Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.

Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving transcriptional heterogeneity. However, its application to studying cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells; this lack of coverage prevents the correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single cells from both genomic and coding DNA, in parallel with unbiased whole-transcriptome analysis. Applying TARGET-seq to 4,559 single cells, we demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasms (MPN) stem and progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct subclones of cancer cells.

Canonical Notch signaling is dispensable for adult steady-state and stress myelo-erythropoiesis.

Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.

Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.

The blood system is maintained by a small pool of haematopoietic stem cells (HSCs), which are required and sufficient for replenishing all human blood cell lineages at millions of cells per second throughout life. Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood, crucial for preventing bleeding--a common and life-threatening side effect of many cancer therapies--and major efforts are focused at identifying the most suitable cellular and molecular targets to enhance platelet production after bone marrow transplantation or chemotherapy. Although it has become clear that distinct HSC subsets exist that are stably biased towards the generation of lymphoid or myeloid blood cells, we are yet to learn whether other types of lineage-biased HSC exist or understand their inter-relationships and how differently lineage-biased HSCs are generated and maintained. The functional relevance of notable phenotypic and molecular similarities between megakaryocytes and bone marrow cells with an HSC cell-surface phenotype remains unclear. Here we identify and prospectively isolate a molecularly and functionally distinct mouse HSC subset primed for platelet-specific gene expression, with enhanced propensity for short- and long-term reconstitution of platelets. Maintenance of platelet-biased HSCs crucially depends on thrombopoietin, the primary extrinsic regulator of platelet development. Platelet-primed HSCs also frequently have a long-term myeloid lineage bias, can self-renew and give rise to lymphoid-biased HSCs. These findings show that HSC subtypes can be organized into a cellular hierarchy, with platelet-primed HSCs at the apex. They also demonstrate that molecular and functional priming for platelet development initiates already in a distinct HSC population. The identification of a platelet-primed HSC population should enable the rational design of therapies enhancing platelet output.

Promoting oral health care among people living in residential aged care facilities: Perceptions of care staff.

OBJECTIVE: This study aimed to look at the practices and perspectives of residential aged care facility (RACF) care staff regarding the provision of oral health care in RACFs. BACKGROUND: Emphasis has been placed on the provision of adequate oral health care in RACFs through the Better Oral Health in Residential Aged Care programme. Endorsed by the Australian government, this programme provided oral health education and training for aged care staff. However, recent evidence suggests that nearly five years after the implementation of this programme, the provision of oral care in RACFs in NSW remains inadequate. MATERIALS AND METHODS: This project utilised an exploratory qualitative design which involved a focus group with 12 RACF care staff. Participants were asked to discuss the current oral health practices in their facility, and their perceived barriers to providing oral health care. RESULTS: The key findings demonstrated current oral health practices and challenges among care staff. Most care staff had received oral health training and demonstrated positive attitudes towards providing dental care. However, some participants identified that ongoing and regular training was necessary to inform practice and raise awareness among residents. Organisational constraints and access to dental services also limited provision of dental care while a lack of standardised guidelines created confusion in defining their role as oral healthcare providers in the RACF. CONCLUSION: This study highlighted the need for research and strategies that focus on capacity building care staff in oral health care and improving access of aged care residents to dental services.

There is no compelling evidence that human neonates imitate.

Keven & Akins (K&A) propose that neonatal "imitation" is a function of newborns' spontaneous oral stereotypies and should be viewed within the context of normal aerodigestive development. Their proposal is in line with the result of our recent large longitudinal study that found no compelling evidence for neonatal imitation. Together, these works prompt reconsideration of the developmental origin of genuine imitation.

Ten days of simulated live high:train low altitude training increases Hbmass in elite water polo players.

OBJECTIVES: Water polo requires high aerobic power to meet the demands of match play. Live high:train low (LHTL) may enhance aerobic capacity at sea level. Before the Olympics, the Australian women's water polo team utilised LHTL in an attempt to enhance aerobic fitness. METHODS: Over 6 months, 11 players completed three normobaric LHTL exposures (block 1:11 days at 3000 m; block 2+3:9 days at 2500 m, 11 days normoxia, 10 days at 2800 m). Haemoglobin mass (Hbmass) was measured through carbon monoxide-rebreathing. Before each block, the relationship between Hbmass and water polo-specific aerobic fitness was investigated using the Multistage Shuttle Swim Test (MSST). Effect size statistics were adopted with likely, highly likely and almost certainly results being >75%, >95%, >99%, respectively. A Pearson product moment correlation was used to characterise the association between pooled data of Hbmass and MSST. RESULTS: Hbmass (mean ± SD, pre 721 ± 66 g) likely increased after block 1 and almost certainly after block 2+3 (% change; 90% confidence limits: block 1: 3.7%; 1.3-6.2%, block 2+3: 4.5%; 3.8-5.1%) and the net effect was almost certainly higher after block 2+3 than before block 1 (pre) by 8.5%; 7.3-9.7%. There was a very large correlation between Hbmass (g/kg) and MSST score (r=0.73). CONCLUSIONS: LHTL exposures of <2 weeks induced approximately 4% increase in Hbmass of water polo players. Extra Hbmass may increase aerobic power, but since match performance is nuanced by many factors it is impossible to ascertain whether the increased Hbmass contributed to Australia's Bronze medal.

Altitude training and haemoglobin mass from the optimised carbon monoxide rebreathing method determined by a meta-analysis.

OBJECTIVE: To characterise the time course of changes in haemoglobin mass (Hbmass) in response to altitude exposure. METHODS: This meta-analysis uses raw data from 17 studies that used carbon monoxide rebreathing to determine Hbmass prealtitude, during altitude and postaltitude. Seven studies were classic altitude training, eight were live high train low (LHTL) and two mixed classic and LHTL. Separate linear-mixed models were fitted to the data from the 17 studies and the resultant estimates of the effects of altitude used in a random effects meta-analysis to obtain an overall estimate of the effect of altitude, with separate analyses during altitude and postaltitude. In addition, within-subject differences from the prealtitude phase for altitude participant and all the data on control participants were used to estimate the analytical SD. The 'true' between-subject response to altitude was estimated from the within-subject differences on altitude participants, between the prealtitude and during-altitude phases, together with the estimated analytical SD. RESULTS: During-altitude Hbmass was estimated to increase by ∼1.1%/100 h for LHTL and classic altitude. Postaltitude Hbmass was estimated to be 3.3% higher than prealtitude values for up to 20 days. The within-subject SD was constant at ∼2% for up to 7 days between observations, indicative of analytical error. A 95% prediction interval for the 'true' response of an athlete exposed to 300 h of altitude was estimated to be 1.1-6%. CONCLUSIONS: Camps as short as 2 weeks of classic and LHTL altitude will quite likely increase Hbmass and most athletes can expect benefit.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.

Reduced T(H)1/T(H)17 CD4 T-cell numbers are associated with impaired purified protein derivative-specific cytokine responses in patients with HIV-1 infection.

BACKGROUND: In HIV-1-infected patients impaired IFN-γ responses to purified protein derivative (PPD) are associated with an increased risk of active tuberculosis. Tuberculosis antigen-specific cells are found in the T(H)1/T(H)17 subset of CD4 T cells, which support HIV-1 replication. Selective loss of T(H)1/T(H)17 cells in patients with HIV-1 infection might contribute to reduced tuberculosis-induced immune responses and an increased susceptibility to active tuberculosis. OBJECTIVES: We sought to investigate the association between T(H)1/T(H)17 cells and PPD-specific cytokine responses in HIV-1-infected patients. METHODS: A cross-sectional study was performed on healthy control subjects, HIV-1-infected patients receiving successful antiretroviral therapy (ART(+)), and ART-naive HIV-1-infected patients (ART(-)). All patients studied had evidence of BCG vaccination. Four discrete CD4 T-cell subsets were assessed by flow cytometry: T(H)1/T(H)17 cells (CXCR3(+)CCR6(+)CCR4(-)), T(H)1 cells (CXCR3(+)CCR6(-)CCR4(-)), T(H)17 cells (CXCR3(-)CCR6(+)CCR4(+)), and T(H)2 cells (CXCR3(-)CCR6(-)CCR4(+)). IFN-γ and IL-2 PPD-specific cytokine responses were assessed in PBMCs by using the enzyme-linked immunospot assay. RESULTS: Twenty-nine healthy control subjects, 34 ART(+) patients, and 26 ART(-) patients were recruited. The number and frequency of T(H)1/T(H)17 and T(H)1/T(H)17 CCR5(+) CD4 T cells were significantly reduced in HIV-1-infected patients. IFN-γ and IL-2 PPD responses were significantly lower in ART(-) patients and were partially reconstituted with successful ART. Loss of T(H)1/T(H)17 CCR5(+) cells was associated with reduced IFN-γ and IL-2 PPD responses. CONCLUSIONS: Selective loss of T(H)1/T(H)17 cells may be a risk factor for the development of active tuberculosis in patients with HIV-1 infection and might be a useful biomarker in the development of tuberculosis vaccines.

In utero origin of myelofibrosis presenting in adult monozygotic twins.

The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs.

Carbon monoxide uptake kinetics of arterial, venous and capillary blood during CO rebreathing.

The uptake and distribution of CO throughout the circulatory system during two different methods of CO rebreathing (2 min 'Schmidt' and 40 min 'Burge' methods) were determined in nine healthy volunteers. Specifically, the impact of (i) differences in circulatory mixing time (t(mix)), (ii) CO diffusion to myoglobin (Mb) and (iii) CO wash-out was assessed on calculated haemoglobin mass (Hb(mass)). Arterial (a), muscle venous (vm) and capillary samples (c) were obtained simultaneously at 0, 1, 2, 3.5, 5, 7.5, 10, 12.5, 15, 20, 30 and 40 min for determination of carboxyhaemoglobin (HbCO). Carbon monoxide wash-out was measured from expired air following rebreathing. The rate of CO diffusion to Mb was calculated using the change in HbCO after t(mix), and the rate of CO wash-out. In both methods, HbCOa and HbCOc followed a near-identical time course, peaking within the first 2 min and decreasing thereafter. The HbCOvm increased slowly and was significantly lower at 1, 2 and 3.5 min in both methods (P < 0.01). The HbCOa peaked significantly higher in the Schmidt method (P = 0.03). Circulatory mixing had occurred by 10 min in most but not all subjects. The rate of CO wash-out was 0.25 ± 0.13 ml min⁻¹ in the Schmidt and 0.25 ± 0.16 ml min⁻¹ in the Burge method. The rate of CO diffusion to Mb was 0.22 ± 0.11 and 0.16 ± 0.13 ml min⁻¹ (P = 0.63) in Schmidt and Burge methods, respectively. Inhalation of a CO bolus during the Schmidt method results in faster HbCOa uptake but does not greatly shorten t(mix) or influence rates of CO wash-out and flux to Mb. The calculated Hb(mass) depends substantially on the plateau level of HbCO; therefore, it is paramount to ensure HbCO is mixed completely prior to blood sampling, as well as accounting for potential within-subject alterations of CO exhalation and CO flux to Mb.

Oral health in residential aged care: Perceptions of nurses and management staff.

Aim: This study aimed to explore the perceptions of residential aged care nursing and management staff regarding oral care, to develop strategies to improve the oral health of aged care residents. Design: A qualitative approach was used. Methods: Two focus groups were conducted with nursing and management staff at two residential aged care facilities and transcripts were thematically analysed. Results: All staff had an awareness of the importance of oral health; however, they highlighted the significant challenges in the current system that affect implementation of oral health training and practice guidelines in the residential aged care facility. High staff turnover, time constraints, difficulties in accessing dental services and working together with residents, their families and external staff were barriers to providing oral health care. Staff highlighted the need for formalized clinical guidelines and processes and efficient dental referral pathways to create a more cohesive system of care.

Time course of haemoglobin mass during 21 days live high:train low simulated altitude.

The aim of this study was to determine the time course of changes in haemoglobin mass (Hb(mass)) in well-trained cyclists in response to live high:train low (LHTL). Twelve well-trained male cyclists participated in a 3-week LHTL protocol comprising 3,000 m simulated altitude for ~14 h/day. Prior to LHTL duplicate baseline measurements were made of Hb(mass), maximal oxygen consumption (VO(2max)) and serum erythropoietin (sEPO). Hb(mass) was measured weekly during LHTL and twice in the week thereafter. There was a 3.3% increase in Hb(mass) and no change in VO(2max) after LHTL. The mean Hb(mass) increased at a rate of ~1% per week and this was maintained in the week after cessation of LHTL. The sEPO concentration peaked after two nights of LHTL but there was only a trivial correlation (r = 0.04, P = 0.89) between the increase in sEPO and the increase in Hb(mass). Athletes seeking to gain erythropoietic benefits from moderate altitude need to spend >12 h/day in hypoxia.

Validity of the Online Athlete Management System to Assess Training Load.

PURPOSE: To validate the quantification of training load (session rating of perceived exertion [s-RPE]) in an Australian Olympic squad (women's water polo), assessed with the use of a modified RPE scale collected via a newly developed online system (athlete management system). METHODS: Sixteen elite women water polo players (age = 26 [3] y, height = 1.78 [0.05] m, and body mass = 75.5 [7.1] kg) participated in the study. Thirty training sessions were monitored for a total of 303 individual sessions. Heart rate was recorded during training sessions using continuous heart-rate telemetry. Participants were asked to rate the intensity of the training sessions on the athlete management system RPE scale, using an online application within 30 min of completion of the sessions. Individual relationships between s-RPE and both Banister training impulse (TRIMP) and Edwards' method were analyzed. RESULTS: Individual correlations with s-RPE ranged between r = .51 and .79 (Banister TRIMP) and r = .54 and .83 (Edwards' method). The percentages of moderate and large correlation were 81% and 19% between s-RPE method and Banister TRIMP, and 56% and 44% between s-RPE and Edwards' method. CONCLUSIONS: The online athlete management system for assessing s-RPE was shown to be a valid indicator of internal training load and can be used in elite sport.

Nonhematological mechanisms of improved sea-level performance after hypoxic exposure.

Altitude training has been used regularly for the past five decades by elite endurance athletes, with the goal of improving performance at sea level. The dominant paradigm is that the improved performance at sea level is due primarily to an accelerated erythropoietic response due to the reduced oxygen available at altitude, leading to an increase in red cell mass, maximal oxygen uptake, and competitive performance. Blood doping and exogenous use of erythropoietin demonstrate the unequivocal performance benefits of more red blood cells to an athlete, but it is perhaps revealing that long-term residence at high altitude does not increase hemoglobin concentration in Tibetans and Ethiopians compared with the polycythemia commonly observed in Andeans. This review also explores evidence of factors other than accelerated erythropoiesis that can contribute to improved athletic performance at sea level after living and/or training in natural or artificial hypoxia. We describe a range of studies that have demonstrated performance improvements after various forms of altitude exposures despite no increase in red cell mass. In addition, the multifactor cascade of responses induced by hypoxia includes angiogenesis, glucose transport, glycolysis, and pH regulation, each of which may partially explain improved endurance performance independent of a larger number of red blood cells. Specific beneficial nonhematological factors include improved muscle efficiency probably at a mitochondrial level, greater muscle buffering, and the ability to tolerate lactic acid production. Future research should examine both hematological and nonhematological mechanisms of adaptation to hypoxia that might enhance the performance of elite athletes at sea level.

The effect of acute simulated moderate altitude on power, performance and pacing strategies in well-trained cyclists.

Athletes regularly compete at 2,000-3,000 m altitude where peak oxygen consumption (VO2peak) declines approximately 10-20%. Factors other than VO2peak including gross efficiency (GE), power output, and pacing are all important for cycling performance. It is therefore imperative to understand how all these factors and not just VO2peak are affected by acute hypobaric hypoxia to select athletes who can compete successfully at these altitudes. Ten well-trained, non-altitude-acclimatised male cyclists and triathletes completed cycling tests at four simulated altitudes (200, 1,200, 2,200, 3,200 m) in a randomised, counter-balanced order. The exercise protocol comprised 5 x 5-min submaximal efforts (50, 100, 150, 200 and 250 W) to determine submaximal VO2 and GE and, after 10-min rest, a 5-min maximal time-trial (5-minTT) to determine VO2peak and mean power output (5-minTT(power)). VO2peak declined 8.2 +/- 2.0, 13.9 +/- 2.9 and 22.5 +/- 3.8% at 1,200, 2,200 and 3,200 m compared with 200 m, respectively, P < 0.05. The corresponding decreases in 5-minTT(power) were 5.8 +/- 2.9, 10.3 +/- 4.3 and 19.8 +/- 3.5% (P < 0.05). GE during the 5-minTT was not different across the four altitudes. There was no change in submaximal VO2 at any of the simulated altitudes, however, submaximal efficiency decreased at 3,200 m compared with both 200 and 1,200 m. Despite substantially reduced power at simulated altitude, there was no difference in pacing at the four altitudes for athletes whose first trial was at 200 or 1,200 m; whereas athletes whose first trial was at 2,200 or 3,200 m tended to mis-pace that effort. In conclusion, during the 5-minTT there was a dose-response effect of hypoxia on both VO2peak and 5-minTT(power) but no effect on GE.

Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

Comprehensive Longitudinal Study Challenges the Existence of Neonatal Imitation in Humans.

Human children copy others' actions with high fidelity, supporting early cultural learning and assisting in the development and maintenance of behavioral traditions [1]. Imitation has long been assumed to occur from birth [2-4], with influential theories (e.g., [5-7]) placing an innate imitation module at the foundation of social cognition (potentially underpinned by a mirror neuron system [8, 9]). Yet, the very phenomenon of neonatal imitation has remained controversial. Empirical support is mixed and interpretations are varied [10-16], potentially because previous investigations have relied heavily on cross-sectional designs with relatively small samples and with limited controls [17, 18]. Here, we report surprising results from the most comprehensive longitudinal study of neonatal imitation to date. We presented infants (n = 106) with nine social and two non-social models and scored their responses at 1, 3, 6, and 9 weeks of age. Longitudinal analyses indicated that the infants did not imitate any of the models, as they were just as likely to produce the gestures in response to control models as they were to matching models. Previous positive findings were replicated in limited cross-sections of the data, but the overall analyses confirmed these findings to be mere artifacts of restricted comparison conditions. Our results undermine the idea of an innate imitation module and suggest that earlier studies reporting neonatal imitation were methodologically limited.