Peripheral HLA-G/ILT-2 immune checkpoint axis in acute and convalescent COVID-19 patients.

The immunosuppressive non-classical human leukocyte antigen-G (HLA-G) can elicits pro-viral activities by down-modulating immune responses. We analysed soluble forms of HLA-G, IL-6 and IL-10 as well as on immune effector cell expression of HLA-G and its cognate ILT-2 receptor in peripheral blood obtained from hospitalised and convalescent COVID-19 patients. Compared with convalescents (N = 202), circulating soluble HLA-G levels (total and vesicular-bound molecules) were significantly increased in hospitalised patients (N = 93) irrespective of the disease severity. During COVID-19, IL-6 and IL-10 levels were also elevated. Regarding the immune checkpoint expression of HLA-G/ILT-2 on peripheral immune effector cells, the frequencies of membrane-bound HLA-G on CD3+ and CD14+ cells were almost identical in patients during and post COVID-19, while the frequency of ILT-2 receptor on CD3+ and CD14+ cells was increased during acute infection. A multi-parametric correlation analysis of soluble HLA-G forms with IL-6, IL-10, activation markers CD25 and CD154, HLA-G, and ILT-2 expression on immune cells revealed a strong positive correlation of soluble HLA-G forms with membrane-bound HLA-G molecules on CD3+/CD14+ cells only in convalescents. During COVID-19, only vesicular-bound HLA-G were positively correlated with the activation marker CD25 on T cells. Thus, our data suggest that the elevated levels of soluble HLA-G in COVID-19 are due to increased expression in organ tissues other than circulating immune effector cells. The concomitant increased expression of soluble HLA-G and ILT-2 receptor frequencies supports the concept that the immune checkpoint HLA-G/ILT-2 plays a role in the immune-pathogenesis of COVID-19.

Long-term cellular immune response in immunocompromised unvaccinated COVID-19 patients undergoing monoclonal antibody treatment.

Immunocompromised patients are at increased risk for a severe course of COVID-19. Treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection with anti-SARS-CoV-2 monoclonal antibodies (mAbs) has become widely accepted. However, the effects of mAb treatment on the long-term primary cellular response to SARS-CoV-2 are unknown. In the following study, we investigated the long-term cellular immune responses to SARS-CoV-2 Spike S1, Membrane (M) and Nucleocapsid (N) antigens using the ELISpot assay in unvaccinated, mAb-treated immunocompromised high-risk patients. Anti-SARS-CoV-2 mAb untreated though vaccinated COVID-19 immunocompromised patients, vaccinated SARS-CoV-2 immunocompromised patients without COVID-19 and vaccinated healthy control subjects served as control groups. The cellular immune response was determined at a median of 5 months after SARS-CoV-2 infection. Our data suggest that immunocompromised patients develop an endogenous long-term cellular immune response after COVID-19, although at low levels. A better understanding of the cellular immune response will help guide clinical decision making for these vulnerable patient cohorts.

COVID-19 in Elderly, Immunocompromised or Diabetic Patients-From Immune Monitoring to Clinical Management in the Hospital.

The novel, highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic of acute respiratory illness worldwide and remains a huge threat to the healthcare system's capacity to respond to COVID-19. Elderly and immunocompromised patients are at increased risk for a severe course of COVID-19. These high-risk groups have been identified as developing diminished humoral and cellular immune responses. Notably, SARS-CoV-2 RNA remains detectable in nasopharyngeal swabs of these patients for a prolonged period of time. These factors complicate the clinical management of these vulnerable patient groups. To date, there are no well-defined guidelines for an appropriate duration of isolation for elderly and immunocompromised patients, especially in hospitals or nursing homes. The aim of the present study was to characterize at-risk patient cohorts capable of producing a replication-competent virus over an extended period after symptomatic COVID-19, and to investigate the humoral and cellular immune responses and infectivity to provide a better basis for future clinical management. In our cohort, the rate of positive viral cultures and the sensitivity of SARS-CoV-2 antigen tests correlated with higher viral loads. Elderly patients and patients with diabetes mellitus had adequate cellular and humoral immune responses to SARS-CoV-2 infection, while immunocompromised patients had reduced humoral and cellular immune responses. Our patient cohort was hospitalized for longer compared with previously published cohorts. Longer hospitalization was associated with a high number of nosocomial infections, representing a potential hazard for additional complications to patients. Most importantly, regardless of positive SARS-CoV-2 RNA detection, no virus was culturable beyond a cycle threshold (ct) value of 33 in the majority of samples. Our data clearly indicate that elderly and diabetic patients develop a robust immune response to SARS-CoV-2 and may be safely de-isolated at a ct value of more than 35.

Moving beyond informal action: sustainable energy and the humanitarian response system.

Energy and humanitarian action have long been uneasy bedfellows. In the field, many humanitarian practitioners lack the time or remit to engage with a complex issue such as energy, and the topic to date has received relatively little attention from the private, development and academic sectors. This paper hopes to provide more clarity on energy in forced displacement settings by analysing how energy is interwoven with the humanitarian cluster system. This paper has two aims: (1) to assess existing evidence in the sector and explain the links between energy and each of the humanitarian clusters and (2) to provide recommendations on how humanitarian response efforts can transition from informal action to a comprehensive response on sustainable energy provision. This paper is the first to investigate the role of energy using the cluster system as a framework and contributes to a rapidly evolving field of research and practice on energy in humanitarian contexts. Our analysis demonstrates that energy is not fully integrated within humanitarian programme planning. Further, it highlights pathways for improving humanitarian outcomes enabled by improved energy practices. We identify ten ways clusters can integrate action on energy to support crisis-affected communities.

Extinction of cognitive associations is preserved in patients with cerebellar disease.

In the present study extinction and renewal of cognitive associations were assessed in two experiments in participants with focal and degenerative cerebellar disease. Using a predictive learning task, participants had to learn by trial and error the relationships between food items and the occurrence of stomach trouble in a hypothetical patient. In the first experiment, focus was on renewal effects. Participants with chronic cerebellar stroke (n = 14; mean age 50.9 ± 12 years), participants with degenerative cerebellar disease (n = 16; mean age 58 ± 12 years), age-, sex-, and education matched controls (n = 20; mean age 53.7 ± 10.8 years) and young controls (n = 19; mean age 23.2 ± 2.7 years) were tested. Acquisition and extinction of food-stomach trouble associations took part in two different contexts (represented by restaurants). In a subsequent test phase, food stimuli were presented in both contexts and no feedback was given. This allowed testing for renewal of the initially acquired associations in the acquisition context. Acquisition and extinction learning were not significantly different between groups. Significant renewal effects were present in young controls only. In the second experiment, focus was on extinction. To control for age effects, 19 young participants with chronic surgical lesions of the cerebellum (mean age 25.6 ± 6.1 years), and 24 age-, sex- and education-matched healthy controls were tested. Acquisition and extinction of food-stomach trouble associations took part in the same context. In the extinction phase, the relationship with stomach trouble was reversed in some of the food items. Acquisition and extinction learning were not significantly different between groups. The main finding of the present study was preserved extinction of learned cognitive associations in participants with chronic cerebellar disease. Findings agree with previous observations in the literature that cognitive abnormalities are frequently absent or weak in adults with cerebellar disease. This does not exclude a contribution of the cerebellum to extinction of learned associations. For example, findings may be different in more challenging cognitive tasks, and in participants with acute cerebellar disease with no time for compensation.

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification.

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

Obesity management in Australian primary care: where has the general practitioner gone?

Obesity is a chronic condition with significant health and economic consequences that requires more effective management in Australia. General practitioners (GPs) currently act as care co-ordinators in line with national guidelines for overweight and obesity. Australian patients indicate that they would appreciate more involvement from their GP in the management of obesity, and this is in line with international findings. Not all patients have access to specialist obesity services or affordable allied health care because of location, cost and time, particularly in rural and remote areas where there is a greater prevalence of obesity. Empowering GPs to use their skills as expert generalists to manage obesity is an option that should be explored to improve access for all individuals. GPs will require evidence-based tools to assist them in structuring obesity management within their own general practice environment.

Obesity management by general practitioners: the unavoidable necessity.

Nearly 62% of primary care patients are overweight or obese, and obesity is now a National Health Priority Area. Weight management interventions in primary care currently generate little more than 1 kg of weight loss per patient over a 2-year period. Consequently, further strategies are required to improve the effectiveness of weight management in primary care. The National Health and Medical Research Council (NHMRC) have released updated guidelines for the management of overweight and obese patients in primary care. However, there is some disconnect between establishment of guidelines and their implementation in practice. Barriers to GPs using guidelines for the management of obesity include low self-efficacy, perceived insufficient time in consultations and the challenge of raising the topic of a patient's weight. Nonetheless, patients prefer to receive weight management support from GPs rather than other health professionals, suggesting that the demand on GPs to support patients in weight management will continue. GPs cannot afford to side-line obesity management, as obesity is likely to be the most prevalent modifiable risk factor associated with patients' long-term health. Without further strategies to support GPs in their management of patients' weight, obesity will continue to be an expensive and long-term public health issue.

Impact of the Medicare Chronic Disease Management program on the conduct of Australian dietitians' private practices.

OBJECTIVE: This study explored private practice dietitians' perceptions of the impact of the Australian Chronic Disease Management (CDM) program on the conduct of their private practice, and the care provided to patients. METHODS: Twenty-five accredited practising dietitians working in primary care participated in an individual semistructured telephone interview. Interview questions focussed on dietitians' perceptions of the proportion of patients receiving care through the CDM program, fee structures, adhering to reporting requirements and auditing. Transcript data were thematically analysed using a process of open coding. RESULTS: Half of the dietitians (12/25) reported that most of their patients (>75%) received care through the CDM program. Many dietitians (19/25) reported providing identical care to patients using the CDM program and private patients, but most (17/25) described spending substantially longer on administrative tasks for CDM patients. Dietitians experienced pressure from doctors and patients to keep their fees low or to bulk-bill patients using the CDM program. One-third of interviewed dietitians (8/25) expressed concern about the potential to be audited by Medicare. Recommendations to improve the CDM program included increasing the consultation length and subsequent rebate available for dietetic consultations, and increasing the number of consultations to align with dietetic best-practice guidelines. CONCLUSIONS: The CDM program creates challenges for dietitians working in primary care, including how to sustain the quality of patient-centred care and yet maintain equitable business practices. To ensure the CDM program appropriately assists patients to receive optimal care, further review of the CDM program within the scope of dietetics is required.

Comparing the rehydration potential of different milk-based drinks to a carbohydrate-electrolyte beverage.

The aim of this study was to compare the rehydration potential of a carbohydrate-electrolyte beverage with several varieties of milk following exercise-induced fluid losses. Fifteen male participants (age 24.9 ± 5.5 years, height 179.3 ± 4.9 cm, body mass 75.8 ± 6.6 kg (mean ± SD)) lost 2.0% ± 0.2% body mass through intermittent cycling before consuming a different beverage on 4 separate occasions. Drinks included cow's milk (286 kJ·100 mL(-1)), soy milk (273 kJ·100 mL(-1)), a milk-based liquid meal supplement (Sustagen Sport (Nestle); 417 kJ·100 mL(-1)), and a sports drink (Powerade (Coca Cola Ltd); 129 kJ·100 mL(-1)). Beverages were consumed over 1 h in volumes equivalent to 150% of body mass loss. Body mass, blood and urine samples, and measures of gastrointestinal tolerance were obtained before and hourly for 4 h after beverage consumption. Net body mass at the conclusion of each trial was significantly less with Powerade (-1.37 ± 0.3 kg) than with cow's milk (-0.92 ± 0.48 kg), soy milk (-0.78 ± 0.37 kg), and Sustagen Sport (-0.48 ± 0.39 kg). Net body mass was also significantly greater for Sustagen Sport compared with cow's milk trials, but not soy milk. Upon completion of trials, the percentage of beverage retained was Sustagen Sport 65.1% ± 14.7%, soy milk 46.9% ± 19.9%, cow's milk 40.0% ± 24.9%, and Powerade 16.6% ± 16.5%. Changes in plasma volume and electrolytes were unaffected by drink treatment. Subjective ratings of bloating and fullness were higher during all milk trials compared with Powerade whereas ratings of overall thirst were not different between beverages. Milk-based drinks are more effective rehydration options compared with traditional sports drinks. The additional energy, protein, and sodium in a milk-based liquid meal supplement facilitate superior fluid recovery following exercise.

Glucose deprivation, oxidative stress and peroxisome proliferator-activated receptor-alpha (PPARA) cause peroxisome proliferation in preimplantation mouse embryos.

Ex vivo two-cell mouse embryos deprived of glucose in vitro can develop to blastocysts by increasing their pyruvate consumption; however, zygotes when glucose-deprived cannot adapt this metabolic profile and degenerate as morulae. Prior to their death, these glucose-deprived morulae exhibit upregulation of the H+-monocarboxylate co-transporter SLC16A7 and catalase, which partly co-localize in peroxisomes. SLC16A7 has been linked to redox shuttling for peroxisomal beta-oxidation. Peroxisomal function is unclear during preimplantation development, but as a peroxisomal transporter in embryos, SLC16A7 may be involved and influenced by peroxisome proliferators such as peroxisome proliferator-activated receptor-alpha (PPARA). PCR confirmed Ppara mRNA expression in mouse embryos. Zygotes were cultured with or without glucose and with the PPARA-selective agonist WY14643 and the developing embryos assessed for expression of PPARA and phospho-PPARA in relation to the upregulation of SLC16A7 and catalase driven by glucose deprivation, indicative of peroxisomal proliferation. Reactive oxygen species (ROS) production and relationship to PPARA expression were also analysed. In glucose-deprived zygotes, ROS was elevated within 2 h, as were PPARA expression within 8 h and catalase and SLC16A7 after 12-24 h compared with glucose-supplied embryos. Inhibition of ROS production prevented this induction of PPARA and SLC16A7. Selective PPARA agonism with WY14643 also induced SLC16A7 and catalase expression in the presence of glucose. These data suggest that glucose-deprived cleavage stage embryos, although supplied with sufficient monocarboxylate-derived energy, undergo oxidative stress and exhibit elevated ROS, which in turn upregulates PPARA, catalase and SLC16A7 in a classical peroxisomal proliferation response.

Characterization and regulation of monocarboxylate cotransporters Slc16a7 and Slc16a3 in preimplantation mouse embryos.

Concurrent with compaction, preimplantation mouse embryos switch from the high pyruvate consumption that prevailed during cleavage stages to glucose consumption against a constant background of pyruvate uptake. However, zygotes exposed to and subsequently deprived of glucose can form blastocysts by increasing pyruvate uptake. This metabolic switch requires cleavage-stage exposure to glucose and is one aspect of metabolic differentiation that normally occurs in vivo. Monocarboxylates, such as pyruvate and lactate, are transported across membranes via the SLC16 family of H(+)-monocarboxylate cotransporter (MCT) proteins. Thus, the increase in pyruvate uptake in embryos developing without glucose must involve changes in activity and localization of MCT. In mouse embryos, continued expression of Slc16a1 (MCT1) requires glucose supply. Messenger RNA for Slc17a7 (MCT2) and Slc16a3 (MCT4) has been detected in mouse preimplantation embryos; however, protein function, localization, and regulation of expression at the basis of these net pyruvate uptake changes remain unclear. The expression and localization of SLC16A7 and SLC16A3 have therefore been examined to clarify their respective roles in embryos derived from the reproductive tract and cultured under varied conditions. SLC16A3 appears localized to the plasma membrane until the morula stage and also maintains a nuclear distribution throughout preimplantation development. However, continued Slc16a3 mRNA expression is dependent on prior exposure to glucose. SLC16A7 localizes to apical cortical regions with punctate, vesicular expression throughout blastomeres, partially colocalizing in peroxisomes with peroxisomal catalase (CAT). In contrast to SLC16A3 and SLC16A1, SLC16A7 and CAT demonstrate upregulation in the absence of glucose. These striking differences between the two isoforms in expression localization and regulation suggest unique roles for each in monocarboxylate transport and pH regulation during preimplantation development, and implicate peroxisomal SLC16A7 as an important redox regulator in the absence of glucose.

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development.

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. In the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (Km 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. In expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.