A Novel Monoclonal Antibody 1D2 That Broadly Inhibits Clinically Important Aspergillus Species.

Aspergillus fumigatus is a ubiquitous airborne fungus, is the predominant cause (>90%) of invasive aspergillosis (IA) in immunosuppressed patients and has a high mortality. New approaches to prevention and treatment are needed because of the poor efficacy, toxicity and side effects of the current anti-Aspergillus drugs on patients. Thus, we aim to explore a new avenue to combat Aspergillus infection by using a novel monoclonal antibody (mAb) 1D2 against a glycoprotein on the cell wall of Aspergillus. The ability of this mAb to inhibit attachment, germination, and growth of Aspergillus conidia and hyphae in vitro were examined. A dose-dependent growth inhibition of Aspergillus conidia in the presence of mAb 1D2 was found. The mAb 1D2 inhibited attachment of Aspergillus conidia to an untreated slide surface and fibronectin-treated surface compared to an unrelated mAb 6B10. When conidia were exposed to 1D2 concomitantly with inoculation into culture media, the mAb prevented the swelling and germination of conidia. This inhibitory ability of 1D2 was less apparent if it was added two hours after inoculation. Damage to hyphae was also observed when 1D2 was added to Aspergillus hyphae that had been incubated in media overnight. These in vitro results indicate that mAb 1D2 broadly inhibits clinically important Aspergillus species and has a promising therapeutic effect both as prophylaxis to inhibit an Aspergillus infection as well as a treatment.

Monoclonal Antibodies and Invasive Aspergillosis: Diagnostic and Therapeutic Perspectives.

Invasive aspergillosis (IA) is a life-threatening fungal disease that causes high morbidity and mortality in immunosuppressed patients. Early and accurate diagnosis and treatment of IA remain challenging. Given the broad range of non-specific clinical symptoms and the shortcomings of current diagnostic techniques, most patients are either diagnosed as "possible" or "probable" cases but not "proven". Moreover, because of the lack of sensitive and specific tests, many high-risk patients receive an empirical therapy or a prolonged treatment of high-priced antifungal agents, leading to unnecessary adverse effects and a high risk of drug resistance. More precise diagnostic techniques alongside a targeted antifungal treatment are fundamental requirements for reducing the morbidity and mortality of IA. Monoclonal antibodies (mAbs) with high specificity in targeting the corresponding antigen(s) may have the potential to improve diagnostic tests and form the basis for novel IA treatments. This review summarizes the up-to-date application of mAb-based approaches in assisting IA diagnosis and therapy.

Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice.

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) transports cortisol and other steroids. High-affinity CBG (haCBG) undergoes proteolysis of the reactive center loop (RCL) by neutrophil elastase (NE) altering conformation to low-affinity CBG (laCBG). Elevated temperature reduces CBG:cortisol binding affinity. Surface plasmon resonance was used to determine binding profiles of 19 steroids to haCBG and laCBG at 25, 37, and 39°C mimicking pyrexia and pH 7.4 and 7.0 mimicking acidosis, pathophysiological conditions relevant to sepsis. An expected 4-8-fold reduction in affinity for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone, and prednisolone occurred with NE-mediated haCBG-to-laCBG conversion. CBG:cortisol binding affinity was further reduced 3.5-fold at 39°C relative to 37°C, binding affinity was also reduced by acidosis for both haCBG and laCBG. Using a conformational antibody generated against the RCL, we confirmed RCL antibody binding was eliminated by NE cleavage, but preserved in pyrexia and acidosis. Molecular modeling studies performed at 40°C confirmed a critical role for Trp371, positioned within the steroid-binding pocket, in ligand binding. These studies demonstrated CBG binding affinity to range of steroids is ligand specific and is reduced with NE-mediated haCBG-to-laCBG transition. Reduced CBG:cortisol binding occurs with increased temperature and in acidosis. Increased flexibility of the Trp371 side chain is proposed in the thermo-coupling mechanism of cortisol release. The synergy of NE cleavage, pyrexia, and acidosis on CBG:cortisol binding may serve to enhance cortisol delivery to the interstitial space in inflammation.

Enhancement of Culture of Legionella longbeachae from Respiratory Samples by Use of Immunomagnetic Separation and Antimicrobial Decontamination.

Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae A polyclonal antibody specific for L. longbeachae was produced from New Zealand White rabbits and coupled to tosyl-activated magnetic beads. Stored L. longbeachae qPCR-positive respiratory samples were retrieved from -80°C storage for testing. One portion of test samples was mixed with GVPC and the antibody bead complex, separated, washed, and cultured on modified Wadowsky and Yee agar (MWY) agar. Another portion was exposed to HCl-KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (P = 0.001). Growth of contaminants was rare. The mean qPCR threshold cycle values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7; 95% CI, ±2.48; P = 0.16). The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination, but this should be confirmed in a prospective study of fresh specimens.

High binding site occupancy of corticosteroid-binding globulin by progesterone increases fetal free cortisol concentrations.

OBJECTIVE: Corticosteroid-binding globulin (CBG) binds and transports cortisol in the circulation in high cortisol-binding affinity (haCBG) and low affinity (laCBG) forms, the latter resulting from enzyme cleavage to target cortisol delivery at sites of inflammation. CBG also has substantial progesterone binding affinity, 3-fold less than cortisol. Progesterone and cortisol are important in the maintenance of pregnancy and in fetal development, respectively. The interactions of cortisol, progesterone and CBG affinity forms have not been studied together. We examined the interaction between progesterone and cortisol with CBG during fetal development. STUDY DESIGN: A retrospective cohort analysis of 351 neonates born between January and December 2012 at the Women's and Children's Hospital, Adelaide, South Australia. Cord blood serum samples were collected immediately following delivery. Clinical data was provided by hospital records. Total cortisol, free cortisol, total progesterone, total CBG and haCBG were measured by immunoassay. RESULTS: Cord blood total and free cortisol, and progesterone concentrations increased with gestational age. Cord blood progesterone concentrations were 100-fold luteal and 10-fold those in late pregnancy maternal circulation. The proportion of haCBG to total CBG was similar to that in healthy non-pregnant adults. However, free cortisol comprised approximately 15% of total cortisol, 3-fold higher than that in adults. CONCLUSION: In a manner unique to fetal life, very high progesterone concentrations are capable of elevating free cortisol concentrations through competition with cortisol at CBG's hormone binding site, without altered binding affinity through CBG cleavage or altered CBG hormone-binding affinity. High circulating fetal progesterone concentrations compete for CBG binding with cortisol, leading to a 3-fold increase in the free cortisol fraction in cord blood. Higher free-to-bound cortisol may alter fetal cortisol distribution facilitating cortisol's roles such as neurodevelopment in concert with dehydroepiandrosterone (sulfate) and lung maturation, or support cortisol action at times of low ambient cortisol. This mechanism may underlie the known association between cortisol, progesterone and CBG, and be relevant principally in the fetal circulation due to the high progesterone concentrations encountered.

Glycosylation may influence sex hormone-binding globulin measurements.

A discordance between sex hormone-binding globulin (SHBG) measurements by 2-site ELISAs was investigated using pairings of various "in house" SHBG antibodies together with a concordant control. The 2-site monoclonal ELISAs used the same base coat (11F11) and discordance was observed with one top coat monoclonal antibody (7H9) and also when a polyclonal SHBG antibody was paired with the basecoat antibody (11F11). Sialidase treatment of the discordant sample and purified SHBG revealed increased levels using 7H9 whereas there was no change in SHBG in the concordant sample. Conversely, following sialidase treatment, the discordant sample showed no change in SHBG measured using the other monoclonal antibody pairings whereas the SHBG levels in the concordant sample declined following sialidase using the same monoclonal antibody pairings. This implicated glycosylation as a factor in antibody recognition and synthetic peptides spanning the two N-linked and one O-linked glycosylation regions showed that SHBG recognition by monoclonal antibody 7H9 could be disrupted by a peptide spanning the O-linked glycosylation site. Hence rather than immunoassay discordance being attributed to heterophile antibodies or other circulating antibodies here it can be likely attributed to glycosylation affecting antibody recognition and hence the measurement of SHBG.

Effect of genetic factors on the response to vitamin D3 supplementation in the VIDARIS randomized controlled trial.

OBJECTIVES: Supplementation provides the best means of improving vitamin D status; however, individual responses vary partly owing to genetics. The aim of this study was to determine whether 28 single nucleotide polymorphisms (SNPs) in six key vitamin D pathway genes (GC, DHCR7, CYP2 R1, CYP24 A1, CYP27 B1, VDR) were associated with differences in response to supplementation. METHODS: Participants (N = 313; n = 160 vitamin D, n = 153 placebo) were part of VIDARIS (Vitamin D and Acute Respiratory Infections Study), a double-blind, randomized controlled trial involving oral monthly supplementation of either vitamin D3 (200 000 IU each for the first 2 mo, thereafter 100 000 IU monthly) or placebo for 18 mo. Circulating 25-hydroxyvitamin D (25[OH]D) concentrations at baseline and 2, 6, 12, and 18 mo, and vitamin D binding protein (Gc-globulin) and calculated free 25(OH)D concentrations at baseline and 2 mo were obtained. Multiple regression was used to model associations between genetic variants and 25(OH)D, Gc-globulin, and free 25(OH)D concentrations. RESULTS: SNPs within GC, CYP2 R1, and CYP27 B1 were associated with 25(OH)D concentrations following supplementation. However, only two GC gene SNPs (rs2282679, rs1155563) were significant after adjustment for multiple testing. This effect disappeared after more than 2 mo of supplementation. None of the SNPs were significantly associated with Gc-globulin concentrations; however, there was a significant interaction with one SNP in DHCR7 (rs12785878), which was associated with reduced free 25(OH)D concentrations in the supplemented arm. CONCLUSION: Only variants of GC were associated with 25(OH)D concentrations after supplementation. This effect was modest and disappeared after >2 mo of supplementation, suggesting it may be time/dose-dependent and saturable.

N-Glycosylation influences human corticosteroid-binding globulin measurements.

OBJECTIVE: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. METHODS AND SUBJECTS: Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. RESULTS: We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. CONCLUSIONS: Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

Fine mapping of Rha2 in barley reveals candidate genes for resistance against cereal cyst nematode.

KEY MESSAGE: The cereal cyst nematode resistance locus Rha2 was mapped to a 978 kbp region on the long arm of barley chromosome 2H. Three candidate genes are discussed. The cereal cyst nematode (CCN) Heterodera avenae is a soil-borne obligate parasite that can cause severe damage to cereals. This research involved fine mapping of Rha2, a CCN resistance locus on chromosome 2H of barley. Rha2 was previously mapped relative to restriction fragment length polymorphisms (RFLPs) in two mapping populations. Anchoring of flanking RFLP clone sequences to the barley genome assembly defined an interval of 5077 kbp. Genotyping-by-sequencing of resistant and susceptible materials led to the discovery of potentially useful single nucleotide polymorphisms (SNPs). Assays were designed for these SNPs and applied to mapping populations. This narrowed the region of interest to 3966 kbp. Further fine mapping was pursued by crossing and backcrossing the resistant cultivar Sloop SA to its susceptible ancestor Sloop. Evaluation of F2 progeny confirmed that the resistance segregates as a single dominant gene. Genotyping of 9003 BC2F2 progeny identified recombinants. Evaluation of recombinant BC2F3 progeny narrowed the region of interest to 978 kbp. Two of the SNPs within this region proved to be diagnostic of CCN resistance across a wide range of barley germplasm. Fluorescence-based and gel-based assays were developed for these SNPs for use in marker-assisted selection. Within the candidate region of the reference genome, there are nine high-confidence predicted genes. Three of these, one that encodes RAR1 (a cysteine- and histidine-rich domain-containing protein), one that is predicted to encode an acetylglutamate kinase and one that is predicted to encode a tonoplast intrinsic protein, are discussed as candidate genes for CCN resistance.

Total and high-affinity corticosteroid-binding globulin depletion in septic shock is associated with mortality.

CONTEXT: Corticosteroid-binding globulin (CBG) and albumin transport circulating cortisol. Cleavage of high-affinity CBG (haCBG) by neutrophil elastase at inflammatory sites causes cortisol release into tissues, facilitating immunomodulatory effects. OBJECTIVE: To determine whether depletion of haCBG is related to mortality in septic shock. DESIGN: A single-center prospective observational cohort study of patients recruited with critical illness or septic shock, using serum samples collected at 0, 8, 24, 48 and 72 hours. Serum total and haCBG, and total and free cortisol were assayed directly. Glucocorticoid treatment was an exclusion criterion. Mortality was assessed at 28 days from Intensive Care Unit admission. RESULTS: Thirty septic shock (SS) and 42 nonseptic critical illness (CI) patients provided 195 serum samples. SS/CI patients had lower total CBG, haCBG and low-affinity CBG (laCBG) than controls. Total CBG and haCBG were significantly lower in septic shock patients who died than in those that survived (P < 0.009, P = 0.021, respectively). Total and free cortisol were higher in septic than nonseptic individuals. Free/total cortisol fractions were higher in those with low haCBG as observed in septic shock. However, cortisol levels were not associated with mortality. Albumin levels fell in sepsis but were not related to mortality. CONCLUSIONS: Low circulating haCBG concentrations are associated with mortality in septic shock. These results are consistent with an important physiological role for haCBG in cortisol tissue delivery in septic shock.

Neutrophil elastase-cleaved corticosteroid-binding globulin is absent in human plasma.

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.

A monoclonal antibody sandwich ELISA for vitamin D-binding protein (VDBP) is unaffected by Gc-globulin phenotype peptides and actin and demonstrates reduced levels in sepsis and non-sepsis intensive care patients.

The measurement of vitamin D-binding protein (VDBP) by immunoassay has been confounded by variable antibody recognition of the Gc1s, Gc1F and Gc2 phenotypes. This has led to spurious conclusions regarding vitamin D status in different ethnic groups. In order to overcome these problems there is a requirement for VDBP antibodies that are unaffected by phenotype status. Here we report the generation and testing of three monoclonal antibodies to VDBP which recognise linear epitopes and are unaffected by vast molar excesses of synthetic peptides spanning these phenotypic domains. These IgG1 kappa antibodies were purified and biotinylated to allow suitable pairings to develop a sandwich ELISA for circulating VDBP. The VDBP ELISA is unaffected by actin and confirms that VDBP levels are significantly reduced in sepsis patients and non-sepsis intensive care patients compared to normal healthy subjects. Levels of VDBP along with total 25OH vitamin D3 can be used to calculate free 25OH vitamin D3 levels and these compare well with consensus values determined independently. The VDBP ELISA meets acceptable performance criteria and as such can be used in conjunction with total 25OH vitamin D3 to determine the free 25OH vitamin D3 status in various cohorts.

Spectral Photon-Counting Molecular Imaging for Quantification of Monoclonal Antibody-Conjugated Gold Nanoparticles Targeted to Lymphoma and Breast Cancer: An In Vitro Study.

The purpose of the present study was to demonstrate an in vitro proof of principle that spectral photon-counting CT can measure gold-labelled specific antibodies targeted to specific cancer cells. A crossover study was performed with Raji lymphoma cancer cells and HER2-positive SKBR3 breast cancer cells using a MARS spectral CT scanner. Raji cells were incubated with monoclonal antibody-labelled gold, rituximab (specific antibody to Raji cells), and trastuzumab (as a control); HER2-positive SKBR3 breast cancer cells were incubated with monoclonal antibody-labelled gold, trastuzumab (specific antibody to HER2-positive cancer cells), and rituximab (as a control). The calibration vials with multiple concentrations of nonfunctionalised gold nanoparticles were used to calibrate spectral CT. Spectral imaging results showed that the Raji cells-rituximab-gold and HER2-positive cells-trastuzumab-gold had a quantifiable amount of gold, 5.97 mg and 0.78 mg, respectively. In contrast, both cell lines incubated with control antibody-labelled gold nanoparticles had less gold attached (1.22 mg and 0.15 mg, respectively). These results demonstrate the proof of principle that spectral molecular CT imaging can identify and quantify specific monoclonal antibody-labelled gold nanoparticles taken up by Raji cells and HER2-positive SKBR3 breast cancer cells. The present study reports the future potential of spectral molecular imaging in detecting tumour heterogeneity so that treatment can be tuned accordingly, leading to more effective personalised medicine.

Differential Effects of Estrogen on Corticosteroid-Binding Globulin Forms Suggests Reduced Cleavage in Pregnancy.

Corticosteroid-binding globulin (CBG) is secreted as high-affinity CBG (haCBG), which may be cleaved by tissue proteases to low-affinity CBG (laCBG), releasing free cortisol. Pregnancy and the estrogen-based combined oral contraceptive pill (COCP) increase CBG concentrations twofold to threefold. The relative effects of these two hyperestrogenic states on the CBG affinity forms are unknown. We performed an observational study in 30 pregnant women, 27 COCP takers and 23 controls. We analyzed circulating total CBG, haCBG, laCBG, and free and total cortisol concentrations. In pregnancy, total CBG and haCBG were increased compared to controls (both P < 0.0001); however, laCBG concentrations were similar. In COCP takers, total CBG and haCBG were increased [802 ± 41 vs compared to controls (both P < 0.0001)], but laCBG was also increased (P = 0.03). Pregnancy and use of COCP were associated with a comparable rise in haCBG, but laCBG was lower in pregnancy (P < 0.0001). These results were consistent with an estrogen-mediated increase in CBG synthesis in both hyperestrogenemic states but with reduced CBG cleavage in pregnancy relative to the COCP, perhaps due to pregnancy-induced CBG glycosylation. Speculatively, increased circulating haCBG concentrations in pregnancy may provide an increased reservoir of CBG-bound cortisol to prepare for the risk of puerperal infection or allow for cortisol binding in the face of competition from increased circulating progesterone concentrations.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation.

The physiological response to cold-water immersion following a mixed martial arts training session.

Combative sport is one of the most physically intense forms of exercise, yet the effect of recovery interventions has been largely unexplored. We investigated the effect of cold-water immersion on structural, inflammatory, and physiological stress biomarkers following a mixed martial arts (MMA) contest preparation training session in comparison with passive recovery. Semiprofessional MMA competitors (n = 15) were randomly assigned to a cold-water immersion (15 min at 10 °C) or passive recovery protocol (ambient air) completed immediately following a contest preparation training session. Markers of muscle damage (urinary myoglobin), inflammation/oxidative stress (urinary neopterin + total neopterin (neopterin + 7,8-dihydroneopterin)), and hypothalamic-pituitary axis (HPA) activation (saliva cortisol) were determined before, immediately after, and 1, 2, and 24 h postsession. Ratings of perceived soreness and fatigue, counter movement jump, and gastrointestinal temperature were also measured. Concentrations of all biomarkers increased significantly (p < 0.05) postsession. Cold water immersion attenuated increases in urinary neopterin (p < 0.05, d = 0.58), total neopterin (p < 0.05, d = 0.89), and saliva cortisol after 2 h (p < 0.05, d = 0.68) and urinary neopterin again at 24 h (p < 0.01, d = 0.57) in comparison with passive recovery. Perceived soreness, fatigue, and gastrointestinal temperatures were also lower for the cold-water immersion group at several time points postsession whilst counter movement jump did not differ. Combative sport athletes who are subjected to impact-induced stress may benefit from immediate cold-water immersion as a simple recovery intervention that reduces delayed onset muscle soreness as well as macrophage and HPA activation whilst not impairing functional performance.