T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control.

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet the mechanisms of lymphocyte transit are not well defined. Here we show that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression by effector CD8+ T cells. Only high-affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells, therefore, exit the tumor, which limits the pool of CD8+ T cells available to exert tumor control. CXCR4 inhibition or loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. These data indicate that strategies to limit T cell egress might be an approach to boost the quantity and quality of intratumoral T cells and thereby response to immunotherapy.

Effect of high-fat diet on isometric, concentric and eccentric contractile performance of skeletal muscle isolated from female CD-1 mice.

Despite evidence inferring muscle and contractile mode-specific effects of high-fat diet (HFD), no study has yet considered the impact of HFD directly on eccentric muscle function. The present work uniquely examined the effect of 20-week HFD on the isometric, concentric and eccentric muscle function of isolated mouse soleus (SOL) and extensor digitorum longus (EDL) muscles. CD-1 female mice were randomly split into a control (n = 16) or HFD (n = 17) group and for 20 weeks consumed standard lab chow or HFD. Following this period, SOL and EDL muscles were isolated and assessments of maximal isometric force and concentric work loop (WL) power were performed. Each muscle was then subjected to either multiple concentric or eccentric WL activations. Post-fatigue recovery, as an indicator of incurred damage, was measured via assessment of concentric WL power. In the EDL, absolute concentric power and concentric power normalised to muscle mass were reduced in the HFD group (P < 0.038). HFD resulted in faster concentric fatigue and reduced eccentric activity-induced muscle damage (P < 0.05). For the SOL, maximal isometric force was increased, and maximal eccentric power normalised to muscle mass and concentric fatigue were reduced in the HFD group (P < 0.05). HFD effects on eccentric muscle function are muscle-specific and have little relationship with changes in isometric or concentric function. HFD has the potential to negatively affect the intrinsic concentric and eccentric power-producing capacity of skeletal muscle, but a lack of a within-muscle uniform response indicates disparate mechanisms of action which require further investigation.

Proteomic Analysis of Whole Blood Using Volumetric Absorptive Microsampling for Precision Medicine Biomarker Studies.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Activation of the myosin motors in fast-twitch muscle of the mouse is controlled by mechano-sensing in the myosin filaments.

Myosin motors in resting muscle are inactivated by folding against the backbone of the myosin filament in an ordered helical array and must be released from that conformation to engage in force generation. Time-resolved X-ray diffraction from single fibres of amphibian muscle showed that myosin filament activation could be inhibited by imposing unloaded shortening at the start of stimulation, suggesting that filaments were activated by mechanical stress. Here we improved the signal-to-noise ratio of that approach using whole extensor digitorum longus muscles of the mouse contracting tetanically at 28°C. Changes in X-ray signals associated with myosin filament activation, including the decrease in the first-order myosin layer line associated with the helical motor array, increase in the spacing of a myosin-based reflection associated with packing of myosin tails in the filament backbone, and increase in the ratio of the 1,1 and 1,0 equatorial reflections associated with movement of motors away from the backbone, were delayed by imposing 10-ms unloaded shortening at the start of stimulation. These results show that myosin filaments are predominantly activated by filament stress, as in amphibian muscle. However, a small component of filament activation at zero load was detected, implying an independent mechanism of partial filament activation. X-ray interference measurements indicated a switch-like change in myosin motor conformation at the start of force development, accompanied by transient disordering of motors in the regions of the myosin filament near its midpoint, suggesting that filament zonal dynamics also play a role in its activation. KEY POINTS: Activation of myosin filaments in extensor digitorum longus muscles of the mouse is delayed by imposing rapid shortening from the start of stimulation. Stress is the major mechanism of myosin filament activation in these muscles, but there is a small component of filament activation during electrical stimulation at zero stress. Myosin motors switch rapidly from the folded inhibited conformation to the actin-attached force-generating conformation early in force development.

Age-related changes in isolated mouse skeletal muscle function are dependent on sex, muscle, and contractility mode.

The present study aimed to simultaneously examine the age-related, muscle-specific, sex-specific, and contractile mode-specific changes in isolated mouse skeletal muscle function and morphology across multiple ages. Measurements of mammalian muscle morphology, isometric force and stress (force/cross-sectional area), absolute and normalized (power/muscle mass) work-loop power across a range of contractile velocities, fatigue resistance, and myosin heavy chain (MHC) isoform concentration were measured in 232 isolated mouse (CD-1) soleus, extensor digitorum longus (EDL), and diaphragm from male and female animals aged 3, 10, 30, 52, and 78 wk. Aging resulted in increased body mass and increased soleus and EDL muscle mass, with atrophy only present for female EDL by 78 wk despite no change in MHC isoform concentration. Absolute force and power output increased up to 52 wk and to a higher level for males. A 23-36% loss of isometric stress exceeded the 14-27% loss of power normalized to muscle mass between 10 wk and 52 wk, although the loss of normalized power between 52 and 78 wk continued without further changes in stress (P > 0.23). Males had lower power normalized to muscle mass than females by 78 wk, with the greatest decline observed for male soleus. Aging did not cause a shift toward slower contractile characteristics, with reduced fatigue resistance observed in male EDL and female diaphragm. Our findings show that the loss of muscle quality precedes the loss of absolute performance as CD-1 mice age, with the greatest effect seen in male soleus, and in most instances without muscle atrophy or an alteration in MHC isoforms.

The impact of a high-fat diet in mice is dependent on duration and age, and differs between muscles.

Prolonged high-fat diets (HFDs) can cause intramyocellular lipid (IMCL) accumulation that may negatively affect muscle function. We investigated the duration of a HFD required to instigate these changes, and whether the effects are muscle specific and aggravated in older age. Muscle morphology was determined in the soleus, extensor digitorum longus (EDL) and diaphragm muscles of female CD-1 mice from 5 groups: young fed a HFD for 8 weeks (YS-HFD, n=16), young fed a HFD for 16 weeks (YL-HFD, n=28) and young control (Y-Con, n=28). The young animals were 20 weeks old at the end of the experiment. Old (70 weeks) female CD-1 mice received either a normal diet (O-Con, n=30) or a HFD for 9 weeks (OS-HFD, n=30). Body mass, body mass index and intramyocellular lipid (IMCL) content increased in OS-HFD (P≤0.003). In the young mice, this increase was seen in YL-HFD and not YS-HFD (P≤0.006). The soleus and diaphragm fibre cross-sectional area (FCSA) in YL-HFD was larger than that in Y-Con (P≤0.004) while OS-HFD had a larger soleus FCSA compared with that of O-Con after only 9 weeks on a HFD (P<0.001). The FCSA of the EDL muscle did not differ significantly between groups. The oxidative capacity of fibres increased in young mice only, irrespective of HFD duration (P<0.001). High-fat diet-induced morphological changes occurred earlier in the old animals than in the young, and adaptations to HFD were muscle specific, with the EDL being least responsive.

An Acetylenic Lipid from the New Zealand Ascidian Pseudodistoma cereum: Exemplification of an Improved Workflow for Determination of Absolute Configuration of Long-Chain 2-Amino-3-alkanols.

An acetylenic 2-amino-3-alcohol, distaminolyne B (2), isolated from the New Zealand ascidian Pseudodistoma cereum, is reported. The isolation and structure elucidation of 2 and assignment of 2S,3S absolute configuration (AC) using the exciton coupled circular dichroism technique are described. Using a methodologically facile workflow, the same AC was also established by analysis of specific rotation, terminal methyl C-1 δC chemical shift, and NH δH and J values of the N,O-diacetate derivative.

Investigating a dose-response relationship between high-fat diet consumption and the contractile performance of isolated mouse soleus, EDL and diaphragm muscles.

PURPOSE: Recent evidence has demonstrated an obesity-induced, skeletal muscle-specific reduction in contractile performance. The extent and magnitude of these changes in relation to total dose of high-fat diet consumption remains unclear. This study aimed to examine the dose-response relationship between a high-fat diet and isolated skeletal muscle contractility. METHODS: 120 female CD1 mice were randomly assigned to either control group or groups receiving 2, 4, 8 or 12 weeks of a high-calorie diet (N = 24). At 20 weeks, soleus, EDL or diaphragm muscle was isolated (n = 8 in each case) and isometric force, work loop power output and fatigue resistance were measured. RESULTS: When analysed with respect to feeding duration, there was no effect of diet on the measured parameters prior to 8 weeks of feeding. Compared to controls, 8-week feeding caused a reduction in normalised power of the soleus, and 8- and 12-week feeding caused reduced normalised isometric force, power and fatigue resistance of the EDL. Diaphragm from the 12-week group produced lower normalised power, whereas 8- and 12-week groups produced significantly lower normalised isometric force. Correlation statistics indicated that body fat accumulation and decline in contractility will be specific to the individual and independent of the feeding duration. CONCLUSION: The data indicate that a high-fat diet causes a decline in muscle quality with specific contractile parameters being affected in each muscle. We also uniquely demonstrate that the amount of fat gain, irrespective of feeding duration, may be the main factor in reducing contractile performance.

Red blood cells: The primary reservoir of macrophage migration inhibitory factor in whole blood.

Red blood cells are widely accepted to be inert carriers of oxygen and haemoglobin, but there is growing evidence that they play a much more critical role in immune function. Macrophage migration inhibitory factor (MIF) is a key cytokine in disease with additional oxido-reductase activity, which aids in managing oxidative stress. Although two studies have reported the presence of MIF in red blood cells, no study has quantified the levels of this protein. In this study, freshly isolated plasma, platelets, leukocytes, and red blood cells from healthy individuals were collected and the concentration of MIF was determined using an enzyme linked immunosorbent assay. This analysis demonstrated that MIF in red blood cells was present at 25 µg per millilitre of whole blood, which is greater than99% of the total MIF and 1000-fold higher concentration than plasma. This result was supported by electrophoresis and Western blot analysis, which identified MIF in its monomer structural form following sample processing. Furthermore, by assessing the level of tautomerase activity in red blood cell fractions in the presence of a MIF inhibitor, it was determined that the red blood cell-derived MIF was also functionally active. Together, these findings have implications on the effect of haemolysis during sample preparation and provide some clue into the inflammatory processes that occur following haemolysis in vivo. These results support the hypothesis that red blood cells are a major reservoir of this inflammatory protein and may play a role in inflammation.

Blockade of the IL-1R1/TLR4 pathway mediates disease-modification therapeutic effects in a model of acquired epilepsy.

We recently discovered that forebrain activation of the IL-1 receptor/Toll-like receptor (IL-1R1/TLR4) innate immunity signal plays a pivotal role in neuronal hyperexcitability underlying seizures in rodents. Since this pathway is activated in neurons and glia in human epileptogenic foci, it represents a potential target for developing drugs interfering with the mechanisms of epileptogenesis that lead to spontaneous seizures. The lack of such drugs represents a major unmet clinical need. We tested therefore novel therapies inhibiting the IL-1R1/TLR4 signaling in an established murine model of acquired epilepsy. We used an epigenetic approach by injecting a synthetic mimic of micro(mi)RNA-146a that impairs IL1R1/TLR4 signal transduction, or we blocked receptor activation with antiinflammatory drugs. Both interventions when transiently applied to mice after epilepsy onset, prevented disease progression and dramatically reduced chronic seizure recurrence, while the anticonvulsant drug carbamazepine was ineffective. We conclude that IL-1R1/TLR4 is a novel potential therapeutic target for attaining disease-modifications in patients with diagnosed epilepsy.

Evaluation of a remote hybrid staffing model for investigational drug service pharmacists.

DISCLAIMER: In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. PURPOSE: This project compared the time to complete investigational prescription drug order verification by investigational drug service (IDS) pharmacists in an onsite vs offsite (hybrid) staffing model, evaluating the impact of remote work on verification time. METHODS: Parenteral IDS drug orders from August 2019 through June 2022 were analyzed. Electronic medical records were timestamped for each order, first verification, second verification, and medication administration. The project was divided into four 6-month time periods to allow for 2 independent comparisons of onsite vs hybrid staffing models. A survey was given retrospectively to IDS pharmacists for the last hybrid timeframe to assess work satisfaction and burnout. RESULTS: A total of 4,398 orders were evaluated. The time from order entry to first verification was 14 minutes (n = 1,248) during the first onsite time period compared to 13 minutes (n = 1,042) during the first hybrid period (P = 0.003). The time for order verification was similar between the second onsite and hybrid periods (12 minutes [n = 1,041] vs 10 minutes [n= 1,067], respectively; P < 0.0001). The staff satisfaction survey for the hybrid model showed high levels of job satisfaction and no self-reported burnout. CONCLUSION: The remote hybrid staffing model did not result in a meaningful change in the time from order placement to medication administration within the IDS pharmacy. Hybrid staffing also resulted in greater job satisfaction among IDS pharmacists. This study highlights the benefits of implementing remote work practices in the pharmacy practice setting.

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Introduction: Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. Methods: We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Results: Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. Discussion: We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Proteome Analysis of Whole Blood Collected by Volumetric Absorptive Microsampling.

Proteomic biomarker discovery and analysis from human biofluids using liquid chromatography-mass spectrometry (LC-MS) is an area of intense biomedical research. There is a growing interest to analyze microsampled patient blood specimens as this is potentially more patient-friendly enabling at-home and bedside self-collection of small blood volumes. However, there are limited studies applying LC-MS proteomic analysis of whole blood as it is dominated by red blood cell proteins such as hemoglobin which suppresses the detection of other less abundant proteins. Volumetric absorptive microsampling (VAMS) devices overcome this issue in part by providing a trapping matrix which allows depletion of abundant blood cell proteins through washing, prior to proteolysis and LC-MS. This approach allows the analysis of proteins from erythrocytes, leukocytes, and plasma and leads to deeper proteomic coverage compared to conventional plasma proteomics, increasing the prospects to discover novel biomarker proteins.

Piloting a new model of personalised care for people with fibromyalgia in primary care with secondary care multidisciplinary support.

OBJECTIVE: An estimated 5.5 million people in England have high-impact chronic pain, which is severe pain associated with significant disability. Current models of healthcare often fail to address their broad range of symptoms and address their complex non-medical needs. METHODS: A pilot project was designed with the aim of improving the quality of care and addressing unmet needs of patients high-impact musculoskeletal (MSK) pain through providing a personalised approach to their pain and wider psychosocial needs. The model comprised a longer initial appointment with a general practitioner, a later follow-appointment, and support of the multidisciplinary team (MDT) (informally and through a formal MDT meeting) with both primary care clinicians and specialists based in secondary care. RESULTS: Forty six patients were seen using this model, with prominent themes of consultations including self-management, social needs, mental health and understanding their diagnosis. Evaluation of the pilot demonstrated improvements in MSK and non-MSK symptoms, together with improved patient confidence in self-management and knowledge and understanding of their condition. Multidisciplinary working proved to be invaluable in addressing patients' wider needs but also upskilling and supporting primary care clinicians. Primary care staff also found it to be a satisfying way to care for patients, and developed increasing skills and confidence in supporting patients with chronic pain. CONCLUSION: This model of care appears to be an effective way to help primary care teams to provide more holistic personalised care to a group of patients who are highly complex and so often forgotten.

Pediatric Provider and Staff Burnout in Federally Qualified Community Health Centers.

This study examined changes in provider and staff burnout in 4 Boston-area federally qualified community health centers (FQHCs) participating in a pediatric behavioral health integration project. Utilizing the Maslach Burnout Inventory-Human Services Survey for Medical Personnel (MBI-HSSMP), the study found that emotional exhaustion among primary care providers ( M = 20.5; confidence interval [CI], 17.8-23.2) was higher ( P = .001) than that among behavioral health clinicians ( M = 13.6; CI, 10.4-16.8) and ( P = .00005) community health workers ( M = 10.8; CI, 7.3-14.2). Emotional exhaustion among staff increased ( P = .04) from baseline ( M = 16.8; CI, 15.0-18.6) to follow-up ( M = 20.8; CI, 17.5-24.2), but burnout at follow-up was lower than national averages. FQHCs are integral in caring for marginalized patients; therefore, supporting a stable workforce by minimizing burnout is essential.

Analysis of in vitro secretion profiles from adipose-derived cell populations.

BACKGROUND: Adipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs). Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF) is becoming increasingly common. METHODS: In this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs) at passage 2. In addition, we produced an 'in silico' dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the 'in silico' dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of < 0.05 was considered statistically different. To assess the overall changes that may occur as a result of co-culture we compared the proteomes of SVF and SVF co-cultured with adipocytes using iTRAQ quantitative mass spectrometry. RESULTS: A co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes. CONCLUSIONS: The use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the SVF with adipocytes may be considered as an alternative to MSCs or fresh SVF alone.

Gonadal sex patterns p21-induced cellular senescence in mouse and human glioblastoma.

Males exhibit higher incidence and worse prognosis for the majority of cancers, including glioblastoma (GBM). Disparate survival may be related to sex-biased responses to treatment, including radiation. Using a mouse model of GBM, we show that female cells are more sensitive to radiation, and that senescence represents a major component of the radiation therapeutic response in both sexes. Correlation analyses revealed that the CDK inhibitor p21 and irradiation induced senescence were differentially regulated between male and female cells. Indeed, female cellular senescence was more sensitive to changes in p21 levels, a finding that was observed in wildtype and transformed murine astrocytes, as well as patient-derived GBM cell lines. Using a novel Four Core Genotypes model of GBM, we further show that sex differences in p21-induced senescence are patterned during early development by gonadal sex. These data provide a rationale for the further study of sex differences in radiation response and how senescence might be enhanced for radiation sensitization. The determination that p21 and gonadal sex are required for sex differences in radiation response will serve as a foundation for these future mechanistic studies.

Age-Related Skeletal Muscle Dysfunction Is Aggravated by Obesity: An Investigation of Contractile Function, Implications and Treatment.

Obesity is a global epidemic and coupled with the unprecedented growth of the world's older adult population, a growing number of individuals are both old and obese. Whilst both ageing and obesity are associated with an increased prevalence of chronic health conditions and a substantial economic burden, evidence suggests that the coincident effects exacerbate negative health outcomes. A significant contributor to such detrimental effects may be the reduction in the contractile performance of skeletal muscle, given that poor muscle function is related to chronic disease, poor quality of life and all-cause mortality. Whilst the effects of ageing and obesity independently on skeletal muscle function have been investigated, the combined effects are yet to be thoroughly explored. Given the importance of skeletal muscle to whole-body health and physical function, the present study sought to provide a review of the literature to: (1) summarise the effect of obesity on the age-induced reduction in skeletal muscle contractile function; (2) understand whether obesity effects on skeletal muscle are similar in young and old muscle; (3) consider the consequences of these changes to whole-body functional performance; (4) outline important future work along with the potential for targeted intervention strategies to mitigate potential detrimental effects.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle.

Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.