Dysregulation of leukocyte trafficking in ageing: Causal factors and possible corrective therapies.

Ageing is a universal biological phenomenon that is accompanied by the development of chronic, low-grade inflammation and remodelling of the immune system resulting in compromised immune function. In this review, we explore how the trafficking of innate and adaptive immune cells under homeostatic and inflammatory conditions is dysregulated in ageing. We particularly highlight the age-related changes in the expression of adhesion molecules and chemokine receptor/ligands, and the accumulation of senescent cells that drive modulated leukocyte trafficking. These age-related changes to leukocyte trafficking are multifactorial and specific to leukocyte subset, tissue, type of vascular bed, and inflammatory status. However, dysregulated leukocyte trafficking ultimately affects immune responses in older adults. We therefore go on to discuss approved drugs, including anti-integrins, anti-chemokines and statins, as well as novel therapeutics that may be used to target dysregulated leukocyte trafficking in ageing, improve immune responses and delay the onset of age-related diseases.

Dysregulation of the actin scavenging system and inhibition of DNase activity following severe thermal injury.

BACKGROUND: Circulating cell-free DNA (cfDNA) is not found in healthy subjects, but is readily detected after thermal injury and may contribute to the risk of multiple organ failure. The hypothesis was that a postburn reduction in DNase protein/enzyme activity could contribute to the increase in cfDNA following thermal injury. METHODS: Patients with severe burns covering at least 15 per cent of total body surface area were recruited to a prospective cohort study within 24 h of injury. Blood samples were collected from the day of injury for 12 months. RESULTS: Analysis of blood samples from 64 patients revealed a significant reduction in DNase activity on days 1-28 after injury, compared with healthy controls. DNase protein levels were not affected, suggesting the presence of an enzyme inhibitor. Further analysis revealed that actin (an inhibitor of DNase) was present in serum samples from patients but not those from controls, and concentrations of the actin scavenging proteins gelsolin and vitamin D-binding protein were significantly reduced after burn injury. In a pilot study of ten military patients with polytrauma, administration of blood products resulted in an increase in DNase activity and gelsolin levels. CONCLUSION: The results of this study suggest a novel biological mechanism for the accumulation of cfDNA following thermal injury by which high levels of actin released by damaged tissue cause a reduction in DNase activity. Restoration of the actin scavenging system could therefore restore DNase activity, and reduce the risk of cfDNA-induced host tissue damage and thrombosis.

ANTECEDENTES: El ADN libre de las células circulantes (circulating cell-free DNA, cfDNA) no se encuentra en sujetos sanos, pero se detecta fácilmente después de una lesión térmica y puede contribuir al riesgo de fallo multiorgánico. La hipótesis fue que una disminución en la actividad de la proteína/enzima ADNasa tras la lesión térmica podría contribuir a la elevación del cfDNA que ocurre tras la misma. MÉTODOS: Los pacientes con quemaduras graves con una extensión ≥ 15% del área de superficie corporal total (total body surface area, TBSA) se incluyeron en un estudio prospectivo de cohortes durante las primeras 24 horas posteriores a la lesión. Se recogieron muestras de sangre desde el día de la lesión hasta los 12 meses posteriores a la misma. RESULTADOS: El análisis de muestras de sangre de 64 pacientes reveló una reducción significativa de la actividad de la ADNasa en los días 1 a 28 después de la lesión, en comparación con los controles sanos. Los niveles de proteína ADNasa no se vieron afectados, lo que sugiere la presencia de un inhibidor enzimático. Un análisis adicional reveló que la actina (un inhibidor de la ADNasa) estaba presente en las muestras de suero de los pacientes, pero no en los controles, y las concentraciones de la gelsolina, proteína que causa la disociación de la actina, y la proteína de unión a la vitamina D se redujeron significativamente después de la lesión térmica. En un estudio piloto de 10 pacientes con politrauma por lesiones militares, la administración de hemoderivados produjo un aumento en la actividad de la ADNasa y de los niveles de gelsolina. CONCLUSIÓN: Este estudio sugiere un nuevo mecanismo biológico para la acumulación de cfDNA después de una lesión térmica, por el cual los altos niveles de actina liberada por el tejido dañado causarían una reducción en la actividad de la ADNasa. La restauración del sistema eliminador de actina podría, por lo tanto, restaurar la actividad de la ADNasa y reducir el riesgo de daño tisular y trombosis en el huésped inducido por el cfDNA.

Relationships Between Markers of Inflammation and Muscle Mass, Strength and Function: Findings from the Hertfordshire Cohort Study.

We investigated the longitudinal relationships between inflammation markers and the following outcomes in a UK cohort study: appendicular lean mass (ALM); walking speed; level and change in grip strength; and sarcopenia defined by the European Working Group on Sarcopenia in Older People. Analyses were based on 336 community-dwelling older men and women (aged 59-70 years) who participated in the Hertfordshire Cohort Study (HCS). Inflammation markers were ascertained at baseline using enzyme-linked immunosorbent assay techniques and Bio-Plex Pro Assays. Grip strength was measured at baseline and follow-up [median follow-up time: 10.8 years (inter-quartile range 10.2-11.6)] and change in grip strength was ascertained using a residual change approach. At follow-up, ALM was ascertained using dual-energy X-ray absorptiometry, customary walking speed was measured and sarcopenia status was ascertained. Gender-adjusted linear and Poisson regression was used to examine the associations between inflammation markers and outcomes with and without adjustment for anthropometric and lifestyle factors. Higher C-reactive protein was associated (p < 0.04) with lower grip strength and accelerated decline in grip strength from baseline to follow-up. Higher cortisol was associated with lower ALM (p < 0.05). Higher interleukin-8 (IL-8) was associated with lower ALM (p < 0.05) and increased risk of sarcopenia [fully-adjusted relative risk per SD increase in IL-8: 1.37 (95% CI 1.10, 1.71), p = 0.005]. All associations were robust in fully-adjusted analyses. Inflammation markers were associated with measures of muscle mass, strength and function in HCS. Further work is required to replicate these associations and to delineate the underlying mechanisms.

Cardiorespiratory fitness not sedentary time or physical activity is associated with cardiometabolic risk in active older adults.

Sedentary time (ST) and moderate-to-vigorous physical activity (MVPA) are associated with cardiometabolic health. Cardiorespiratory fitness (CRF) is also implicated but often overlooked in health recommendations. This study assessed the relationships between ST, MVPA, CRF, and cardiometabolic health in highly active older individuals. 125 healthy amateur cyclists aged 55 to 79 years had their ST and MVPA levels assessed by actigraphy over a 7-day period. CRF was assessed using a maximal effort cycle ergometry test to determine VO2max with results normalized to both body mass and fat-free mass measured by DXA. Markers of cardiometabolic risk (blood glucose, triglycerides, cholesterol, HDL, LDL, Insulin, HOMA IR, blood pressure, and body fat) were assessed and used to determine cumulative cardiometabolic risk. Multiple linear regression was used to assess ST, MVPA, and CRF associations with cardiometabolic health with the relationship between activity levels and CRF determined. CRF was associated with training volume (P = .003), but not ST or MVPA. A high CRF was associated with lower cumulative cardiometabolic risk, body fat percentage, triglyceride, and HDL levels (P < .05 in all cases). MVPA was negatively associated with body fat percentage, while ST was not associated with any marker of cardiometabolic risk when adjusting for activity levels. An association between CRF and cardiometabolic risk even in a group of older individuals with high fitness levels highlights the importance that CRF may have in maintaining health.

The individual and combined effects of obesity- and ageing-induced systemic inflammation on human skeletal muscle properties.

BACKGROUND/OBJECTIVES: The purpose of this study was to determine whether circulating pro-inflammatory cytokines, elevated with increased fat mass and ageing, were associated with muscle properties in young and older people with variable adiposity. SUBJECTS/METHODS: Seventy-five young (18-49 yrs) and 67 older (50-80 yrs) healthy, untrained men and women (BMI: 17-49 kg/m2) performed isometric and isokinetic plantar flexor maximum voluntary contractions (MVCs). Volume (Vm), fascicle pennation angle (FPA), and physiological cross-sectional area (PCSA) of the gastrocnemius medialis (GM) muscle were measured using ultrasonography. Voluntary muscle activation (VA) was assessed using electrical stimulation. GM specific force was calculated as GM fascicle force/PCSA. Percentage body fat (BF%), body fat mass (BFM), and lean mass (BLM) were assessed using dual-energy X-ray absorptiometry. Serum concentration of 12 cytokines was measured using multiplex luminometry. RESULTS: Despite greater Vm, FPA, and PCSA (P<0.05), young individuals with BF% ⩾40 exhibited 37% less GM specific force compared to young BF%<40 (P<0.05). Older adults with BF% ⩾40 showed greater isokinetic MVC compared to older BF%<40 (P=0.019) but this was reversed when normalised to body mass (P<0.001). IL-6 correlated inversely with VA in young (r=-0.376; P=0.022) but not older adults (p>0.05), while IL-8 correlated with VA in older but not young adults (r⩾0.378, P⩽0.027). TNF-alpha correlated with MVC, lean mass, GM FPA and maximum force in older adults (r⩾0.458; P⩽0.048). CONCLUSIONS: The age- and adiposity-dependent relationships found here provide evidence that circulating pro-inflammatory cytokines may play different roles in muscle remodelling according to the age and adiposity of the individual.

Where have all the blue flowers gone: pollinator responses and selection on flower colour in New Zealand Wahlenbergia albomarginata.

Although pollinators are thought to select on flower colour, few studies have experimentally decoupled effects of colour from correlated traits on pollinator visitation and pollen transfer. We combined selection analysis and phenotypic manipulations to measure the effect of petal colour on visitation and pollen export at two spatial scales in Wahlenbergia albomarginata. This species is representative of many New Zealand alpine herbs that have secondarily evolved white or pale flowers. The major pollinators, solitary bees, exerted phenotypic selection on flower size but not colour, quantified by bee vision. When presented with manipulated flowers, bees visited flowers painted blue to resemble a congener over white flowers in large, but not small, experimental arrays. Pollen export was higher for blue flowers in large arrays. Pollinator preference does not explain the pale colouration of W. albomarginata, as commonly hypothesized. Absence of bright blue could be driven instead by indirect selection of correlated characters.

Association of SARC-F Score and Rockwood Clinical Frailty Scale with CT-Derived Muscle Mass in Patients with Aortic Aneurysms.

OBJECTIVES: Patients with aortic aneurysms (AA) are often co-morbid and susceptible to frailty. Low core muscle mass has been used as a surrogate marker of sarcopenia and indicator of frailty. This study aimed to assess association between core muscle mass with sarcopenia screening tool SARC-F and Clinical Frailty Scale (CFS) in patients with AA. METHODS: Prospective audit of patients in pre-operative aortic clinic between 01/07/2019-31/01/2020 including frailty assessment using Rockwood CFS and sarcopenia screening using SARC-F questionnaire. Psoas and sartorius muscle area were measured on pre-operative CT scans and adjusted for height. Association was assessed using Spearman's rank correlation coefficient. RESULTS: Of 84 patients assessed, median age was 75 years [72,82], 84.5% were men, 65.5% were multimorbid and 63.1% had polypharmacy. Nineteen percent were identified as frail (CFS score >3) and 6.1% positively screened for sarcopenia (SARC-F score 4 or more). Median psoas area (PMA) at L3 was 5.6cm2/m2 [4.8,6.6] and L4 was 7.4cm2/m2 [6.3,8.6]. Median sartorius area (SMA) was 1.8 cm2/m2 [1.5,2.2]. CFS demonstrated weak but statistically significant negative correlation with height-adjusted PMA at L3 (r=-0.25, p=0.034) but not at L4 (r=-0.23, p=0.051) or with SMA (r=-0.22, p=0.065). No association was observed between SARC-F score and PMA or SMA (L3 PMA r=-0.015, p=0.9; L4 PMA r=-0.0014, p= 0.99; SMA r=-0.051, p=0.67). CONCLUSION: CFS showed higher association with CT-derived muscle mass than SARC-F. Comprehensive pre-operative risk-stratification tools which incorporate frailty assessment and body composition analysis may assist in decision making for surgery and allow opportunity for pre-habilitation.

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum.

The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.

Lactoferrin is a survival factor for neutrophils in rheumatoid synovial fluid.

OBJECTIVES: Lactoferrin is an iron-binding protein that is released from activated neutrophils at sites of inflammation and has anti-microbial as well as anti-inflammatory properties. This study set out to determine whether lactoferrin can delay neutrophil apoptosis and could act as a survival factor for neutrophils in SF. METHODS: Human peripheral blood and SF neutrophils were incubated with iron-free lactoferrin and apoptosis determined after 9 h. SF from patients with RA was added to isolated neutrophils, with or without immunodepletion of lactoferrin, and effects on neutrophil apoptosis determined. Levels of lactoferrin in SF were assessed and related to disease duration and markers of disease activity. RESULTS: Iron-free lactoferrin significantly delayed apoptosis of peripheral blood neutrophils, in a concentration-dependent manner after 9 h in culture (P < 0.04). Lactoferrin could also delay apoptosis of neutrophils isolated from SF of patients with RA. SF from patients with established RA delayed apoptosis of peripheral blood neutrophils and this effect was significantly reduced by depletion of lactoferrin (P < 0.03). Lactoferrin levels in SF from patients with established RA did not correlate with disease severity, but did correlate with markers of inflammation (CRP) and with the presence of RF. SF from patients with arthritis of <12 weeks duration did not contain significant levels of lactoferrin. CONCLUSION: Lactoferrin contributes to extended neutrophil survival in the rheumatoid joint in the established phase of RA but not in very early arthritis.

Toxin entry: how reversible is the secretory pathway?

A number of proteins produced by plants and bacteria are extremely toxic to eukaryotic cells. Their potency arises from their ability to catalyse the modification of crucial cellular components. Only a few toxin molecules are required to kill a cell, but to do so they must first reach the cytosol. How such proteins are translocated across the target cell membrane is poorly understood, but we argue here that some toxins may travel the secretory pathway in reverse, passing all the way from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol.

Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm.

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined. This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase. The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase. This fraction also had NADH diaphorase activity and contained cytochromes b(5) and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm(3) and sedimented separately from the ribosomes; electron micrographs showed that it was comprised of smooth membranes. When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1.16 g/cm(3) and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum. Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes.

Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells.

Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.

An activated protein kinase C alpha gives a differentiation signal for hematopoietic progenitor cells and mimicks macrophage colony-stimulating factor-stimulated signaling events.

Highly enriched, bipotent, hematopoietic granulocyte macrophage colony-forming cells (GM-CFC) require cytokines for their survival, proliferation, and development. GM-CFC will form neutrophils in the presence of the cytokines stem cell factor and granulocyte colony-stimulating factor, whereas macrophage colony-stimulating factor leads to macrophage formation. Previously, we have shown that the commitment to the macrophage lineage is associated with lipid hydrolysis and translocation of protein kinase C alpha (PKCalpha) to the nucleus. Here we have transfected freshly prepared GM-CFC with a constitutively activated form of PKCalpha, namely PKAC, in which the regulatory domain has been truncated. Greater than 95% of the transfected cells showed over a twofold increase in PKCalpha expression with the protein being located primarily within the nucleus. The expression of PKAC caused macrophage development even in the presence of stimuli that normally promote only neutrophilic development. Thus, M-CSF-stimulated translocation of PKCalpha to the nucleus is a signal associated with macrophage development in primary mammalian hematopoietic progenitor cells, and this signal can be mimicked by ectopic PKAC, which is also expressed in the nucleus.

Dissociation of caspase-mediated events and programmed cell death induced via HLA-DR in follicular lymphoma.

Human leukocyte antigens (HLA) class II antigen-mediated apoptosis has been documented in antigen-presenting cells and B lymphoproliferations. Characteristics of the apoptosis include rapidity and selectivity for mature cells. Follicular lymphomas are particularly refractory to apoptosis. The B-cell lymphoma Ramos shares characteristics of this subgroup and is insensitive to apoptosis via simple HLA-DR engagement. However, oligomerization of HLA-DR antigens induced caspase activation followed by phosphatidylserine externalization, activation of PKC-delta and cleavage of nuclear lamin B. Mitochondrial injury was also detected. However, inhibition of caspase activation simply delayed the apoptotic phenotype but neither protected against cell death nor prevented mitochondrial injury. The data in this report demonstrate that the requirements for the initiating signal (oligomerization versus engagement) as well as the molecular pathways varies between different B lymphoproliferations despite their common expression of HLA-DR. Finally, blockade of caspase activation in parallel with HLA-DR mAb stimulation could provide a potent autovaccination stimulus by leading to necrotic death of B-cell lymphomas.

Go outside and see the proteasome. Protein degradation.

Newly synthesized proteins that fail to fold or assemble properly in the endoplasmic reticulum are degraded. Recent work on several endoplasmic reticulum membrane proteins has shown that the cytosolic proteasome plays a role in their degradation.

Glycoprotein degradation: do sugars hold the key?

Secretory glycoproteins that fail to fold or assemble correctly are retained in the endoplasmic reticulum and eventually degraded. Recent evidence shows that trimming of their N-linked oligosaccharide chains plays a key role in targeting glycoproteins for destruction.

Retrograde transport: going against the flow.

Certain protein toxins act by catalytically modifying substrates in the cytosol of mammalian cells. To reach this compartment, these proteins undergo retrograde transport from the cell surface, via the Golgi complex, to the endoplasmic reticulum.

Increased Phosphorylation of a 26-kD Pollen Protein Is Induced by the Self-Incompatibility Response in Papaver rhoeas.

We have investigated whether specific protein phosphorylation events are induced in Papaver rhoeas pollen as a consequence of the self-incompatibility (SI) response. Pollen grown in vitro in the presence of 32P-orthophosphate was challenged with biologically active recombinant S proteins, and pollen proteins were extracted and analyzed. The results provide strong evidence that the increased phosphorylation of a 26-kD protein of pl 6.2, p26, is specifically induced by the SI response. This phosphorylation event occurs in living pollen tubes and was observed specifically when pollen was challenged with S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible or incompatible heat-denatured S proteins. Further characterization demonstrated that p26 comprises two phosphoproteins, p26.1 and p26.2, that are found in soluble and microsomal fractions, respectively. Increased phosphorylation of p26.1 is implicated in the SI response and appears to be Ca2+ and calmodulin dependent. These data argue for the involvement of a Ca2+-dependent protein kinase requiring calmodulin-like domains, whose activation comprises an intracellular signal mediating the SI response in P. rhoeas pollen.

The influence of sex steroid hormones on the response to trauma and burn injury.

Trauma and related sequelae result in disturbance of homeostatic mechanisms frequently leading to cellular dysfunction and ultimately organ and system failure. Regardless of the type and severity of injury, gender dimorphism in outcomes following trauma have been reported, with females having lower mortality than males, suggesting that sex steroid hormones (SSH) play an important role in the response of body systems to trauma. In addition, several clinical and experimental studies have demonstrated the effects of SSH on the clinical course and outcomes following injury. Animal studies have reported the ability of SSH to modulate immune, inflammatory, metabolic and organ responses following traumatic injury. This indicates that homeostatic mechanisms, via direct and indirect pathways, can be maintained by SSH at local and systemic levels and hence result in more favourable prognosis. Here, we discuss the role and mechanisms by which SSH modulates the response of the body to injury by maintaining various processes and organ functions. Such properties of sex hormones represent potential novel therapeutic strategies and further our understanding of current therapies used following injury such as oxandrolone in burn-injured patients.

Changes in novel haematological parameters following thermal injury: A prospective observational cohort study.

The mortality caused by sepsis is high following thermal injury. Diagnosis is difficult due to the ongoing systemic inflammatory response. Previous studies suggest that cellular parameters may show promise as diagnostic markers of sepsis. The aim of this study was to evaluate the effect of thermal injury on novel haematological parameters and to study their association with clinical outcomes. Haematological analysis was performed using a Sysmex XN-1000 analyser on blood samples acquired on the day of the thermal injury to 12 months post-injury in 39 patients (15-95% TBSA). Platelet counts had a nadir at day 3 followed by a rebound thrombocytosis at day 21, with nadir values significantly lower in septic patients. Measurements of extended neutrophil parameters (NEUT-Y and NEUT-RI) demonstrated that septic patients had significantly higher levels of neutrophil nucleic acid content. A combination of platelet impedance count (PLT-I) and NEUT-Y at day 3 post-injury exhibited good discriminatory power for the identifying septic patients (AUROC = 0.915, 95% CI [0.827, 1.000]). Importantly, the model had improved performance when adjusted for mortality with an AUROC of 0.974 (0.931, 1.000). A combination of PLT-I and NEUT-Y show potential for the early diagnosis of sepsis post-burn injury. Importantly, these tests can be performed rapidly and require a small volume of whole blood highlighting their potential utility in clinical practice.