Factors Affecting Measurement of Salivary Cortisol and Secretory Immunoglobulin A in Field Studies of Athletes.

AIMS: Biological and lifestyle factors, such as daily rhythm, caffeine ingestion, recent infection, and antibiotic intake, have been shown to influence measurements of salivary cortisol (SC) and secretory immunoglobulin A (sIgA). Current methodology in unsynchronized, field-based biomarker studies does not take these effects into account. Moreover, very little is known about the combined effects of biological and lifestyle factors on SC and sIgA. This study supports development of a protocol for measuring biomarkers from saliva collected in field studies by examining the individual and combined effects of these factors on SC and sIgA. METHOD: At three time points (start of the pre-season; start of playing season; and end of playing season), saliva samples were collected from the entire squad of 45 male players of an elite Australian Football club (mean age 22.8 ± 3.5 years). At each time, point daily rhythm and lifestyle factors were determined via a questionnaire, and concentrations of both SC and sIgA via an enzyme linked immuno-sorbent (ELISA) assay of saliva samples. In addition, player times to produce 0.5 mL of saliva were recorded. RESULTS: Analysis of covariance of the data across the three time points showed that daily rhythm had a more consistent effect than the lifestyle factors of caffeine ingestion, recent infection, and antibiotic intake on SC, but not on sIgA. Data for sIgA and SC concentrations were then adjusted for the effects of daily rhythm and lifestyle factors, and correlational analysis of the pooled data was used to examine the relative effects of these two sources of influence on sIgA and SC. With the exception of time to produce saliva, the biological measures of stress were affected by players' daily rhythms. When daily rhythm was taken into account the group of lifestyle factors did not have an additional effect. DISCUSSION: It is recommended that future studies measuring SC and sIgA make additional adjustments for the daily rhythm, in particular time since first sight of daylight, as small measurement errors of biomarkers can confound discrimination among study participants.

Selenoprotein P analysis in human plasma: a discrepancy between HPLC fractionation of human plasma with heparin-affinity chromatography and SDS-PAGE with immunoblot analysis.

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.

The effects of anti-histone H1 antibody on immune cells responsible for rejection reaction.

We previously demonstrated the immunosuppressive activity of anti-histone H1 autoreactive antibodies (Ab) transiently induced in serum of a rat tolerogenic orthotopic liver transplantation (OLT) model. In the present study, we investigated the effects of anti-histone H1 Ab on dendritic cells (DCs), T-cells, lymphokine-activated killer (LAK) cells, and human natural killer (NK) cells. The effects of anti-histone H1 Ab on Concanavalin A (ConA) blast, on rat DC cytokine profiles and phenotypes, and on T-cells, LAK cells, and human NK cells were examined by flow cytometry and RT-PCR. The cytotoxicity of LAK and NK cells pretreated with anti-histone H1 Ab was assayed. The addition of anti-histone H1 Ab to ConA blast inhibited the proliferation of 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE)-labeled lymphocytes without toxicity but increased the population of CD4+CD25+ T-cells. DCs treated with anti-histone H1 Ab expressed lower levels of CD80/CD86, IL-1beta, and IL-6. The addition of anti-histone H1 Ab to LAK culture decreased the percentages of NKR-P1 populations and down-regulated levels of inducible nitric oxide synthase (iNOS), IL-2, and INF-gamma in RT-PCR. The cytotoxicity of LAK and NK cells was lower when pretreated with anti-histone H1 Ab than when pretreated with control IgG. We found that the blockade of histone H1 modulated DCs toward tolerogenic status, decreased the cytotoxicity of LAK and NK cells, and induced CD4+CD25+ T-cells. These results suggest that the use of anti-histone H1 Abs might be a useful strategy for the development of a form of immunosuppression.

Proteomic analysis of selected prognostic factors of breast cancer.

The incidence of breast cancer is on the rise but as yet there is no guaranteed beneficial treatment for many of the sufferers. The treatments specific for breast and other hormone-sensitive cancers work well at times, however, the population of women that they will benefit is relatively small. Many are limited to surgical, chemotherapy, and radiotherapy options. Here, using two-dimensional electrophoresis (2-DE) in conjunction with a silver stain and Western blotting approach, we attempt to locate selected known prognostic markers for breast cancer. With these results, we can exclude these proteins from the future search for potential pharmaceutical targets, using the same techniques. The proteins that were located include the estrogen receptor-alpha, beta-casein, cytokeratin 7, calponin and bax. For each protein an estimated M(r) and pI was gained. Each protein was found in multiple variants. By locating these proteins the number of unknown proteins found on the 2-DE gel has been reduced, helping the future search for novel markers that are shown as being differentially expressed between healthy and cancerous tissue samples.

Prolongation of heart allograft survival of rats treated by a Th2 inhibitor.

In terms of Th1/Th2 balance in response to signals given during donor antigen presentation, induction of tolerance is more often correlated with Th2-type than with Th1-type reactions. However, in our study, heart allograft survival was prolonged by treatment of rats with a Th2 inhibitor. Suplatast tosilate (IPD; Taiho; Tokyo, Japan) is a novel immunoregulator that suppresses IgE production and eosinophil infiltration through selective inhibition of interleukin (IL)-4 and IL-5 synthesis by Th2-like cells but not IFN-gamma production in Th1 cells. Five LEW rats of DA heart grafts were treated with IPD (100 microg/day, p.o.) for 10 days. Heart allograft survival of all IPD-treated cases was prolonged more than 14 days while the beating of heart grafts in control groups was stopped within 9 days. In an in vitro study, the cell proliferation both in Con A blast and in mixed lymphocyte reaction assay was suppressed by IPD in dose-dependent manner. We could at least in part conclude that Th2 inhibition might temporarily suppress heart allograft rejection.

Role of nitric oxide in posthypoxic contractile dysfunction of diabetic cardiomyopathy.

We investigated the role of nitric oxide synthase (NOS) in the contractile dysfunction of diabetic cardiomyopathy, comparing streptozotocin-treated (60 mg/kg) diabetic Wistar rats with matched non-diabetic controls. Isolated isovolumic heart function was studied during normoxia and in response to brief hypoxia-reoxygenation. Diabetic hearts had significantly lower left-ventricular pressure and slower isovolumic relaxation than controls (relaxation time constant, T 40.2+/-2.3 vs. 27.7+/-0.9 ms; P<0.05) and a blunted response to hypoxia. These abnormalities were unaffected by NOS inhibition. Upon reoxygenation after brief hypoxia, diabetic hearts exhibited substantial worsening of LV relaxation compared to normal hearts (T 69.1+/-3.3 vs. 56.6+/-7.9 ms; P<0.05). This post-hypoxic diastolic dysfunction was significantly attenuated either by the non-selective NOS inhibitor L-NAME, the iNOS inhibitor L-NIL, or the reactive-oxygen-species (ROS) scavenger thiourea. Only diabetic hearts expressed iNOS protein, whereas eNOS expression was similar in both groups. In conclusion, diabetic hearts exhibit markedly abnormal post-hypoxic relaxation, which is attributable to both ROS and NO derived from iNOS.

LSF-1 may modulate the indirect allorecognition pathway to delay allograft rejection.

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the non-rejector combination of DA donors into PVG recipients. In the present study, a polyclonal anti-LSF-1 antibody was used to detect the lymphoid cell populations expressing LSF-1 epitopes in several transplant models. In this study we examined cell samples acquired from rats that had undergone OLT in syngeneic, rejecting or non-rejecting combinations. Flow cytometry indicated that the polyclonal antibody reacts specifically with an epitope present on a CD4 CD11b positive cell of recipient origin. In the first 3 weeks after transplant there is a large increase in the number of these cells isolated from the spleens and livers of rats in tolerogenic OLT model, which correlates with prolongation of allograft survival. In the rejector and isograft models of OLT there is a minimal change in the expression of the LSF-1 N-terminal pep epitope. These results suggest that these recipient CD4 CD11b cells may have a critical role in the formation of transplant tolerance by interfering with the indirect pathway of allorecognition via as yet undetermined mechanisms. The increased expression of the LSF-1 N-terminal pep epitope on liver leukocytes 14 days after partial hepatectomy indicates a natural role for LSF-1 in the process of liver regeneration after insult or injury.