Genetically engineering glycolysis in T cells increases their antitumor function.

BACKGROUND: T cells play a central role in the antitumor response. However, they often face numerous hurdles in the tumor microenvironment, including the scarcity of available essential metabolites such as glucose and amino acids. Moreover, cancer cells can monopolize these resources to thrive and proliferate by upregulating metabolite transporters and maintaining a high metabolic rate, thereby outcompeting T cells. METHODS: Herein, we sought to improve T-cell antitumor function in the tumor vicinity by enhancing their glycolytic capacity to better compete with tumor cells. To achieve this, we engineered human T cells to express a key glycolysis enzyme, phosphofructokinase, in conjunction with Glucose transporter 3, a glucose transporter. We co-expressed these, along with tumor-specific chimeric antigen or T-cell receptors. RESULTS: Engineered cells demonstrated an increased cytokine secretion and upregulation of T-cell activation markers compared with control cells. Moreover, they displayed superior glycolytic capacity, which translated into an improved in vivo therapeutic potential in a xenograft model of human tumors. CONCLUSION: In summary, these findings support the implementation of T-cell metabolic engineering to enhance the efficacy of cellular immunotherapies for cancer.

Metabolic modeling of single Th17 cells reveals regulators of autoimmunity.

Metabolism is a major regulator of immune cell function, but it remains difficult to study the metabolic status of individual cells. Here, we present Compass, an algorithm to characterize cellular metabolic states based on single-cell RNA sequencing and flux balance analysis. We applied Compass to associate metabolic states with T helper 17 (Th17) functional variability (pathogenic potential) and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known Th17/regulatory T cell (Treg) differences, which we validated by metabolic assays. Compass also predicted that Th17 pathogenicity was associated with arginine and downstream polyamine metabolism. Indeed, polyamine-related enzyme expression was enhanced in pathogenic Th17 and suppressed in Treg cells. Chemical and genetic perturbation of polyamine metabolism inhibited Th17 cytokines, promoted Foxp3 expression, and remodeled the transcriptome and epigenome of Th17 cells toward a Treg-like state. In vivo perturbations of the polyamine pathway altered the phenotype of encephalitogenic T cells and attenuated tissue inflammation in CNS autoimmunity.

Targeting nucleotide metabolism as the nexus of viral infections, cancer, and the immune response.

Virus-infected cells and cancers share metabolic commonalities that stem from their insatiable need to replicate while evading the host immune system. These similarities include hijacking signaling mechanisms that induce metabolic rewiring in the host to up-regulate nucleotide metabolism and, in parallel, suppress the immune response. In both cancer and viral infections, the host immune cells and, specifically, lymphocytes augment nucleotide synthesis to support their own proliferation and effector functions. Consequently, established treatment modalities targeting nucleotide metabolism against cancers and virally infected cells may result in restricted immune response. Encouragingly, following the introduction of immunotherapy against cancers, multiple studies improved our understanding for improving antigen presentation to the immune system. We propose here that understanding the immune consequences of targeting nucleotide metabolism against cancers may be harnessed to optimize therapy against viral infections.

T Cell Activation Depends on Extracellular Alanine.

T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation.

Adaptation of Human iPSC-Derived Cardiomyocytes to Tyrosine Kinase Inhibitors Reduces Acute Cardiotoxicity via Metabolic Reprogramming.

Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance.

Defective respiration and one-carbon metabolism contribute to impaired naïve T cell activation in aged mice.

T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naïve T cell activation. We observed a decrease in the number and activation of naïve T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naïve T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.

Maintenance of CD4 T cell fitness through regulation of Foxo1.

Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor ß-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.

Collaboration Patterns and Processes Between Dentists and Dental Laboratories When Planning and Fabricating Implant-Supported Restorations.

INTRODUCTION: The success of implant-supported restorations is dependent on proper treatment planning, effective communication within the clinical team, and the use of appropriate methods and materials in the dental laboratory. The objective of this study was to determine collaboration trends between dentists and laboratories and to assess the common methods and materials involved in fabricating implant-supported restorations. METHODS: Questionnaires were distributed to dental laboratories and technicians. Seventy questionnaires were answered and were included in the data analyses. RESULTS: Most of the impressions (87%) were taken using an individual custom-made open tray. In 83% of impressions, screw-retained transfer units were used, and in 61% of cases, the units were splinted. Bite registration was recorded in 91% of the cases. In 80% of cases, articulator setup was done. When matching the shade of a restoration in the anterior, 57% of the technicians do so in collaboration with the dentist, and 39% match the shade independently. Type of restoration and abutment selection were done mainly by the technicians. Abutment selection was reported to be carried out by 72% of the technicians. CONCLUSIONS: Generally, dentists and technicians follow the standards recommended in the contemporary literature, especially, in major procedures such as impression taking, bite registration, and articulator setup. However, principal decisions, such as abutment and color shade are done mainly by technicians.

Consequences of experience and specialist training on the fabrication of implant-supported prostheses: A survey.

STATEMENT OF PROBLEM: Fabricating fixed restorations on implants requires that dentists stay up-to-date with the scientific publications. MATERIAL AND METHODS: A questionnaire containing 29 questions was sent to dentists, members of the Israeli Society of Prosthodontics, and university faculty members. Differences were evaluated using the chi-square and Fisher exact tests and Mann-Whitney U and Wilcoxon tests for questions enquiring into the frequency of use of certain methods (α=.05). RESULTS: A total of 155 questionnaires were completed by 84 general dental practitioners and 71 specialists and residents. Statistical differences (P<.05) were found in the course of the fabrication of the implant-supported prostheses in the diagnostic waxing, trial restoration, and impression-making processes. Trial restorations were often used by residents and specialists before starting an esthetic restoration. Diagnostic waxing was used when treating patients with complex esthetic needs, mainly by prosthodontists (P=.019). A custom tray for impression making was used more by residents and specialists than by general dental practitioners (P<.001). The open tray technique for impression making of single or up to 3 implants was performed mainly by residents, specialists, and general dental practitioners with professional experience of fewer than 15 years. General dental practitioners used primarily plastic or metal trays for impression making. CONCLUSIONS: Significantly, impression techniques and the use of custom open trays, diagnostic waxing, and trial restorations were performed by residents, specialists, and dentists with fewer than 15 years of experience.

Suppression by TFR cells leads to durable and selective inhibition of B cell effector function.

Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)-mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we found that TFR cells induced a distinct suppressive state in TFH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by TFR cells was durable and persisted even in the absence of TFR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome TFR cell-mediated suppression and inhibited TFR cells and stimulated B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating the function of TFR cells and antibody production.

Mitochondrial Biogenesis and Proteome Remodeling Promote One-Carbon Metabolism for T Cell Activation.

Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival.

The Retentive Strength of Cemented Zirconium Oxide Crowns after Dentin Pretreatment with Desensitizing Paste Containing 8% Arginine and Calcium Carbonate.

The effect of dentin pretreatment with Desensitizing Paste containing 8% arginine and calcium carbonate on the retention of zirconium oxide (Y-TZP) crowns was tested. Forty molar teeth were mounted and prepared using a standardized protocol. Y-TZP crowns were produced using computer-aided design and computer-aided manufacturing (CAD-CAM) technology. The 40 prepared teeth were either pretreated with Desensitizing Paste or not pretreated. After two weeks, each group was subdivided into two groups, cemented with either Resin Modified Glass Ionomer Cement (RMGIC) or Self Adhesive Resin Cement (SARC)). Prior to cementation, the surface areas of the prepared teeth were measured. After aging, the cemented crown-tooth assemblies were tested for retentive strength using a universal testing machine. The debonded surfaces of the teeth and crowns were examined microscopically at 10× magnification. Pretreating the dentin surfaces with Desensitizing Paste prior to cementation did not affect the retention of the Y-TZP crowns. The retentive values for RMGIC (3.04 ± 0.77 MPa) were significantly higher than those for SARC (2.28 ± 0.58 MPa). The predominant failure modes for the RMGIC and SARC were adhesive cement-dentin and adhesive cement-crown, respectively. An 8.0% arginine and calcium carbonate in-office desensitizing paste can be safely used to reduce post-cementation sensitivity without reducing the retentive strength of Y-TZP crowns.

One Piece Immediately Loaded Implants Versus 1 Piece or 2 Pieces Delayed: 3 Years Outcome.

PURPOSE: To compare time-dependent outcome of immediately loaded 1-piece (1P) implants with delayed loaded 1P and 2-piece (2P) implants. MATERIALS AND METHODS: A cohort of 33 patients divided into 3 groups: group A, 13 patients, 49 immediately placed and loaded 1P implants; group B, 11 patients, immediately placed and delayed loaded 1P implants; and group C, 10 patients, 39 2P implants delayed placed and loaded in a two-stage procedure. Marginal bone loss (MBL) was analyzed using x-ray radiography every 6 months, 1 year, and 3 years. RESULTS: A statistically significant mean MBL was observed between baseline, 6 months, 1 year, and 3 years in all groups. There was no statistical significant difference in MBL between immediate and delayed loaded 1P implants. MBL around mandibular implants was lower compared with maxillary implants. CONCLUSIONS: 2P implants showed less MBL compared with 1P implants in both maxilla and mandible. There was no statistical difference in MBL between immediate and delayed loaded 1P implants. Immediate loaded implants show more MBL in maxilla.

Mitochondrial metabolism in T cell activation and senescence: a mini-review.

The aging immune system is unable to optimally respond to pathogens and generate long-term immunological memory against encountered antigens. Amongst the immune components most affected by aging are T lymphocytes. T lymphocytes are cells of the cell-mediated immune system, which can recognize microbial antigens and either directly kill infected cells or support the maturation and activation of other immune cells. When activated, T cells undergo a metabolic switch to accommodate their changing needs at every stage of the immune response. Here we review the different aspects of metabolic regulation of T cell activation, focusing on the emerging role of mitochondrial metabolism, and discuss changes that may contribute to age-related decline in T cell potency. Better understanding of the role of mitochondrial metabolism in immune cell function could provide insights into mechanisms of immune senescence with the potential for developing novel therapeutic approaches to improve immune responses in aged individuals.

Evaluation of heat conduction in dental implants after exposure to hot beverages.

STATEMENT OF PROBLEM: It is unknown if the consumption of hot beverages after implant placement poses a danger of overheating at the bone-implant interface. PURPOSE: The purpose of this study was to investigate the effect of simulated consumption of hot beverages on the heat transfer to different dental implant types, implant sizes, and the presence of an interim restoration. MATERIAL AND METHODS: A model that consisted of 2 plastic containers was constructed to simulate the oral cavity and endosseous region of the jaw. One-piece and 2-piece implants with abutments were placed into a block of bovine mandibular bone without any healing tissue, surrounded by water maintained at 37°C in the lower compartment. The abutments, which extended into the upper container, were covered with water heated to 60°C to simulate consumption of a hot substance and then were cooled down spontaneously to 37°C during 100 to 600 seconds. Five thermocouple electrodes with an accuracy of ±0.1°C were attached to each test specimen and to a computer with data recording and analysis software to record temperature changes. Repeated measures ANOVA (α=.05)was performed to determine the effect of each major factor. RESULTS: Heat conduction from the abutment exposed to hot liquid was significantly higher in the cervical as opposed to the apical areas of the implants. Implant type (1 piece), diameter (wider), and the absence of an interim coping had a significant effect on the maximum temperature measured and on the temperature change rate. CONCLUSIONS: Abutment exposure to hot liquids resulted in heat conduction to the cervical region of the implant, which could be biologically harmful in healing tissues. Heat conduction was mitigated by implant design and diameter, and by the presence of an interim prosthesis. Results may differ in clinical models.

The effect of one-step vs. two-step impression techniques on long-term accuracy and dimensional stability when the finish line is within the gingival sulcular area.

PURPOSE: To evaluate the effect of two putty-wash impression techniques on the long-term accuracy and dimensional stability of poly(vinyl siloxane) (PVS) in the gingival sulcus area. MATERIALS AND METHODS: Impressions were taken from a master cast to simulate molar crown preparation. A space around the abutment served as the gingival sulcus. Fifteen impressions using the one- and two-step impression techniques were taken using Express Regular, Express Fast, and President impression materials with custom trays. Using a Toolmaker's microscope, the long (LD) and short distances (SD) of the abutment and the planar distance between two parallel lines (PL) at the circumference of the cast were taken at 0.5, 2, 24, 48, 72, 96, 120, and 144 hours after mixing. ANOVA was performed, with the discrepancy between the distances of the impressions and the master cast as the dependent variable. RESULTS: The differences when different materials and impression techniques were used were significant (p < 0.001) for LD, SD, and PL, as was the interaction between the material, time, and technique (p < 0.001). SD discrepancies were higher than those of LD for all materials and times. The two-step impression technique was more accurate, with smaller discrepancies than the one-step impression technique. For all materials, the PL discrepancy was deemed acceptable (less than 0.5%) for all tested times. President had higher discrepancies than the other materials. CONCLUSIONS: When using the two-step putty-wash impression technique, pouring of the impressions may be postponed up to 30 hours; however, when using the one-step impression technique, pouring should be performed within 2 hours.

One-year survival rate outcomes of innovative dental implants: a prospective clinical study.

PURPOSE: The aim of this research was to evaluate an innovative implant design for different placement and loading protocols. The unique implant is a combination of tapered and cylindrical shape, which is aimed to enhance initial stability and long-term osseointegration. MATERIALS AND METHODS: Four hundred and sixty implants were placed in 141 patients under different placement and loading protocols in similarity to those encountered in a dental office. Implants were followed and evaluated for 1 year to assert the survival rate of the newly introduced implant. RESULTS: The results showed a total of 97.4% survival rate, ranging from 92% to 98.6% depending on the different protocols. There was no statistical difference between the different protocol groups. CONCLUSION: The new implant design showed good results for 1 year of follow-up, comparable with the literature, and could be a good choice for every implant-based procedure.

Bone microstrain values of 1-piece and 2-piece implants subjected to mechanical loading.

PURPOSE: The purpose of this study was to measure and compare the strain levels in peri-implant bone as generated by 1-piece (1P) and 2-piece (2P) implant systems. MATERIALS AND METHODS: The implants (1P and 2P) were placed into bovine bone according to the manufacturer's protocol. Four linear strain gauges were placed around each implant neck and apex. Each model was loaded in static loading by a material testing machine in ascending forces ranging from 20 to 120 N. Microstrains (µ[Latin Small Letter Open E]) generated in the surrounding bone were measured by a strain gauge and recorded. RESULTS: Recorded microstrains were significantly higher for 1P implants than for 2P implants. Average recorded microstrain values were significantly lower in the neck (71.6 and 17.3 µs) compared with the apical (132 and 60 µs) regions of 1P and 2P implants, respectively (P < 0.0001). CONCLUSIONS: Within the limitations of this study, highest microstrains were generated in apical regions regardless of implant design, but the 2P implant ap-peared to provide a stress-damping effect in both the cervical and apical regions compared with the 1P implant.

A 10-year retrospective clinical evaluation of immediately loaded tapered maxillary implants.

PURPOSE: The aim of this study was to compare the effects of immediate loading (IL) and delayed loading (DL) on peri-implant crestal bone loss around maxillary implants after long-term functioning. MATERIALS AND METHODS: A retrospective chart review was conducted to assess the outcomes of 110 tapered, multithreaded implants placed for the treatment of one or more missing and/or unsalvageable teeth in the maxilla of 23 patients. Implants were assigned to either the DL or IL database according to loading time. Marginal bone changes were calculated using standardized radiographs taken at implant placement (baseline) and during annual follow-ups. RESULTS: One implant failed in the DL group. After a mean follow-up of 111 months in the DL group and 119 months in the IL group, cumulative implant survival was 99.09% (DL = 98.11%, IL = 100%). No observable bone loss was evident in 83.49% of the surviving implants. Cumulative success rates were 100% for the IL group and 98.11% for the DL group. CONCLUSIONS: Immediately loaded maxillary implants showed long-term results comparable to delayed loaded maxillary implants.

Dimensional stability of polyvinyl siloxane impression material reproducing the sulcular area.

The dimensional stability of a thin intra sulcular impression material reproducing the preparation finish line was evaluated. Impressions were taken of a stainless-steel master model of a simulated abutment with a 'gingival sulcus' using Express regular, Express fast and Aquasil. The putty-wash two-step technique was applied with spacer thicknesses of 0.5, 1 and 1.5 mm. Mid mesiodistal and bucco-lingual measurements were taken directly from the sulcular impression material after 0.5, 2, 24, 48 and 72 h via a Toolmaker's microscope. The discrepancies between the measurements of the impression material and the master model were calculated. The discrepancies changed significantly over time (p<0.001). The use of a 0.5 mm spacer resulted in a negative deviation from the model (2-46 µm), minimally after 2 h. The use of 1 and 1.5 mm spacers showed a positive deviation from the model (21-52 µm) and both are equally recommended. Investment can be postponed until 72 h.