Lack of association between beta 2-adrenergic receptor polymorphisms and juvenile idiopathic arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is a chronic autoimmune arthropathy. Beta 2-adrenergic receptors are a link between the sympathetic nervous system and the immune system. Associations between variants in the gene encoding the beta 2-adrenergic receptor (ADRB2) and autoimmune disorders such as rheumatoid arthritis (RA) have been demonstrated. We aimed to investigate ADRB2 variants for association with JIA. METHODS: Genotypes and haplotypes of two ADRB2 variants (G16R and Q27E) were determined in 348 children with JIA and 448 healthy controls by direct molecular haplotyping using melting-curve analysis of a fluorescently labelled loci-spanning probe. Case-control analysis was performed to investigate whether ADRB2 variants were associated with JIA. RESULTS: No association was found between JIA and alleles, genotypes, or haplotypes of ADRB2. Specifically, the haplotype that demonstrated a strong association with RA (R16/Q27) was not associated with JIA. None of the variants demonstrated association after stratification by JIA subtypes or gender. CONCLUSIONS: Our results indicate that ADRB2 variants are not associated with JIA or any of the major JIA subtypes. These observations suggest that, although they share several clinical and pathological features, JIA and RA have unique genetic associations.

A promising new treatment for solar lentigines.

The purpose of this open-label study was to determine the adverse event rate of topical 4HA/tretinoin when used twice daily for up to 24 weeks with concomitant sunscreen in the treatment of solar lentigines and related hyperpigmented lesions. There were two treatment areas: bilateral dorsal forearms, including the back of the hands; and the face, including the forehead and cheek areas. Each treatment area had a target lesion at least 5 mm in diameter and was moderately darker than the surrounding skin. A nine-point bipolar scale was used for evaluation of Target Lesion Pigmentation (0 = extremely lighter than pigment of the surrounding skin, 4 = equal with pigment of surrounding skin, 8 = extremely darker than pigment of surrounding skin). The other solar lentigines present in the treatment areas also had to have an overall pigmentation grade of at least Grade 6. Twice daily applications to individual lesions in each treatment area were made for up to 24 weeks followed by a 4-week follow-up phase. Sunscreen applications (sunscreen with sun protection factor (SPF) 25 or greater) were made every morning and reapplied after six hours if additional sun exposure was expected. Clinical evaluations were performed at weeks 0, 4, 8, 16, 24 and 28. The clinical signs of Target Lesion Pigmentation and Overall Lesion Pigmentation were evaluated at each visit. A total of 96 subjects were enrolled at four study centers; 77 (80%) subjects completed the study. Treatment-related adverse events (AEs) for 4HA/tretinoin included erythema, burning/stinging/tingling, desquamation, pruritus, skin irritation, halo hypopigmentation and hypopigmentation. Five (5%) subjects discontinued from the study due to adverse events considered to be related to study medication. When used with sunscreen of SPF 25 or greater, 4HA/tretinoin was safe and well tolerated and did not produce any unexpected or unusual adverse events.

Application of haplotype pair analysis for the identification of hemizygous loci.

An expectation maximisation based prediction algorithm was created to identify unusual haplotypes in patient samples that may be caused by small intragenic deletions. In this approach, unphased SNP genotypes are compared to pairs of canonical haplotypes to identify potentially hemizygous regions. This method was successfully applied to identify five deletions in the 3' region of BRCA1.

Coordination sphere flexibility of active-site models for Fe-only hydrogenase: studies in intra- and intermolecular diatomic ligand exchange.

A series of dinuclear complexes, (mu-SRS)Fe(2)(CO)(6) (R = -CH(2)CH(2)-, -CH(2)CH(2)CH(2)-, -CH(2)-C(6)H(4)-CH(2)-; edt, pdt, and o-xyldt, respectively) has been examined for specific characteristics that might relate to structural similarity with the active site of Fe-only hydrogenases. Variable-temperature proton NMR studies display the fluxionality of the iron-dithiocyclohexane unit in (mu-pdt)Fe(2)(CO)(6) while in the (mu-o-xyldt)Fe(2)(CO)(6) compound, the bridge is fixed. Temperature-dependent (13)C NMR spectral studies establish intramolecular CO site exchange localized on discrete Fe(CO)(3) units in all complexes, which is influenced by steric effects of the mu-SRS unit. Kinetic studies of intermolecular CO/CN(-) ligand-exchange reactions establish associative or I(a) mechanisms in sequential steps to form the dicyano dianion, (mu-SRS)[Fe(CO)(2)(CN)](2)(=) with 100% selectivity. Theoretical calculations (DFT) of transition states in the intramolecular site-exchange processes lead to a rationale for the interesting cooperativity in the CN(-)/CO intermolecular ligand-exchange process. The hinge motion of the three light atom S-to-S bridge is related to a possible heterolytic H(2) activation/production process in the enzyme.

Hereditary hemochromatosis since discovery of the HFE gene.

BACKGROUND: Hereditary hemochromatosis is an inherited disorder of iron metabolism that is characterized by excessive iron deposition in major organs of the body. Chronic increased iron absorption leads to multiorgan dysfunction. Since the discovery of the gene responsible for the majority of cases, research has progressed rapidly to identify the gene product, the effects of mutations, and the implications for different populations. The protein product of the HFE gene is a transmembrane glycoprotein, termed HFE, that modulates iron uptake. Mutations in the HFE protein compromise its function and produce disease symptoms. Two mutations, C282Y and H63D, have been linked to the majority of disease cases. APPROACH: We reviewed the recent literature for the molecular basis of hereditary hemochromatosis. Genotypic information was combined with biochemical and clinical phenotypic information to achieve a better understanding of the disease mechanism. CONTENT: This review provides a comprehensive discussion of known mutations in the HFE gene and their phenotypic expression. Diagnostic criteria using molecular genetic techniques in conjunction with traditional biochemical tests are provided. Current methods and limitations of molecular testing are examined in detail. A strategy for population screening and an algorithm for diagnosis that incorporates molecular testing are presented. Treatment by therapeutic phlebotomy and the use of blood obtained from hemochromatosis patients are discussed. SUMMARY: Although the disease mechanism has not been completely elucidated, phenotypic and penetrance data are becoming available. Controversy still exists concerning the role of genetic testing in diagnosis and population screening.

Quantification of HER2/neu gene amplification by competitive pcr using fluorescent melting curve analysis.

BACKGROUND: Molecular detection methods for HER2/neu gene amplification include fluorescence in situ hybridization (FISH) and competitive PCR. We designed a quantitative PCR system utilizing fluorescent hybridization probes and a competitor that differed from the HER2/neu sequence by a single base change. METHODS: Increasing twofold concentrations of competitor were coamplified with DNA from cell lines with various HER2/neu copy numbers at the HER2/neu locus. Competitor DNA was distinguished from the HER2/neu sequence by a fluorescent hybridization probe and melting curve analysis on a fluorescence-monitoring thermal cycler. The percentages of competitor to target peak areas on derivative fluorescence vs temperature curves were used to calculate copy number. RESULTS: Real-time monitoring of the PCR reaction showed comparable relative areas throughout the log phase and during the PCR plateau, indicating that only end-point detection is necessary. The dynamic range was over two logs (2000-250 000 competitor copies) with CVs < 20%. Three cell lines (MRC-5, T-47D, and SK-BR-3) were determined to have gene doses of 1, 3, and 11, respectively. Gene amplification was detected in 3 of 13 tumor samples and was correlated with conventional real-time PCR and FISH analysis. CONCLUSION: Use of relative peak areas allows gene copy numbers to be quantified against an internal competitive control in < 1 h.

Mutation detection using fluorescent hybridization probes and melting curve analysis.

The LightCycler is a real-time PCR instrument that combines a thermocycler and a micro-volume fluorimeter. LightCycler technology is gaining popularity due to its ability to detect mutations quickly and accurately. Multiple base alterations are discriminated using hybridization probes and fluorescent melting curves. This review focuses on mutation detection and base discrimination by fluorescent hybridization probes. Assay designs for single base mutation detection and complex multiplex reactions are discussed. Types of mutations detected and reported applications are reviewed. Guidelines using melting curve analysis for the clinical laboratory are presented.

Simultaneous detection of C282Y and H63D hemochromatosis mutations by dual-color probes.

BACKGROUND: Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm). METHODS AND RESULTS: The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine. CONCLUSIONS: Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.

Comparison of automated short tandem repeat and manual variable number of tandem repeat analysis of chimerism in bone marrow transplant patients.

Hematopoietic chimerism can be monitored in bone marrow transplant patients at DNA polymorphic sites. In this study, allele detection and quantification by ethidium bromide-stained agarose gels were compared with automated fluorescent sizing on an artificially mixed system and on chimeric post-transplant whole blood and sorted cell populations. A panel of five variable number of tandem repeats (VNTRs) were amplified and quantified visually on an ethidium bromide-stained gel. The ten short tandem repeats (STRs) were amplified as a multiplex polymerase chain reaction (PCR) and fluorescently detected on a DNA sequencer. Fluorescent band intensities were converted to fluorescent peak areas for allele quantification. Using mixed DNA of different proportions, both STRs and VNTRs showed linearity and appeared equally sensitive. However, case studies showed STRs to be more sensitive (<5%) than VNTRs (<10%). The STRs more accurately quantified the minor DNA component at low concentrations.

A review of the effects of nicotine on schizophrenia and antipsychotic medications.

OBJECTIVE: Research on the impact of nicotine on schizophrenia and antipsychotic medications was reviewed to determine ways to improve treatment planning for patients with schizophrenia who smoke and to evaluate smoking cessation programs for this population. METHODS: All major research databases were searched. The review focuses on reports published since 1990. RESULTS: Smoking improves processing of auditory stimuli (sensory gating) by patients with schizophrenia and may lessen negative symptoms by increasing dopamine in the nucleus accumbens and the prefrontal and frontal cortex. Use of traditional antipsychotics may result in patients' smoking more, whereas patients taking atypical antipsychotics may smoke less. Patients who smoke metabolize antipsychotics faster than nonsmoking patients. Smoking cessation programs for outpatients with schizophrenia report a success rate of about 12 percent after six months. No studies of cessation programs for chronically ill inpatients with schizophrenia have been published. Several hospitals have implemented smoking bans with equivocal results. CONCLUSIONS: Nicotine affects both schizophrenia and antipsychotic medications. Neurobiological and psychosocial factors reinforce the high use of nicotine by patients with schizophrenia

A rapid practical RT-PCR-based approach for the detection of the PML/RAR alpha fusion transcript in acute promyelocytic leukemia.

The t(15;17) and its molecular equivalent, PML/RAR alpha gene fusion, is strongly associated with acute promyelocytic leukemia (APL). Since treatment response to all-trans retinoic acid correlates directly with PML/RAR alpha, expeditious documentation is critical to patient care. We have designed an extremely rapid, practical, polymerase chain reaction (PCR)-based method using a rapid air thermal cycler to detect type A, B, and B-variant fusion patterns of PML/RAR alpha. We examined 15 cases of APL and 13 cases of leukemias other than APL with a nested reverse-transcription PCR assay. Three APL samples were type A, 11 were type B, and 1 was a B variant based on gel band patterns. PCR products exhibited positive probe hybridization signals and had sequences containing type A, B, or B-variant fusion patterns. PCR amplification of PML/RAR alpha was complete in 22 minutes, and the entire test required 4 1/2 hours. This method permits exceptional turnaround time and is an alternative to cytogenetics and slower PCR assays.

Implications of a novel cryptic splice site in the BRCA1 gene.

This study was designed to determine the significance of a single intronic base change (IVS5-12 G-->A) found in a family with a history of breast cancer. This change is predicted to form a cryptic splice site resulting in the addition of 11 nucleotides to the BRCA1 transcript. The BRCA1 gene of the relatives and control individuals was sequenced and analyzed using RT-PCR, ASO hybridization, and size fractionation. All patients showed an 11 nucleotide insert at the intron 5/exon 6 boundary. This variant is likely to form a short protein product incapable of the hypothesized tumor suppressor functions of the BRCA1 gene. This information is important for providing counseling for families with this cryptic splice site and a family history of breast cancer.

Applicability of Iceland spar as a stone model standard for lithotripsy devices.

The identification of a universal stone model standard would enable reproducible fragmentation data useful for the design, evaluation, and comparison of various lithotripsy devices. The clinical benefits of such a stone model include the elucidation of setting parameters that would optimize fragmentation strategies. Iceland spar is a pure form of calcite (CaCO3) that was subjected to experimental disintegration by electrohydraulic lithotripsy and extracorporeal shockwave lithotripsy. Iceland spar was fragmented with both lithotripsy methods in a reproducible fashion. The degree of fragmentation was directly related to alterations in either power or shock frequency. Iceland spar is radiopaque, inexpensive, easily obtained, homogenous in composition, and sizable. Iceland spar meets a variety of stone model criteria, warranting its continued investigation as a potential stone model standard.

Conservative management of transitional cell carcinoma of the kidney and ureter.

We report six patients with upper tract transitional cell neoplasms who were treated by an endourologic approach. Two patients had undergone nephroureterectomy previously, whereas one patient had a single functioning kidney. Two patients were deemed candidates for endourologic procedures secondary to significant comorbid disease precluding major open surgery. The final patient had a low-grade distal ureteral lesion and desired a conservative approach.Of six patients, two were rendered tumor free without recurrence 48 months and 60 months after surgery. Two patients had multiple superficial recurrences managed endoscopically and are currently disease free after 12 and 48 months, respectively. One patient died from metastatic transitional cell cancer 18 months postoperatively whereas one died perioperatively secondary to bleeding and multiple organ failure.Of five patients who tolerated endoscopic therapy, four were disease free with a mean follow-up of 30 months. Endoscopic treatment of upper urinary tract transitional cell carcinoma is feasible, although the risk of disease recurrence and progression may be significant.

Monoclonal antibodies to glioma-derived growth factor(s).

Several monoclonal antibodies (mAb) reactive against a high-molecular-weight growth factor from human glioblastoma cell lines have been produced by immunizing mice with partially purified preparations from conditioned media. Antibody-secreting colonies were selected by their capacity to bind 35S-labeled glioma cell protein and by reactivity in indirect enzyme-linked immunoadsorbent assay (ELISA), using high-molecular-weight gel filtration fractions and preparative isoelectric focusing fractions containing growth factor activities. Two of the select mAbs (20F3 and 12A12) depleted mitogenic activity (> 50% inhibition, p < 0.05) from gel filtration fractions by immunoprecipitation, but could not neutralize mitogenic activity directly. Mitogenic activity recovered from affinity columns prepared with mAb 20F3 eluted at 48% and 52% acetonitrile from HPLC C4 reversed-phase columns. Immunoprecipitation of 35S-labeled cell lysates with 20F3 followed by resolution with SDS-PAGE autoradiography revealed one unique protein of 170 kD. Established glioma cell line D-54 MG showed perinuclear and cytoplasmic staining with mAb 20F3. mAb 20F3 should prove useful in purification and characterization of these glioma-derived growth factor(s).

Endopyelotomy: patient selection, results, and complications.

The popularity of minimally invasive surgical techniques, such as endopyelotomy, has increased markedly among urologists in recent years. While it was initially thought that this procedure was best utilized in patients with secondary UPJ obstruction, recent evidence suggests that endopyelotomy should be considered in the majority of cases. The primary contraindication to endoscopic incision of the UPJ is a long stricture, although a large redundant renal pelvis and the presence of crossing lower pole vessels are considered by some to be relative contraindications as well. Although the majority of surgeons have used a percutaneous, antegrade approach to endopyelotomy, successful results also have been reported with a ureteroscopic, retrograde technique. With the development of modified ureterotomes and balloon-cutting devices, the retrograde approach eventually may become the preferred method since no skin incision or external drainage are needed. The role of endopyelotomy in children remains undefined. While successful results have been reported in infants, the relative morbidity and long-term success of open pyeloplasty in this age group are excellent, thus limiting the relative advantage of an endoscopic approach. However, there may be a role for endopyelotomy in older children and in those patients with secondary obstruction who have failed open surgery. From a technical standpoint, there are several minor variations in surgical technique and postoperative management that are important. The success rate of endopyelotomy using a cold knife or small electrocautery probe appears to be comparable, and the use of cautery may allow for precise control of minor bleeding thus decreasing the risk of complications. However, larger electrodes may induce greater tissue reaction leading to fibrosis and should be avoided. Postoperatively, most authors prefer a tapered double-pigtail stent which allows for adequate internal drainage while avoiding excessive pressure within the distal ureter. While successful results have been reported with stenting intervals of only four days, it is generally recommended that the stent be left in place for a minimum of six weeks following endoscopic incision of the UPJ. Overall, endopyelotomy is associated with shortened hospitalization, more rapid return to normal activity levels, and decreased morbidity compared with open pyeloplasty. The success rates reported with endopyelotomy approach those achieved with open surgery, and it is likely that an endoscopic approach to UPJ obstruction will assume an increasingly greater role in the future.

Management of acute ureteral obstruction in pregnancy utilizing ultrasound-guided placement of ureteral stents.

Of 6,275 pregnancies seen at our institution over a two-year period, 5 patients required operative intervention for acute urinary obstruction unresponsive to medical management. Ultrasonography was able to definitively diagnose the presence of an obstructing calculus in 4 of 5 patients. Using ultrasound guidance, 7 indwelling ureteral stents were successfully placed with local anesthesia supplemented by intravenous sedation. Complications consisted of distal stent migration in 1 patient. This method of management was successful for symptomatic nephrolithiasis in a pregnant renal transplant patient. Endoscopic placement of ureteral stents under ultrasound guidance is an effective, safe method of urinary decompression, with no radiation risks imparted to the mother or fetus. Definitive therapy then can be safely deferred to the post-partum period.