CD8+ T lymphocytes enhance the anabolic effect of intermittent parathyroid hormone on cementoblasts.

Root resorption is usually inflammatory in nature and has a tight link with immune system. Intermittent parathyroid hormone (iPTH) could promote cementum regeneration. The cross-talk of immune cells and cementoblasts may play an important role in the regeneration which stayed to be elucidated. In this study, a CD8+ T cells-OCCM-30 cells coculture system was established in vitro to investigate whether CD8+ T cells could enhance the anabolic effect of iPTH on cementoblasts and to find out the potential link of the effect with Wnt signal pathway. Determined by real-time PCR and Western Blot, we found an amplified cementogenesis in the OCCM-30 cells from coculture system, including increased mRNA and protein expression of Alp, Opn and Runx2, ALP activity and mineralization. We also found iPTH could increase the expression of Wnt10b in CD8+ T cells by ELISA. In addition, Wnt10b would promote the proliferation of OCCM-30 cells, while the effect on differentiation was various in different culture medium. These results demonstrated that the stimulating effect of iPTH on cementoblasts could be mediated through an interaction with CD8+ T cells, and T-cell-induced Wnt10b might be a key mechanism in the mediation.

Screening hybridomas for anabolic androgenic steroids by steroid analog antigen microarray.

BACKGROUND: Currently, dozens of anabolic androgenic steroids (AAS) are forbidden in the World Anti-Doping Agency Prohibited List, however, despite extensive investigation, there are still lots of AAS without corresponding monoclonal antibodies. RESULTS: A steroid analog antigen microarray made up of ten AAS was fabricated to screen the hybridoma and it was found an original unsuccessful clone turned out to be a candidate anti-boldenone antibody, without any cross-reactions with endogenous AAS or 44 different AAS standard reference materials tested. CONCLUSION: Our findings suggested that steroid analog antigen microarray could be a promising tool to screen and characterize new applications of antibodies for structure analogs, and this also exhibits the potential to fast identify effective epitopes of hybridomas in a single assay.

Systemic administration of sclerostin monoclonal antibody accelerates fracture healing in the femoral osteotomy model of young rats.

Genetic studies have demonstrated that sclerostin was a key negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment enhanced bone healing in experimental fracture healing. The purpose was to investigate the effects of systemic Scl-Ab administration on open fracture healing in young rats. Unilateral femoral fractures were generated in eight-week-old Sprague-Dawley rats. Rats were treated with vehicle or Scl-Ab for 6weeks. Fracture healing was evaluated by western blotting, immunohistochemistry, histology, radiography, micro-CT, and biomechanical testing. In addition, the bone mass of intact femur was also evaluated by micro-CT. The results showed that, at 1 and 2weeks after fracture, proliferating cell nuclear antigen (PCNA) score and bone morphogenetic protein-2 (BMP-2) expression in the Scl-Ab group were significantly increased compared with the control group. A decrease in cartilage in the Scl-Ab group was also observed after fracture, and this was accompanied by more rapider fracture healing. At 4 and 6weeks, there were significant increases in bone mass and mechanical properties in the calluses from Scl-Ab group compared with control group. In addition, Scl-Ab treatment also showed significant anabolic effects in intact femur. In conclusion, systemic Scl-Ab administration has a significant enhancement in a rat femoral osteotomy model. These results support the therapeutic potential of Scl-Ab as a noninvasive strategy to enhance open fracture healing.

Affinity-based biosensors in sport medicine and doping control analysis.

Affinity-based biosensors (ABBs) have started to be considered in sport medicine and doping control analysis because they are cheap, easy to use and sufficiently selective analytical devices, characterized by a reversible interaction with the analyte under investigation allowing the use of the same sensor for multiple analyses. In this review we describe the main categories of substances reported in the World Anti-Doping Agency Prohibited List and how ABBs may contribute to their detection. Although several ABBs proposed in the last few years display limit of detections that are in principle matching the World Anti-Doping Agency requirements, their application in the framework of 'traditional' antidoping tests seems quite unlikely, mainly because of the still insufficient selectivity especially in the case of 'pseudo-endogenous' compounds, and on the lack of complete information regarding potential matrix effects in real samples and following their routine use. At the same time, ABBs could contribute to fill a significant information gap concerning complementary evidence that can be obtained from their use 'on the spot', as well as to preselect a risk population of individuals to be targeted for a full antidoping test; while in sport medicine they could contribute to obtaining analytical information of physiological relevance from the measurement of specific parameters or markers before, during and after physical exercise.

Electronic anabolic steroid recognition with carbon nanotube field-effect transistors.

A proof of concept of the electronic detection of two anabolic steroids, stanozolol (Stz) and methylboldenone (MB), was carried out using two specific antibodies and arrays of carbon nanotube field-effect transistors (CNTFETs). Antibodies specific for Stz and MB were prepared and immobilized on the carbon nanotubes (CNTs) using two different approaches: direct noncovalent bonding of antibodies to the devices and bonding the antibodies covalently to a polymer previously attached to the CNTFETs. The results indicated that CNTFETs bonded to specific antibodies covalently or noncovalently are able to detect the presence of steroids. Statistically significant changes in the threshold voltage and drain current were registered in the transistors, allowing the steroids to be recognized. On the other hand, it was determined that the specific antibodies do not detect other steroids other than Stz and MB, such as nandrolone (ND) because, in this case, statistically significant changes in the transistors were not detected. The polymer prevents the aggregation of antibodies on the electrodes and decreases the transistor hysteresis. Nevertheless, it is not able to avoid the nonspecific adsorption of streptavidin, meaning that nonspecific adsorption on CNTs remains a problem and that this methodology is only useful for purified samples. Regarding the detection mechanism, in addition to charge transfer, Schottky barrier, SB, modification, and scattering potential reported by other authors, an electron/hole trapping mechanism leading to hysteresis modification has been determined. The presence of polymer seems to hinder the modulation of the electrode-CNT contact.

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

Preparation of steroid antibodies and parallel detection of multianabolic steroid abuse with conjugated hapten microarray.

A conjugated hapten microarray based on miniature immunoassay for fast and multiplex detection of anabolic steroids is reported for the first time. This preliminary study investigated the possibility of using a microarray technology as a multisteroid detection assay. The microarray system used eight monoclonal antibodies raised against three steroid conjugates, 4-androsten-4-chloro-17beta-ol-3-one, 1,5alpha-androsten-1beta-methyl-17beta-ol-3-one, and 5beta-androsten-1-en-17beta-ol-3-one, which were conjugated to BSA by the active ester method. In addition to 4 commercial conjugated haptens, 18 steroid-BSA conjugates were synthesized and from all these a conjugated hapten microarray was fabricated. The analyzed substances included 42 types of anabolic steroid reference materials and 28 positive urine samples. Of these, 24 anabolic steroids and 12 positive urines were successfully detected.

Potential for the effects of anabolic steroid abuse in the immune and neuroendocrine axis.

Some of the effects that high-dose anabolic steroid abuse have and could have on the interactions between the immune and neuroendocrine systems are reviewed. Considering the past demonstrations on the actions of normal steroids on endocrine and immune responses, it is apparent that pharmacologically high doses of both normal and derivatized androgens (anabolic steroids) could have a significant effect. Indeed, some of the pathologies attributed to anabolic steroid abuse point to disturbances in the intimate connection between neuroendocrine and immune function and interaction. We attempt to review both the direct and indirect effects of this abuse, not only on this interaction but also on certain immune functions in particular.

Comparison and evaluation of the specificity and binding capacity of commercial and in house affinity columns used in sample preparation for analysis of growth-promoting drugs.

Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including beta-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of alpha-nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.