Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

Exercise intensity and the protection from postprandial vascular dysfunction in adolescents.

Acute exercise transiently improves endothelial function and protects the vasculature from the deleterious effects of a high-fat meal (HFM). We sought to identify whether this response is dependent on exercise intensity in adolescents. Twenty adolescents (10 male, 14.3 ± 0.3 yr) completed three 1-day trials: 1) rest (CON); 2) 8 × 1 min cycling at 90% peak power with 75 s recovery [high-intensity interval exercise (HIIE)]; and 3) cycling at 90% of the gas exchange threshold [moderate-intensity exercise (MIE)] 1 h before consuming a HFM (1.50 g/kg fat). Macrovascular and microvascular endothelial function was assessed before and immediately after exercise and 3 h after the HFM by flow-mediated dilation (FMD) and laser Doppler imaging [peak reactive hyperemia (PRH)]. FMD and PRH increased 1 h after HIIE [P < 0.001, effect size (ES) = 1.20 and P = 0.048, ES = 0.56] but were unchanged after MIE. FMD and PRH were attenuated 3 h after the HFM in CON (P < 0.001, ES = 1.78 and P = 0.02, ES = 0.59). FMD remained greater 3 h after the HFM in HIIE compared with MIE (P < 0.001, ES = 1.47) and CON (P < 0.001, ES = 2.54), and in MIE compared with CON (P < 0.001, ES = 1.40). Compared with CON, PRH was greater 3 h after the HFM in HIIE (P = 0.02, ES = 0.71) and MIE (P = 0.02, ES = 0.84), with no differences between HIIE and MIE (P = 0.72, ES = 0.16). Plasma triacylglycerol concentration and total antioxidant status concentration were not different between trials. We conclude that exercise intensity plays an important role in protecting the vasculature from the deleterious effects of a HFM. Performing HIIE may provide superior vascular benefits than MIE in adolescent groups.

Do soccer and Zumba exercise improve fitness and indicators of health among female hospital employees? A 12-week RCT.

This randomized controlled study investigated the effectiveness of soccer and Zumba on fitness and health indicators in female participants recruited from a workplace. One hundred seven hospital employees were cluster-randomized to either a soccer group (SG), Zumba group (ZG), or control group (CG). Intervention effects for the two training groups were compared with CG. The training was conducted outside working hours as 2-3 1-h sessions per week for 12 weeks. Peak oxygen uptake (VO2peak ), fat percentage, fat mass, bone mineral content, and plasma osteocalcin were measured before and after the intervention period. Based on intention-to-treat-analyses, SG significantly improved the VO2peak relative to body mass (5%; P = 0.02) and decreased heart rate during 100-W cycle exercise (-7 bpm; P = 0.01), total body fat percentage (-1.1%; P = 0.002), and total body fat mass (-1.0 kg; P = 0.001) compared with CG. ZG significantly improved the VO2peak relative to body mass (5%; P = 0.03) and decreased total fat mass (-0.6 kg; P < 0.05) compared with CG. Plasma osteocalcin increased in SG (21%; P < 0.001) and ZG (10%; P = 0.01) compared with CG. The present study indicates that workplace initiated short-term soccer training as well as Zumba outside working hours may result in fitness and modest health benefits among female hospital employees.

Street football is a feasible health-enhancing activity for homeless men: biochemical bone marker profile and balance improved.

This case-control study investigated the feasibility of street football as a health-enhancing activity for homeless men, specifically the musculoskeletal effects of 12 weeks of training. Twenty-two homeless men participated in the football group (FG) and 10 served as controls (C). Plasma osteocalcin, TRACP5b, leptin, and postural balance were measured, and whole-body DXA scanning was performed. The attendance rate was 75% (2.2 ± 0.7 sessions per week). During 60 min of training, the total distance covered was 5534 ± 610 m, with 1040 ± 353, 2744 ± 671, and 864 ± 224 m covered by high-intensity, low-intensity, and backwards/sideways running, respectively. In FG, osteocalcin increased by 27% from 20.1 ± 11.1 to 25.6 ± 11.8 ng/mL (P = 0.007). Postural balance increased by 39% (P = 0.004) and 46% (P = 0.006) in right and left leg. Trunk bone mineral density increased by 1.0% from 0.959 ± 0.095 to 0.969 ± 0.090 g/cm(2) (P = 0.02). No effects were observed in C. In conclusion, street football appears to be a feasible training activity with musculoskeletal health benefits for homeless men. The attendance rate and the training intensity were high, and 12 weeks of training resulted in a substantial anabolic response in bone metabolism. Postural balance improved markedly, and the overall risk of falling, and hospitalization due to sudden trauma, could be reduced by street football for homeless men.

Hirschsprung's disease in Barbados--a 16-year review.

OBJECTIVE: To assess the surgical outcome of patients with Hirschsprung's disease (HD) who were treated in Barbados, establish referral patterns and identify factors which can lead to better management. DESIGN AND METHODS: All patients with HD who had definitive surgery at the Queen Elizabeth Hospital, Barbados, over a 16-year period (between July 1991 and June 2007) were identified from the hospital records system. Data from patients' notes were collected to establish demographics, including age at referral and definitive surgery, gender presenting symptoms, surgical intervention(s) and definitive procedure. Long term outcome was assessed by the use of a simple questionnaire which was completed by the authors after personal or telephone contact with patients, their parents or guardians or the referring physician. RESULTS: Between July 1991 and June 2007, 27 children were identified with HD. There were 20 males and seven females. Fifteen of the 27 children (55%) were local Barbadians, who were referred earlier when compared to those from the surrounding Caribbean islands. The most common mode of presentation was chronic constipation (13/27; 48%). The majority (69%) of the children were fully continent in the long-term (mean follow-up of 64 months) and passed a bowel motion on an average of 2-3 times per day without the use of laxatives or enemas. CONCLUSION: Children who had definitive surgery for HD in Barbados (using Martin's modified Duhamel procedure) had an overall good long-term outcome comparable to international results. Children referred from the surrounding Caribbean islands were referred at an older age, but their overall outcome was not worse than local Barbadian patients.

Visualisation of Kiss1 Neurone Distribution Using a Kiss1-CRE Transgenic Mouse.

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE-GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss-CRE heterozygous mice with CRE-activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80-90% of tdTomato positive neurones in the ARC were co-labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato-labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre-optic areas and to the lateral hypothalamus.

Short Duration Small Sided Football and to a Lesser Extent Whole Body Vibration Exercise Induce Acute Changes in Markers of Bone Turnover.

We aimed to study whether short-duration vibration exercise or football sessions of two different durations acutely changed plasma markers of bone turnover and muscle strain. Inactive premenopausal women (n = 56) were randomized to complete a single bout of short (FG15) or long duration (FG60) small sided football or low magnitude whole body vibration training (VIB). Procollagen type 1 amino-terminal propeptide (P1NP) was increased during exercise for FG15 (51.6 ± 23.0 to 56.5 ± 22.5 µg·L-1, mean ± SD, P < 0.05) and FG60 (42.6 ± 11.8 to 50.2 ± 12.8 µg·L-1, P < 0.05) but not for VIB (38.8 ± 15.1 to 36.6 ± 14.7 µg·L-1, P > 0.05). An increase in osteocalcin was observed 48 h after exercise (P < 0.05), which did not differ between exercise groups. C-terminal telopeptide of type 1 collagen was not affected by exercise. Blood lactate concentration increased during exercise for FG15 (0.6 ± 0.2 to 3.4 ± 1.2 mM) and FG60 (0.6 ± 0.2 to 3.3 ± 2.0 mM), but not for VIB (0.6 ± 0.2 to 0.8 ± 0.4 mM) (P < 0.05). Plasma creatine kinase increased by 55 ± 63% and 137 ± 119% 48 h after FG15 and FG60 (P < 0.05), but not after VIB (26 ± 54%, NS). In contrast to the minor elevation in osteocalcin in response to a single session of vibration exercise, both short and longer durations of small sided football acutely increased plasma P1NP, osteocalcin, and creatine kinase. This may contribute to favorable effects of chronic training on musculoskeletal health.

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.

Acute ureteric calculus obstruction: unenhanced spiral CT versus HASTE MR urography and abdominal radiograph.

The aim of this study is to compare the performance of unenhanced spiral CT to the combination of HASTE MR urography (MRU) and plain abdominal radiography (KUB) in patients suspected of having acute calculus ureteric obstruction. 64 patients with suspected acute calculus ureteric obstruction were evaluated. The presence of perirenal fluid, presence and level of ureteric obstruction and calculi were assessed on both techniques. 44 of 64 (69%) patients had acute calculus ureteric obstruction based on clinical, radiographic or surgical findings. MRU showed perirenal fluid in acute ureteric obstruction (77%) with a greater sensitivity than CT showed stranding (45%). The combination of fluid and ureteric dilation on MRU showed a sensitivity of 93% (CT 80%), specificity of 95% (CT 85%), and accuracy of 94% (CT 81%). There were 61 findings of either fluid or ureteric dilatation on MRU in 44 acutely obstructed kidneys compared with 37 similar findings on CT (p<0.005). Although there was excellent reproducibility (Kappa=/>0.75) in the finding of perirenal fluid on MRU, there was only fair interobserver agreement (Kappa<0.4) regarding perirenal stranding on CT. MRU/KUB showed ureteric calculi in 21/29 (72%) of patients with calculi seen by CT. Overall, MRU/KUB revealed 2.4 abnormalities per acutely obstructed ureter compared with 1.8 abnormalities detected by CT. MRU/KUB using HASTE sequences can diagnose the presence of acute calculus ureteric obstruction with similar accuracy to spiral CT. The technique has less observer variability and is more accurate than CT in detecting evidence of obstruction such as perirenal fluid.

Demonstration of Skn antigens on mouse epidermal cells by immunofluorescence and flow cytometry.

C57BL/6 (B6) mice that were lethally irradiated and reconstituted with (B6XA/J)F1 spleen cells were immunized against the skin-specific antigens, Skn, by grafting with A/J tail skin. Serum from these mice was shown to contain Skna-specific antibody by a flow cytometric assay using indirect immunofluorescence.

Immunohistochemical identification of Skn antigens in mouse epidermis.

Rejection of murine skin grafts by hematopoietic chimeras that are fully compatible genetically with the skin-graft donor has been attributed to a disparity in skin-selective alloantigens between the irradiated host and the skin graft donor. Monoclonal antibodies recognizing Skna alloantigen were produced and used in indirect immunofluorescent and immunoperoxidase tests with sections of skin to demonstrate and confirm the expression of Skna alloantigen on epidermal cells of Skna genotype and absence of Skna alloantigen from epidermal cells of non-Skna genotype.

Dihydrolipoamide dehydrogenase in the trypanosoma subgenus, trypanozoon.

The enzyme dihydrolipoamide dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydrolipoamide dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their dihydrolipoamide dehydrogenases are indistinguishable. Dihydrolipoamide dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. The T. b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.

The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re-uptake site.

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.

Pseudotumor of the bladder: a late complication of inguinal herniorrhaphy.

Damage to the bladder during inguinal hernia repair is possible especially if the bladder or a bladder diverticulum is involved in the hernia sac. Unrecognized injury to the bladder can lead to late complications. We report a case of pseudotumor in a bladder diverticulum due to long-term retention of a misplaced suture. The literature on bladder injury after inguinal herniorrhaphy and on pseudotumor formation is briefly reviewed. It is important to be aware of a history of inguinal surgery and to obtain definitive histologic evidence of malignancy prior to making the diagnosis of bladder carcinoma. This will avoid unnecessary radical surgery, chemotherapy, or radiation therapy.

Percutaneous nephrolithotomy in infants and preschool age children: experience with a new technique.

OBJECTIVES: To develop a less invasive method for performing percutaneous nephrolithotomy (PCNL) with the intent of decreasing the morbidity of the procedure in young children. METHODS: A novel percutaneous renal access technique ("mini-perc") was developed using an 11F peel-away vascular access sheath. Tract dilation and insertion of the sheath into the collecting system was performed with a single pass over an access wire. PCNL was performed using pediatric instruments and electrohydraulic lithotripsy. Sheath design improvements were implemented that make it specific for pediatric PCNL. RESULTS: Eleven procedures have been performed with the 11F sheath. Patient age ranged from 2 to 6 years (mean 3.4) and weight from 5 to 24 kg (mean 12.5). The average stone burden was 1.2 cm2. Mean procedure time, estimated blood loss, and length of hospitalization were 203 minutes, 25 mL, and 6 days, respectively. Six (85%) of 7 patients are currently stone free with an average follow-up of 12 weeks. No patient required transfusion, developed urosepsis, or had a procedure-related complication. One procedure was performed in an outpatient setting with no postoperative nephrostomy tube. CONCLUSIONS: The 11F "mini-perc" technique was successful in rendering 85% of patients stone free with minimal morbidity. Its advantages over obtaining access with standard 24 to 34F access sheaths include a smaller skin incision, single-step dilation and sheath placement, good working access for pediatric instruments, variable length, and lower cost. In addition, the hypothesized decrease in renal and body wall trauma may result in less pain, reduced severity or risk of complications, and shorter hospital stays including the possibility of performing "tubeless" outpatient PCNLs. Further study is needed to confirm these possibilities.

JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells.

We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.

Laparoscopic retroperitoneal renal biopsy.

Histologic information can be pivotal in making treatment decisions. Ultrasound-guided percutaneous biopsy is the current standard, but if this procedure fails or is considered to be high risk, laparoscopic biopsy may be appropriate. A CT or ultrasound scan is obtained to determine whether there is any condition that would mandate biopsy of a particular kidney. The retroperitoneal space is entered with a visual obturator, and, after CO2 insufflation to 15 to 20 mm Hg, the space is enlarged initially by blunt dissection with the laparoscope. Two-tooth biopsy forceps are used to obtain tissue, and hemostasis is achieved with the argon beam coagulator with care to vent the increased pressure created by the flow of gas. Postoperatively, specific attention is given to blood pressure control. Hemorrhage is the most common serious complication, so any anticoagulation regimen must be reinstituted cautiously.

Laparoscopic infrared imaging.

BACKGROUND: As an adjunct to direct visual imaging, an infrared endoscope was developed to assist in the identification of various anatomic structures and to assess tissue viability during laparoscopic procedures. A camera sensitive to emitted energy in the mid-infrared range (3 to 5 microm) was incorporated into a two-channel visible-light laparoscope. METHODS AND MATERIALS: Laparoscopic procedures were performed in a porcine model, inexperienced laparoscopists being asked to localize and differentiate structures before dissection using the visible-light system and then the infrared system. To determine clinical utility, nine laparoscopic urologic procedures were performed with the assistance of the infrared system. RESULTS: In the clinical evaluation, infrared imaging proved to be useful in differentiating between blood vessels and other anatomic structures. In contrast to the experience with the conventional endoscope, vessel identification, assessment of organ perfusion, and transperitoneal localization of the ureter was successful in all instances using the infrared system. In the porcine model, this system also permitted assessment of bowel perfusion during laparoscopic occlusion of mesenteric vessels and distinguished between the cystic duct and artery. CONCLUSION: Infrared imaging is a potentially powerful adjunct to laparoscopic surgery. It may improve the differentiation and localization of anatomic structures and allow assessment of physiologic features, such as perfusion, not previously attainable with laparoscopic techniques.

Utility of the harmonic scalpel for laparoscopic partial nephrectomy.

Laparoscopic partial nephrectomy (LPN) remains a technically challenging procedure largely because of the lack of methods for obtaining consistent parenchymal hemostasis. The objective of this study was to determine if the extent of resection influences the ability of the harmonic scalpel to achieve hemostasis and to define the cases in which the harmonic scalpel is appropriate for LPN. Thirty LPNs were performed in a 25-kg domestic pig model. The blunt blade of the laparoscopic harmonic scalpel (LaparoSonic Coagulating Shears; Ethicon Endo-Surgery, Cincinnati, OH) at power level 5 was used to divide the parenchyma. Control of the renal hilar vessels was not obtained. Three standardized types of resections were performed: I = peripheral wedge biopsy; II = upper- or lower-pole nephrectomy; and III = heminephrectomy. Bleeding was graded on a scale from 0 to 4: 0 = no hemostasis; 1 = steady bleeding; 2 = moderate bleeding; 3 = parenchymal oozing; and 4 = dry. Hemostasis grades of 2 or less were clinically significant bleeding necessitating supplemental coagulation. The mean hemostasis scores showed a significant (P < 0.02) trend toward inadequate hemostasis with increasing extent of resection: 3.3 for Type I, 3.0 for Type II, and 2.4 for Type III. The percent of kidneys with grade 2 bleeding or worse was 9% for Type I surgery, 25% for Type II, and 57% for Type III. Successful hemostasis with the harmonic scalpel correlates with the extent of parenchymal resection in the porcine model. Most wedge excisions can be done with the harmonic scalpel alone, whereas larger resections necessitate supplemental coagulation. On the basis of this study, heminephrectomies with the harmonic scalpel are not recommended because of the high incidence of significant hemorrhage.

Familial calcium stone disease: TaqI polymorphism and the vitamin D receptor.

BACKGROUND AND OBJECTIVE: Calcium nephrolithiasis has a strong familial component. However, to date, no specific genetic abnormality has been identified. Allelic variation in the vitamin D receptor (VDR) gene has been suggested as a partial explanation of differential calcium absorption or excretion in these patients. Polymorphism of this gene has been associated with altered vitamin D activity and has been implicated in osteoporosis and prostate cancer. We propose that a similar association may be found between familial hypercalciuric stone disease and the VDR. SUBJECTS AND METHODS: Genomic DNA was isolated from 37 controls and 19 patients with hypercalciuria (> 250 mg/24 hours) and a family history of nephrolithiasis. A 740-basepair segment of the VDR gene was amplified by polymerase chain reaction, digested with TaqI endonuclease, and resolved by gel electrophoresis. Alleles were classified as "T" if only one TaqI site was present and "t" if two were present. A simplified strength of family history score (FHS) was computed by adding 2 and 1 points, respectively, for each first- and second-degree relative affected by stone disease. RESULTS: No difference in allelic or genotypic frequencies between the study and control groups was present. In the stone group, a significant association was found between the strength of the family history and the TT genotype. Patients with this genotype had an average FHS of 4.0, whereas the mean FHS for the Tt and tt genotypes was 2.0 and 1.8, respectively (P < 0.05). Nonsignificant trends of the TT genotype toward a higher number of stone episodes (19 v 13 and 3) and higher 24-hour urine calcium excretion (408 v 297 and 353 mg) were also noted in the study group. CONCLUSION: The results suggest that the TT genotype is associated with more aggressive stone disease, both within families and with respect to recurrence. Quantifying the risk of calcium stone disease through DNA markers has potential application in determining the risk of a patient's family members for nephrolithiasis or a patient's risk of recurrence. This information may have therapeutic implications with regard to the rigor of medical therapy and frequency of follow-up.