and potent antibody-dependent cell-mediated cytotoxicity (ADCC) functionality. RNA sequencing analysis supports the augmented effector potential of Vδ2 T cells derived from CD16 high (CD16Hi) donors. Substantial enhancements are further achieved through CAR and IL-15 engineering methodologies. Preclinical investigations in two ovarian cancer models substantiate the effectiveness and safety of engineered CD16Hi Vδ2 T cells. These cells target tumors through multiple mechanisms, exhibit sustained in vivo persistence, and do not elicit graft-versus-host disease. These findings underscore the promise of engineered CD16Hi Vδ2 T cells as a viable therapeutic option for cancer treatment.

Profiling ovarian cancer tumor and microenvironment during disease progression for cell-based immunotherapy design.

Ovarian cancer (OC) is highly lethal due to late detection and frequent recurrence. Initial treatments, comprising surgery and chemotherapy, lead to disease remission but are invariably associated with subsequent relapse. The identification of novel therapies and an improved understanding of the molecular and cellular characteristics of OC are urgently needed. Here, we conducted a comprehensive analysis of primary tumor cells and their microenvironment from 16 chemonaive and 10 recurrent OC patient samples. Profiling OC tumor biomarkers allowed for the identification of potential molecular targets for developing immunotherapies, while profiling the microenvironment yielded insights into its cellular composition and property changes between chemonaive and recurrent samples. Notably, we identified CD1d as a biomarker of the OC microenvironment and demonstrated its targeting by invariant natural killer T (iNKT) cells. Overall, our study presents a comprehensive immuno-profiling of OC tumor and microenvironment during disease progression, guiding the development of immunotherapies for OC treatment, especially for recurrent disease.

Extinction risks and mitigation for a megaherbivore, the giraffe, in a human-influenced landscape under climate change.

Megaherbivores play "outsized" roles in ecosystem functioning but are vulnerable to human impacts such as overhunting, land-use changes, and climate extremes. However, such impacts-and combinations of these impacts-on population dynamics are rarely examined using empirical data. To guide effective conservation actions under increasing global-change pressures, we developed a socially structured individual-based model (IBM) using long-term demographic data from female giraffes (Giraffa camelopardalis) in a human-influenced landscape in northern Tanzania, the Tarangire Ecosystem. This unfenced system includes savanna habitats with a wide gradient of anthropogenic pressures, from national parks, a wildlife ranch and community conservation areas, to unprotected village lands. We then simulated and projected over 50 years how realistic environmental and land-use management changes might affect this metapopulation of female giraffes. Scenarios included: (1) anthropogenic land-use changes including roads and agricultural/urban expansion; (2) reduction or improvement in wildlife law enforcement measures; (3) changes in populations of natural predators and migratory alternative prey; and (4) increases in rainfall as predicted for East Africa. The factor causing the greatest risk of rapid declines in female giraffe abundance in our simulations was a reduction in law enforcement leading to more poaching. Other threats decreased abundances of giraffes, but improving law enforcement in both of the study area's protected areas mitigated these impacts: a 0.01 increase in giraffe survival probability from improved law enforcement mitigated a 25% rise in heavy rainfall events by increasing abundance 19%, and mitigated the expansion of towns and blockage of dispersal movements by increasing abundance 22%. Our IBM enabled us to further quantify fine-scale abundance changes among female giraffe social communities, revealing potential source-sink interactions within the metapopulation. This flexible methodology can be adapted to test additional ecological questions in this landscape, or to model populations of giraffes or other species in different ecosystems.

Genetic evidence of population subdivision among Masai giraffes separated by the Gregory Rift Valley in Tanzania.

The Masai giraffe has experienced a population decline from 70,000 to 35,000 in the past three decades and was declared an endangered subspecies by the IUCN in 2019. The remaining number of Masai giraffe are geographically separated by the steep cliffs of the Gregory Rift escarpments (GRE) in Tanzania and Kenya dividing them into two populations, one west and one east of the GRE. The cliffs of the GRE are formidable barriers to east-west dispersal and gene flow and the few remaining natural corridors through the GRE are occupied by human settlements. To assess the impact of the GRE on Masai giraffe gene flow, we examined whole genome sequences of nuclear and mitochondrial DNA (mtDNA) variation in populations located east (Tarangire ecosystem) and west (Serengeti ecosystem) of the GRE in northern Tanzania. Evidence from mtDNA variation, which measures female-mediated gene flow, suggests that females have not migrated across the GRE between populations in the Serengeti and Tarangire ecosystems in the past ~289,000 years. The analysis of nuclear DNA variation compared to mtDNA DNA variation suggests that male-mediated gene flow across the GRE has occurred more recently but stopped a few thousand years ago. Our findings show that Masai giraffes are split into two populations and fulfill the criteria for designation as distinct evolutionary significant units (ESUs), which we denote as western Masai giraffe and eastern Masai giraffe. While establishing giraffe dispersal corridors across the GRE is impractical, conservation efforts should be focused on maintaining connectivity among populations within each of these two populations. The importance of these efforts is heightened by our finding that the inbreeding coefficients are high in some of these Masai giraffe populations, which could result in inbreeding depression in the small and fragmented populations.

Direct Oral Anticoagulation Versus Warfarin in Left Ventricular Thrombus: Pooled Analysis of Randomized Controlled Trials.

Patients with impaired left ventricular (LV) function can develop LV thrombus, a potentially life-threatening condition due to risk of stroke and embolization. Conventional treatment with vitamin K antagonists (VKAs; e.g., warfarin) puts patients at risk of bleeding, and the use of direct oral anticoagulants (DOACs) appears promising, although data are scant. We searched the published English language literature for randomized controlled trials (RCTs) comparing DOACs with VKAs in LV thrombus. End points were failure to resolve, thromboembolic events (stroke, embolism), bleeding, or any adverse event (composite of thromboembolism or bleeding), or all-cause death. Data were pooled and analyzed in hierarchical Bayesian models. In three eligible RCTs, 141 patients were studied during an average of 4.6 months (53.8 patient-years; n = 71 assigned to DOAC, n = 70 assigned to VKA). A similar number of patients in each treatment arm demonstrated failure to resolve (DOAC: 14/71 vs. VKA: 15/70) and death events (3/71 vs. 4/70). However, patients on DOACs suffered fewer strokes/thromboembolic events (1/71 vs. 7/70; log odds ratio [OR], -2.02 [95% credible interval (CI95 ), -4.53 to -0.31]) and fewer bleeding events (2/71 vs. 9/70; log OR, -1.62 [CI95 , -3.43 to -0.26]), leading to fewer patients on DOACs with any adverse event versus VKAs (3/71 vs. 16/70; log OR, -1.93 [CI95 , -3.33 to -0.75]). In conclusion, pooled analysis of RCT data favors DOACs over VKAs in patients with LV thrombus in terms of both efficacy and safety.

Case report: Resolution of malignant canine mast cell tumor using ketogenic metabolic therapy alone.

Background: Mast cell tumors (MCT) are common neoplasms in dogs and are similar to most other malignant cancers in requiring glucose for growth, regardless of histological grade. Ketogenic metabolic therapy (KMT) is emerging as a non-toxic nutritional intervention for cancer management in animals and humans alike. We report the case of a 7 years-old Pit Bull terrier that presented in 2011 with a cutaneous mast cell tumor under the right nostril. Methods: The patient's parent refused standard of care (SOC) and steroid medication after initial tumor diagnosis due to the unacceptable adverse effects of these treatments. Following tumor diagnosis, the patient's diet was switched from Ol'Roy dog food to raw vegetables with cooked fish. The tumor continued to grow on this diet until July, 2013 when the diet was switched to a carbohydrate free, raw calorie restricted ketogenic diet consisting mostly of chicken and oils. A dog food calculator was used to reduce calories to 60% (40% calorie restriction) of that consumed on the original diet. A total of 444 kilocalories were given twice/day at 12 h intervals with one medium-sized raw radish given as a treat between each meal. Results: The tumor grew to about 3-4 cm and invaded surrounding tissues while the patient was on the raw vegetable, cooked fish diet. The tumor gradually disappeared over a period of several months when the patient was switched to the carbohydrate free calorie restricted ketogenic diet. The patient lost 2.5 kg during the course of the calorie restriction and maintained an attentive and active behavior. The patient passed away without pain on June 4, 2019 (age 15 years) from failure to thrive due to an enlarged heart with no evidence of mast cell tumor recurrence. Conclusion: This is the first report of a malignant cutaneous mast cell tumor in a dog treated with KMT alone. The resolution of the tumor in this canine patient could have been due to the diet-induced energy stress and the restriction of glucose-driven aerobic fermentation that is essential for the growth of most malignant tumors. Further studies are needed to determine if this non-toxic dietary therapeutic strategy could be effective in managing other canine patients with malignant mast cell tumors.

Thermogenic adipose tissue in energy regulation and metabolic health.

The ability to generate thermogenic fat could be a targeted therapy to thwart obesity and improve metabolic health. Brown and beige adipocytes are two types of thermogenic fat cells that regulate energy balance. Both adipocytes share common morphological, biochemical, and thermogenic properties. Yet, recent evidence suggests unique features exist between brown and beige adipocytes, such as their cellular origin and thermogenic regulatory processes. Beige adipocytes also appear highly plastic, responding to environmental stimuli and interconverting between beige and white adipocyte states. Additionally, beige adipocytes appear to be metabolically heterogenic and have substrate specificity. Nevertheless, obese and aged individuals cannot develop beige adipocytes in response to thermogenic fat-inducers, creating a key clinical hurdle to their therapeutic promise. Thus, elucidating the underlying developmental, molecular, and functional mechanisms that govern thermogenic fat cells will improve our understanding of systemic energy regulation and strive for new targeted therapies to generate thermogenic fat. This review will examine the recent advances in thermogenic fat biogenesis, molecular regulation, and the potential mechanisms for their failure.

Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with dreadful clinical outcome. Tyrosine kinase inhibitors and immune checkpoint inhibitors are the only United States Food and Drug Administration-approved therapeutic options for patients with advanced HCC with limited therapeutic success. Ferroptosis is a form of immunogenic and regulated cell death caused by chain reaction of iron-dependent lipid peroxidation. Coenzyme Q10 (CoQ10)/ferroptosis suppressor protein 1 (FSP1) axis was recently identified as a novel protective mechanism against ferroptosis. We would like to explore whether FSP1 could be a potential therapeutic target for HCC. METHODS: FSP1 expression in human HCC and paired non-tumorous tissue samples were determined by reverse transcription-quantitative polymerase chain reaction, followed by clinicopathologic correlation and survival studies. Regulatory mechanism for FSP1 was determined using chromatin immunoprecipitation. The hydrodynamic tail vein injection model was used for HCC induction to evaluate the efficacy of FSP1 inhibitor (iFSP1) in vivo. Single-cell RNA sequencing revealed the immunomodulatory effects of iFSP1 treatment. RESULTS: We showed that HCC cells greatly rely on the CoQ10/FSP1 system to overcome ferroptosis. We found that FSP1 was significantly overexpressed in human HCC and is regulated by kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 pathway. FSP1 inhibitor iFSP1 effectively reduced HCC burden and profoundly increased immune infiltrates including dendritic cells, macrophages, and T cells. We also demonstrated that iFSP1 worked synergistically with immunotherapies to suppress HCC progression. CONCLUSIONS: We identified FSP1 as a novel, vulnerable therapeutic target in HCC. The inhibition of FSP1 potently induced ferroptosis, which promoted innate and adaptive anti-tumor immune responses and effectively suppressed HCC tumor growth. FSP1 inhibition therefore represents a new therapeutic strategy for HCC.

Age-dependent Pdgfrß signaling drives adipocyte progenitor dysfunction to alter the beige adipogenic niche in male mice.

Perivascular adipocyte progenitor cells (APCs) can generate cold temperature-induced thermogenic beige adipocytes within white adipose tissue (WAT), an effect that could counteract excess fat mass and metabolic pathologies. Yet, the ability to generate beige adipocytes declines with age, creating a key challenge for their therapeutic potential. Here we show that ageing beige APCs overexpress platelet derived growth factor receptor beta (Pdgfrß) to prevent beige adipogenesis. We show that genetically deleting Pdgfrß, in adult male mice, restores beige adipocyte generation whereas activating Pdgfrß in juvenile mice blocks beige fat formation. Mechanistically, we find that Stat1 phosphorylation mediates Pdgfrß beige APC signaling to suppress IL-33 induction, which dampens immunological genes such as IL-13 and IL-5. Moreover, pharmacologically targeting Pdgfrß signaling restores beige adipocyte development by rejuvenating the immunological niche. Thus, targeting Pdgfrß signaling could be a strategy to restore WAT immune cell function to stimulate beige fat in adult mammals.

COVID-19 vaccines versus pediatric hospitalization.

Vaccine effectiveness of BNT162b2 and CoronaVac against COVID-19-associated hospitalization and moderate-to-severe disease due to SARS-CoV-2 Omicron BA.2 is studied from the 1.36 million doses administered to 766,601 of 953,400 children aged 3-11 years and adolescents aged 12-18 years in Hong Kong as of April 2022. These vaccines confer substantial protection.