Development of a synthetic peptide-based tartrate-resistant acid phosphatase radioimmunoassay for the measurement of bone resorption in rat serum

Selective markers of bone turnover provide a convenient and reproducible alternative to the complex and expensive histochemical techniques used commonly to study the effect of pharmacological agents and the pathogenesis of bone disease in the ovariectomized (OVX) rat model. One marker, which has been specifically linked to terminally differentiated osteoclasts and, thus, provides useful insight at cellular levels, is type-5 tartrate-resistant acid phosphatase (TRACP). We describe the development of a TRACP radioimmunoassay (RIA), which requires synthetic peptide for antibody development. To develop the RIA, polyclonal antibodies were generated in goats against a synthetic peptide, DPSVRHQRKCY, corresponding to amino acid residues 267–275 of the rat type-5 TRACP sequence. In the RIA, 50 μl of rat serum, 100 μl of goat anti-TRACP antibodies, and 100 μl of tracer were incubated overnight. The antibody-bound fraction was separated, counted, and unknown values were calculated by comparison with the peptide calibrator. Rat serum shows parallelism with the synthetic peptide calibrator used in the RIA. The sensitivity of the RIA was 24.7 μg/l, and the measuring range was 19–2476 μg/l. The average intra-assay coefficients of variation for (CV) two controls were less than 7%. The average dilution and spike recoveries were 107% and 87%, respectively. We applied our peptide-based RIA to study bone resorption in an OVX rat model. TRACP concentrations in serum in 12-week-old OVX Sprague Dawley rats were 14%–22% (P < 0.05) higher than those in the sham-operated rats, and TRACP concentrations in OVX rats treated with estradiol were 24%–32% lower (P < 0.01) than those in the vehicle-treated OVX group. Similarly, as compared with those in OVX rats, TRACP concentrations decreased to those of sham levels in OVX rats receiving 10 μg/kg per day of alendronate for 10 days. In addition, the TRACP levels determined by RIA showed a significant correlation with serum C-telopeptide (type-I collagen) concentrations (r = 0.56; P < 0.001) measured by an enzyme-linked immunosorbent assay (ELISA) developed earlier for the rat model. In conclusion, we have developed a TRACP RIA that could be used to monitor the rate of bone resorption in the rat model. Received: June 18, 2001 / Accepted: October 26, 2001     

Role of Centchroman in Regression of Mastalgia and Fibroadenoma

Objectives Centchroman (Ormeloxifene) is a novel non-steroidal, selective antiestrogen. Because of its selective antiestrogen action, centchroman has been used for treatment of mastalgia and fibroadenoma. Materials and Methods Benign breast disease patients up to 35 years of age attending our surgery outpatient department from August 2003 to September 2004 and fulfilling the inclusion criterion were included in this study. They were started on centchroman 30 mg on alternate days for a period of 3 months and were followed up for 6 months. Results were recorded as per clinical examination, visual analog scale (VAS) for pain, and ultrasonography for breast lump size. Results A total of 60 patients were included in this pilot study, 42 (70%) of whom had mastalgia with or without nodularity, and 18 (30%) had fibroadenoma. Noncyclical pain was recorded in 38 patients (90%), and cyclical pain was recorded in only 4 (10%) patients. A VAS score of 10 was recorded by 33 (80%) patients (severe pain), and the remaining 9 patients (20%) had VAS scores from 7 to 10. Fibroadenoma size ranged from 1.5 to 5 cm., single or multiple in one or both breasts. There was a good response in the mastalgia group, with a decrease in the VAS scoring from 10 to 3 in 90 % of the patients in the first week. Almost all of the patients were painless at the end of one month, with complete disappearance of the nodularity. In the fibroadenoma group there was a mixed response, with complete disappearence in 40%, partial regression in 20%, and no response at all in the remaining 40%. There were very few side effects. Conclusions Centchroman is a safe nonsteroidal drug for the treatment of mastalgia and fibroadenoma. It has shown good results in mastalgia and is a safe drug as compared to the drugs of choice used at present (danazole and bromocriptine). Further randomized studies are in progress and are needed to determine its definitive role in this patient group. Presented at the BSI Prize Session, International Surgical Week “ISW2005,” 41st World Congress of Surgery of ISS/SIC, 21–25 August 2005, Durban, South Africa     

A time course of bone response to jump exercise in C57BL/6J mice

 Exercise, by way of mechanical loading, provides a physiological stimulus to which bone tissue adapts by increased bone formation. The mechanical stimulus due to physical activity depends on both the magnitude and the duration of the exercise. Earlier studies have demonstrated that jump training for 4 weeks produces a significant bone formation response in C57BL/6J mice. An early time point with significant increase in bone formation response would be helpful in: (1) designing genetic quantitative trait loci (QTL) studies to investigate genes regulating the bone adaptive response to mechanical stimulus; and (2) mechanistic studies to investigate early stimulus to bone tissue. Consequently, we investigated the bone structural response after 2, 3, and 4 weeks of exercise with a loading cycle of ten jumps a day. We used biochemical markers and peripheral quantitative computed tomography (pQCT) of excised femur to measure bone density, bone mineral content (BMC), and area. Four-week-old mice were separated into control (n= 6) and jump groups (n= 6), and the latter groups of mice were subjected to jump exercise of 2-week, 3-week, and 4-week duration. Data (pQCT) from a mid-diaphyseal slice were used to compare bone formation parameters between exercise and control groups, and between different time points. There was no statistically significant change in bone response after 2 weeks of jump exercise as compared with the age-matched controls. After 3 weeks of jump exercise, the periosteal circumference, which is the most efficient means of measuring adaptation to exercise, was increased by 3% (P < 0.05), and total and cortical area were increased by 6% (P < 0.05) and 11% (P < 0.01), respectively. Total bone mineral density (BMD) increased by 11% (P < 0.01). The biggest changes were observed in cortical and total BMC, with the increase in total BMC being 12% (P < 0.01). Interestingly, the increase in BMC was observed throughout the length of the femur and was not confined to the mid-diaphysis. Consistent with earlier studies, mid-femur bone mass and area remained significantly elevated in the 4-week exercise group when compared with the control group of mice. The levels of the biochemical markers osteocalcin, skeletal alkaline phosphatase, and C-telopeptide were not significantly different between the exercise and control groups, indicating the absence of any systemic response due to the exercise. We conclude that a shorter exercise regimen, of 3 weeks, induced a bone response that was greater than or equal to that of 4 weeks of jump exercise reported earlier. Received: October 1, 2001 / Accepted: January 18, 2002     

Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection

BackgroundDeveloping a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells’ proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection.MethodsUsing 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets.ResultsAn exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature’s negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell–mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature’s negative predictive value was 90.6% and its positive predictive value was 77.8%.ConclusionsOur findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.     

Acute effect of calcium citrate on serum calcium and cardiovascular function

Calcium supplements have been associated with an increased risk of cardiovascular events. However, the validity of these findings has been questioned. A major concern is that the mechanism underlying an increase in cardiovascular events has not been demonstrated. Calcium initiates cardiac and vascular contraction following influx of calcium into cardiac and smooth muscle from extracellular fluid. We have investigated whether the acute rise in serum calcium following calcium supplement administration is associated with adverse changes in cardiovascular function. In an open interventional study, we recruited 25 volunteers (16 female, age 60.3 ± 6.5 years, body mass index 25.7 ± 2.7 kg/m²) from the community who were not taking calcium supplements. Participants were studied before and 3 hours after a single oral dose of 1000 mg calcium citrate. We assessed well‐validated markers of arterial stiffness (pulse wave velocity [PWV]), arterial wave reflection (augmentation index [AIx]), and myocardial perfusion (subendocardial viability ratio [SEVR]) by pulse wave analysis and endothelial function (reactive hyperemia index [RHI]) by peripheral arterial tonometry. Total and ionized serum calcium were acutely increased by 0.10 ± 0.07 and 0.06 ± 0.03 mmol/L, respectively, 3 hours after calcium citrate administration (p < 0.0001 for both comparisons). Following administration of calcium citrate there was a fall in AIx from a median of 29.7% (23.8% to 34.0%) to 26.4% (22.7% to 34.0%, p = 0.03) and an increase in SEVR from 163% (148% to 174%) to 170% (149% to 185%, p = 0.007). PWV and RHI were not significantly altered. The change in total calcium was negatively correlated with the change in AIx (r = –0.48, p = 0.02). In summary, the acute increase in serum calcium following calcium supplement administration is associated with reduced arterial wave reflection and a marker of increased myocardial perfusion. If maintained long‐term, these changes would be expected to reduce cardiovascular risk. Acute serum calcium–mediated changes in these parameters of cardiovascular function are unlikely to underlie an association between calcium supplementation and cardiovascular events. © 2013 American Society for Bone and Mineral Research     

Contribution of aldose reductase to diabetic hyperproliferation of vascular smooth muscle cells

The objective of this study was to determine whether the polyol pathway enzyme aldose reductase mediates diabetes abnormalities in vascular smooth muscle cell (SMC) growth. Aldose reductase inhibitors (tolrestat or sorbinil) or antisense aldose reductase mRNA prevented hyperproliferation of cultured rat aortic SMCs induced by high glucose. Cell cycle progression in the presence of high glucose was blocked by tolrestat, which induced a G0-G1 phase growth arrest. In situ, diabetes increased SMC growth and intimal hyperplasia in balloon-injured carotid arteries of streptozotocin-treated rats, when examined 7 or 14 days after injury. Treatment with tolrestat (15 mg x kg(-1) x day(-1)) diminished intimal hyperplasia and decreased SMC content of the lesion by 25%. Although tolrestat treatment increased immunoreactivity of the lesion with antibodies raised against protein adducts of the lipid peroxidation product 4-hydroxy trans-2-nonenal, no compensatory increase in lesion fibrosis was observed. Collectively, these results suggest that inhibition of aldose reductase prevents glucose-induced stimulation of SMC growth in culture and in situ. Even though inhibition of aldose reductase increases vascular oxidative stress, this approach may be useful in preventing abnormal SMC growth in vessels of diabetic patients.     

Ulnar nerve compression in Guyon's canal in the presence of a tortuous ulnar artery

A case of ulnar nerve compression in Guyon's canal occurring in the presence of a tortuous ulnar artery in the canal is presented and the pathology and classification of ulnar nerve compression in Guyon's canal discussed.     

Nano-purification of raw semen minimises oxidative stress with improvement in post-thaw quality of buffalo spermatozoa

The study consisted of application of anti-ubiquitin antibodies (Abs)-coated iron oxide-nanoparticles (IONPs) for minimisation of oxidative stress to contemporary live spermatozoa from the raw semen. Round-shaped IONPs (12.09 ± 0.91 nm) after two-stage functionalisation (silanisation and pegylation) were conjugated with Abs. Four aliquots from each of the 24 ejaculates (4 buffalo bulls) formed Control (Group I) and treatment (II, III and IV) groups; each containing 150 ± 25 million dead/damaged spermatozoa. IONPs-Abs complex were added at ratio of 1:1 (0.5 µg/ml), 1:2 (1.0 µg/ml) and 1:4 (2.0 µg/ml), respectively, in Groups II, III and IV. The semen quality parameters showed improvement at lag-stage (post-nano-purification before processing for cryopreservation). The mean post-thaw motility (%) in Group IV was found to be greater (p < .05) than Group I. Moreover, the overall DNA integrity (%) at post-thaw stage was improved in the nano-purified semen samples. The value of malondialdehyde was greater (p < .001) in Group I than Groups II, III and IV. The mean total antioxidant capacity and superoxide dismutase (U/mg protein) activity values in Group IV was greater (p < .05) than Group I. The study results show that IONPs conjugated with anti-ubiquitin Abs at 2.0 µg/ml can be an effective dose for depletion of dead/damaged spermatozoa from buffalo ejaculates to minimise oxidative stress.     

Nonspecific depressant action of γ-aminobutyric acid on somatic reflexes

The effects of γ-aminobutyric acid (GABA) on central integration of somatic reflexes in the cat have been studied by topical application, and by intrathecal or intracerebroventricular injection. Intrathecal injection of GABA inhibited the monosynaptic patellar reflex. The facilitation of the patellar reflex induced by strychnine, leptazol, tubocurarine and tetanus toxin was also inhibited. Polysynaptic facilitation of the patellar reflex induced in the spinal cat by electrical stimulation of the contralateral sciatic nerve was depressed by intrathecal GABA. Similarly, the supraspinal facilitation of the patellar reflex by electrical stimulation of the brain stem reticular formation was inhibited by application of GABA to the floor of the 4th ventricle. The polysynaptic inhibition of the patellar reflex at both levels was intensified by GABA. The flexor (tibialis anterior) reflex was depressed in the same manner as the extensor patellar reflex. The polysynaptic linguomandibular reflex was depressed by intracerebroventricular GABA. The depressant action of GABA at spinal and supraspinal levels of the neuraxis is discussed in relation to the role of GABA as an inhibitory transmitter in the central nervous system.     

Vaccines against advanced melanoma

Research shows that cancers are recognized by the immune system but that the immune recognition of tumors does not uniformly result in tumor rejection or regression. Quantitating the success or failure of the immune system in tumor elimination is difficult because we do not really know the total numbers of encounters of the immune system with the tumors. Regardless of that important issue, recognition of the tumor by the immune system implicitly contains the idea of the tumor antigen, which is what is actually recognized. We review the molecular identity of all forms of tumor antigens (antigens with specific mutations, cancer-testis antigens, differentiation antigens, over-expressed antigens) and discuss the use of these multiple forms of antigens in experimental immunotherapy of mouse and human melanoma. These efforts have been uniformly unsuccessful; however, the approaches that have not worked or have somewhat worked have been the source of many new insights into melanoma immunology. From a critical review of the various approaches to vaccine therapy we conclude that individual cancer-specific mutations are truly the only sources of cancer-specific antigens, and therefore, the most attractive targets for immunotherapy.