The kidney, subclinical thyroid disease and cardiovascular outcomes in older patients.

OBJECTIVE: Thyroid hormones have been implicated to play a role in cardiovascular disease, along with studies linking thyroid hormone to kidney function. The aim of this study is to investigate whether kidney function modifies the association of subclinical thyroid dysfunction and the risk of cardiovascular outcomes. METHODS: In total, 5804 patients were included in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). For the current analysis, 426 were excluded because of overt thyroid disease at baseline or 6 months, 266 because of inconsistent thyroid function at baseline and 6 months, 294 because of medication use that could influence thyroid function, and 16 because of missing kidney or thyroid values. Participants with normal fT4 were classified, based on TSH both at inclusion and 6 months, into three groups: subclinical hypothyroidism (TSH >4.5 mIU/L); euthyroidism (TSH = 0.45-4.5 mIU/L); and subclinical hyperthyroidism (TSH <0.45 mIU/L). Strata of kidney function were made based on estimated glomerular filtration rate into three clinically relevant groups: <45, 45-60, and >60 mL/min/1.73 m2. The primary endpoint consists of death from coronary heart disease, non-fatal myocardial infarction and (non)fatal stroke. RESULTS: Mean age was 75.3 years, and 49.0% patients were male. Mean follow-up was 3.2 years. Of all participants, 109 subjects (2.2%) had subclinical hypothyroidism, 4573 (94.0%) had euthyroidism, and 182 (3.7%) subclinical hyperthyroidism. For patients with subclinical hypothyroidism, euthyroidism, and subclinical hyperthyroidism, primary outcome occurred in 9 (8.3%), 712 (15.6%), and 23 (12.6%) patients, respectively. No statistically significant relationship was found between subclinical thyroid dysfunction and primary endpoint with adjusted hazard ratios of 0.51 (0.24-1.07) comparing subclinical hyperthyroidism and 0.90 (0.58-1.39) comparing subclinical hypothyroidism with euthyroidism. Neither was this relationship present in any of the strata of kidney function, nor did kidney function interact with subclinical thyroid dysfunction in the association with primary endpoint (P interaction = 0.602 for subclinical hyperthyroidism and 0.388 for subclinical hypothyroidism). CONCLUSIONS: In this secondary analysis from PROSPER, we found no evidence that the potential association between thyroid hormones and cardiovascular disease is modified by kidney function in older patients with subclinical thyroid dysfunction.

Subacute renal injury in hypothyroidism: a case report of an unusual phenomenon.

Severe hypothyroidism is known to cause elevation of creatinine and this phenomenon has been reported in clinical settings in the past. Screening for hypothyroidism is not regularly performed in the differential workup for acute kidney injury due to its rare presentation. Therefore, to most physicians hypothyroidism is not known as a cause of acute kidney injury. In this clinical case report, we describe a case of subacute kidney injury in a patient with severe hypothyroidism prior to iodine-131 ablation therapy. Hypothyroidism was not recognized as the cause of elevated creatinine, which in this case led to unnecessary hospital admission and diagnostics. This case report serves as a reminder for clinicians to consider hypothyroidism in the differential diagnosis of (sub)acute kidney injury.

Lung development: number of terminal bronchiolar duct endings and gas exchange surface area in victims of sudden infant death syndrome.

Previous research has demonstrated impaired renal development, particularly with respect to glomerular number, in victims of sudden infant death syndrome (SIDS). The present study used stereological principles to estimate the volume of the upper lobe of the right lung, total number of terminal bronchiolar duct endings (TBDE), and gas exchange surface area of this lobe within a group of human infants. The infants were classified according to cause of death (SIDS or non-SIDS), and further subdivided according to birth-weight: normal birth-weight (NBW) or low birth-weight (LBW). The results demonstrated that TBDE density was significantly reduced in SIDS compared to non-SIDS (P = 0.014), but only reduced from non-SIDS NBW values in the SIDS NBW group (P = 0.044). Total TBDE number was significantly reduced in SIDS from non-SIDS (P = 0.001), and was significantly reduced from non-SIDS NBW values in SIDS NBW (P = 0.023). Mean gas exchange surface area per TBDE was significantly increased in SIDS compared to non-SIDS cases (P = 0.049). The results of the present study indicate developmental delay of the lung in SIDS NBW infants who had previously not been considered growth retarded based on their normal body parameters.

Terminal bronchiolar duct ending number does not increase post-natally in normal infants.

Organ development may be assessed by estimating the total number of functional units within an organ over time; the potential functional capacity of that organ may be represented by the total number of functional units present in the fully developed, mature organ. Relative development of the lung at birth is essential to provide sufficient oxygenation of body tissues and so maintain ex utero life. Estimation of the number of one type of functional unit of the lung - terminal bronchiolar duct endings - provides important information regarding development of the lung. This investigation used stereological techniques, specifically Cavalieri's Principle and the "physical disector", to estimate total number of terminal bronchiolar duct endings in the upper lobe of the right lung of a group of 14 control infants between 0 and 66 weeks post-natal age. Results demonstrate that total terminal bronchiolar duct ending number does not increase significantly over the first 24 weeks of post-natal life in normal infants (P=0.997). The unbiased, design-based techniques used in this paper confirm previous model-based research that indicates that terminal bronchiolar duct ending development is completed before birth.

Audiometric analysis of a Belgian family linked to the DFNA10 locus.

OBJECTIVE: To report the otologic and audiometric characteristics of a nonsyndromic postlingual sensorineural hearing impairment in a Belgian family linked to DFNA10. STUDY DESIGN: Retrospective study of the otologic and audiometric data of 17 genetically affected persons. SETTING: Tertiary referral center. PATIENTS: All members of a Belgian kindred who carried the haplotype linked to the inherited hearing impairment of DFNA10. INTERVENTIONS: Diagnostic otologic and audiometric analysis. MAIN OUTCOME MEASURES: Pure-tone audiometry. RESULTS: To find the frequencies that were most affected by the genetic defect, the excess hearing loss of the 17 patients was calculated per frequency in comparison with the respective p50 and p95 thresholds of the normal population. CONCLUSIONS: The genetically affected persons of a Belgian family shared a progressive symmetric sensorineural hearing loss that started in the first to fourth decade. Thirty-five percent of the affected family members had tinnitus, and only one patient had very mild vestibular complaints. At onset, hearing losses were mainly situated at the midfrequencies. With increasing age, all frequencies became affected. The hearing loss was initially mild, with a spontaneous evolution to a moderate or severe hearing impairment. The progression of the hearing loss for the pure-tone average (between 0.5 and 4 kHz) was 1.08 dB/year for this family, compared with 0.50 dB/year and 0.35 dB/year at the 95th and 50th percentiles of the normal population, respectively.

Renal developmental delay expressed by reduced glomerular number and its association with growth retardation in victims of sudden infant death syndrome and in "normal" infants.

In victims of sudden infant death syndrome (SIDS), renal development has been reported to be significantly impaired. In the present study, we used stereological techniques to estimate volume of kidney cortex and total number of glomeruli in a group of human infants. Infants were classified according to cause of death-SIDS or non-SIDS. Cases were further subdivided according to birth weight-normal birth weight (NBW) or low birth weight (LBW) (we were unable to identify any non-SIDS LBW infants for our study). No significant differences were found between NBW and LBW infants (irrespective of cause of death) for cortical volume, glomerular density, or total glomerular number (p > 0.140). Kidney cortical volume, glomerular density, and total glomerular number were not significantly different between SIDS and non-SIDS infants (p > 0.510). Glomerular number was only significantly less in SIDS infants of LBW (p = 0. 032) than in controls according to the Wilcoxon rank sum test; using the Kruskal-Wallis for one-way analysis, no significant difference was found (p > 0.010). These results contrast with those from previous studies, as a reduction in glomerular number was not noted in SIDS NBW infants, and the mean value for the control (non-SIDS NBW) group was significantly reduced (p < 0.01) from those of previous studies. This indicates that glomerular number reduction is seen in SIDS NBW and non-SIDS NBW cases and is therefore directly associated with growth retardation rather than with SIDS.

Unbiased and efficient estimation of the total number of terminal bronchiolar duct endings in lung: a modified physical disector.

A novel modification of the physical disector is described which was used to estimate the total number of terminal bronchiolar duct endings (TBDEs) in human infant lung. TBDEs are closed three-dimensional space curves of complex shape that are inherently difficult to count from histological sections. However, careful consideration of the microanatomy of the terminal duct endings provides us with the opportunity to define a very simple and unbiased counting rule. To apply the rule in practice we also need to determine a suitable disector height. Owing to the complex shape of the TBDE we had no prior knowledge of what disector height would be suitable for counting the TBDE structures. Exhaustive serial sectioning of complete TBDE structures was carried out and showed that any disector height under 90 microm would give unbiased counts. A further empirical study was then undertaken to determine the most efficient disector height. This was found to be 50 micro. The total number of TBDEs in the upper lobe of the right lung of six human infants aged between 13 and 25 weeks was also estimated. The estimates of numerical density obtained with our modification of the physical disector were multiplied by estimates of lung lobe volume obtained using Cavalieri's Principle. The total number of TBDEs in the lobes ranged from 15 323 to 57 768, with a mean of 40 306. The average coefficient of error of the number estimates was 19%, which was deemed precise enough given the biological coefficient of variation between TBDE number of 36%.

Tophaceous gout: a case of bilateral carpal tunnel syndrome.

Gouty tenosynovitis may present as infection, tendon rupture, nerve compression and/or digital stiffness. We report a case of tophaceous gout which presented as bilateral carpal tunnel syndrome.

Apoptotic fraction in childhood ALL assessed by DNA in situ labelling is ploidy independent.

BACKGROUND: Apoptotic cell fraction and presence or degree of aneuploidy may both affect treatment outcome in childhood acute lymphoblastic leukaemia (ALL), which is largely defined by drug resistance. Independence of the variables is at present not established. Until the development of in situ labelling of cells committed to the apoptotic pathway, the fraction of cells in apoptosis could not be determined objectively. AIM: To determine the relationship between apoptotic cell fraction and karyotype in childhood ALL using in situ labelling. METHODS: 1.3.1. Study Groups and Samples. Diagnostic, pretreatment bone marrow trephine and aspirate samples of 24 consecutive, unselected cases of childhood ALL were included in the study: Normal karyotype (n = 11, 5M,6F), high hyperdiploid aneuploidy (DNA index > 1.5, n = 7, 1M,6F), complex karyotypic anomalies (n = 6, 5M,1F). 1.3.2. Apoptotic Cell Labelling. In situ labelling of the 3'-OH ends of the apoptosis specific DNA (Klenow) fragment (Frag-EL, CalBiochem, USA). 1.3.3. Quantitation. Apoptotic cell fraction was established using 10 systematically random fields of > 20 nuclei. Results were tabled per group. After calculations of means, differences between groups were assessed using t-test. RESULTS: Apoptotic cell fraction, ranging from < 1 to 95%, did not differ statistically significant between the three study groups. CONCLUSION: Apoptotic cell fraction in childhood leukaemia is independent of ploidy status and euploid karyotypic anomalies.

PCNA bearing structures are retained in apoptotic phase of childhood ALL cell cycle.

BACKGROUND: Drug resistance to DNA directed therapy may depend on proliferative as well as apoptotic cell fraction. PCNA/Ki67 ratio excess, possibly reflecting DNA excision repair, is of additional interest to drug resistance in MTT testing. The cell cycle phase/antigen expression pattern in childhood acute lymphoblastic leukemia (ALL) is not known. AIMS: To study the relationship between nuclear expression of PCNA, Ki-67 and Frag-EL positivity in childhood ALL. METHODS: 1.3.1. Study Groups. Diagnostic bone marrow trephine biopsies of 32 consecutive unselected cases of childhood ALL were included in the study. 1.3.2. Immunohistochemistry. Commercially available Moab PCNA (PC10, DAKO, USA), Ki-67 (MM1, NovaCastra, UK) were used to label cycling cells in routinely processed 5 microns paraffin sections. 1.3.3. In-Situ Labelling of Apoptotic Cells. The 3'-OH ends of apoptosis specific DNA fragments were labelled in-situ on subsequent 10 microns sections (Frag-EL, CalBiochem, USA). 1.3.4. Quantitation. After blinding and randomisation, 10 systematic random fields of > 20 nuclei and nuclear size bias correction was used to determine positive nuclei fraction. RESULTS: While the sum of apoptotic and proliferative cell fraction (Ki-67 + Frag-EL%) equalled 100% in 5/32 cases, PCNA expression into at least the early phases of apoptosis ([%PCNA-%K-67] > [100-%Frag-EL] was found in 17/32 cases. CONCLUSIONS: PCNA/Ki67 ratio excess may not reflect DNA excision repair activity but rather slow degradation of antigen bearing structures limiting relevance to drug resistance study.

Apoptosis corrected proliferation fraction in childhood ALL is related to karyotype.

BACKGROUND: Tumour doubling time, a parameter in drug sensitivity testing, reflects both cell proliferation and apoptosis. Variable apoptosis fractions may explain the poor correlation of S-fraction and drug response. DNA aneuploidy (reflecting intrinsic DNA instability) may, by increasing apoptosis, affect drug response. AIM: To assess the relationship between apoptosis corrected proliferation fraction and DNA ploidy in childhood acute lymphoblastic leukemia (ALL). METHODS: 1.3.1. Study Groups. Thirty two consecutive, unselected diagnostic cases of childhood ALL were included in the study. 1.3.2. Karyotype. A normal karyotype was found in 15 cases (7M, 8F, age 8 m-12 yrs); high hyperdiploid aneuploidy (DNA index > 1.5) was found in 7 patients (1M, 7F, age 3-12 yrs) whereas complex karyotypic anomalies, but with 2n or near 2n DNA were present in 10 patients (7M, 3F, age 1 y 7 m -16 yrs). 1.3.3. Proliferation Fraction Assessment. Immunocytochemical demonstration of S-phase associated nuclear expression of the Ki-67 antigen (MM1, NovaCastra, UK). 1.3.4. Apoptosis Fraction Assessment. Binding of a horse radish peroxidase labelled DNA probe for the 3'-OH ends of apoptosis derived Klenow fragments (Frag-EL, CalBiochem, USA). 1.3.5. Quantitation. Computer assisted image analysis (Quantimet 570C), of 10 systematically random fields of a minimum of 20 nuclei each. A nuclear size bias correcting counting frame and rule were used to correct for cell proliferation associated nuclear volume increase and for the expected nuclear volume reduction resulting from apoptosis. RESULTS: Corrected for apoptosis, proliferation fraction was highest (mean 57.5%, range 1-100) in poor prognosis, complex karyotype anomalies. Good prognosis, high hyper diploidy showed significantly lower proliferation rates (mean 24.7%, range 12-40) (p < 0.01, t-test). CONCLUSION: Apoptosis corrected cell proliferation rate in childhood ALL is not independent of karyotype abnormality which may partly explain a relation to therapy response and prognosis.

Proliferation and apoptosis does not affect presenting white cell count in childhood ALL.

BACKGROUND: Treatment response/drug resistance in childhood acute lymphoblastic leukaemia (ALL) is related to presenting white cell count. This relationship might be explained by high proliferation fraction or by absence of significant apoptosis, but is presently unknown. AIMS: To study the relationship between proliferation and apoptosis in childhood ALL. METHODS: 1.3.1. Study Groups. Thirty consecutive, unselected cases of childhood ALL (15M,15F). White cell count varied from 1-200 at presentation. 1.3.2. Proliferation/Apoptosis Fraction. Immunocytochemical detection of Ki-67 (MM1, NovaCastra, UK) on 5 microns paraffin slides, Immunocytochemical detection of apoptosis specific DNA (Klenow) fragments by labelling of 3'-OH ends in 10 microns paraffin sections (Frag-EL, CalBiochem, USA). 1.3.3. Quantitation. Image analysis (Quantimet 570C) using nuclear size bias correcting counting frame and rule. Calculation of proliferation (%Ki-67) fraction and of apoptosis corrected proliferation fraction (%Ki-67/100-%apoptosis). RESULTS: Using both linear, logarithmic regression as well as power analysis, no relationship between variables was detected. CONCLUSION: Presenting white cell count is not related to apoptotic or cell proliferative activity or to net tumour growth defined by the balance between these two processes. The relationship to treatment resistance still requires explanation.

Differential kinetics of drug resistance in human leukaemic cells measured by SCGE/CLSM.

BACKGROUND: New analogues of DNA directed chemotherapy moieties are available for comparative efficacy testing in human neoplastic disease. In addition to MTT testing direct assessment of DNA excision repair activity after direct exposure of marrow cells may provide information on relative DNA effects in vitro. AIMS: To assess the ability of SCGE/high resolution CLSM to detect differences in drug resistance between human neoplastic cell lines in the DNA excision repair response to chemotherapy. METHODS: Eight human leukaemia samples (4 childhood, 4 adult) were exposed to 1 hour of single concentrations of daunorubicin, DaunoXome (courtesy NeXstar Pharmaceuticals Inc, USA), cyclophosphamide and 4-hydroperoxycyclophosphamide (4-HC, courtesy Dr. M. Colvin, Duke University, USA), followed by SCGE/high resolution CLSM with quantitation of total excised DNA. Differences between cases/drug moieties/exposures were analysed. RESULTS: Although generally equal effect dose levels for DaunoXome were lower than for standard daunorubicin, patients/individual neoplastic cells differed considerably in optimal dose levels. Conventional cyclophosphamide in comparison to 4-HC showed inconsistent results indicating considerable differences in the level of drug resistance to the conventional product. CONCLUSIONS: Direct testing for drug resistance patterns in DNA directed drug moieties by SCGE/CLSM reveals individual variability of human malignant cell lines warranting comparison with results of MTT testing and in-vivo patient response.

Demonstration of differences in drug resistance by direct testing of DNA excision repair activity following standard and liposomal daunorubicin exposure in normal paediatric marrow using high resolution CLSM.

BACKGROUND: High resolution Confocal Laser Scanning Microscopy (CLSM) may be applied to testing of drug resistance in vitro in clinical setting. Rapid analysis of DNA damage by precise quantitation of excised DNA in bone marrow samples exposed to potential treatment moieties directly after isolation but the relative sensitivity of the integrated method is as yet untested. AIMS: To test the clinical applicability of SCGE/high resolution CLSM for differences in drug resistance in marrow cells. METHODS: Cells from normal bone marrow samples were exposed for identical periods and at 4 concentrations to either 1 hour of standard Daunorubicin (.5, 1, 1.5, 2 micrograms/ml) or 8 hours DaunoXome (courtesy of NeXstar Inc, USA) (.05, .1, .15, .2 microgram/ml). After 2 and 6 hours recovery, cells were harvested for SCGE, randomization, analysis of tail length, total excised DNA and fragment size distribution using high resolution CLSM. RESULTS: Tail length and fragment size distribution was not, but total excised DNA was significantly increased after 0.1 microgram/ml Liposomal Daunorubicin (DaunoXome) compared to 1.0 microgram/ml Daunorubicin. CONCLUSION: SCGE/high resolution CLSM effectively demonstrated differences in Daunorubicin resistance of human marrow cells to alternative formulations. The method has potential for use in clinical testing of neoplastic cell drug resistance.

Microsatellite instability assessment in prediction of drug resistance in childhood Burkitt's and large cell diffuse malignant non-Hodgkin lymphoma (MNHL).

BACKGROUND: Genomic instability may, especially with DNA directed treatment, be associated with increased therapeutic response; absence may be associated with drug resistance. In childhood MNHL, drug response is variable. At present the degree of presence of microsatellite variation, i.e., intrinsic DNA instability is not known. AIMS: To determine presence and range of microsatellite variability in common childhood MNHL. METHODS: 1.3.1. Study Populations. Consecutive, unselected (1976-96) cases of childhood Large Cell diffuse, N = 16; (9T,7B), age range 1y5m-16y8m; Burkitt's Lymphoma, n = 13, age range 4y2m-14y. Non-malignant/pre-treatment tissue of 20 cases, 13 LC, 7 Burkitt's MNHL. 1.3.2. Molecular Pathology. Routine DNA extraction, amplifications at loci D3S 1304 and D3S1537 (both closely distal to VHL, tumour suppressor gene); ELN gene D7S1870; IFNA D1S243 (1p36) which show microsatellite variation. Isotopic labelling in amplification, non-denaturing gel electrophoresis, autoradiography. RESULTS: Microsatellite variability was found 3/16 LC and 2/13 Burkitt's MNHL. LC MNHL, 4 abnormal areas: n = 1, 3 abnormal areas: n = 1, 2 abnormal areas n = 1; Burkitt's MNHL, 3 abnormal areas: n = 1, 1 abnormal area n = 1. No variability was found in the normal (constitutional) DNA of any of the 20 patients studied. CONCLUSIONS: Microsatellite variability occurred in 5/29 patients with common types of childhood MNHL, indicating a limited contribution to reduced drug resistance through this mechanism.

High resolution confocal laser scanning microscopy analysis of DNA excision repair capability in small volume marrow samples exposed to DNA directed treatment moieties.

BACKGROUND: Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing. AIMS: To optimise sample requirement for SCGE and CLS. METHODS: Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples. RESULTS AND CONCLUSION: Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.

Tophaceous gout: a case of bilateral carpal tunnel syndrome

Gouty tenosynovitis may present as infection, tendon rupture, nerve compression and/or digital stiffness. We report a case of tophaceous gout which presented as bilateral carpal tunnel syndrome.

CAG repeat contraction in the androgen receptor gene in three brothers with mental retardation.

We report on three brothers with mental retardation and a contracted CAG repeat in the androgen receptor (AR) gene. It is known that expansion of the CAG repeat in this gene leads to spinal and bulbar muscular atrophy (SBMA or Kennedy disease); however, contracted repeats have not yet been implicated in disease. As the range of the length of CAG repeats in the AR gene, like those of other genes associated with dynamic mutations, follows a normal distribution, the theoretical possibility of disease at both ends of the distribution should be considered.

Mutations in the KCNQ4 gene are responsible for autosomal dominant deafness in four DFNA2 families.

We have previously found linkage to chromosome 1p34 in five large families with autosomal dominant non-syndromic hearing impairment (DFNA2). In all five families, the connexin31 gene ( GJB3 ), located at 1p34 and responsible for non-syndromic autosomal dominant hearing loss in two small Chinese families, has been excluded as the responsible gene. Recently, a fourth member of the KCNQ branch of the K+channel family, KCNQ4, has been cloned. KCNQ4 was mapped to chromosome 1p34 and a single mutation was found in three patients from a small French family with non-syndromic autosomal dominant hearing loss. In this study, we have analysed the KCNQ4 gene for mutations in our five DFNA2 families. Missense mutations altering conserved amino acids were found in three families and an inactivating deletion was present in a fourth family. No KCNQ4 mutation could be found in a single DFNA2 family of Indonesian origin. These results indicate that at least two and possibly three genes responsible for hearing impairment are located close together on chromosome 1p34 and suggest that KCNQ4 mutations may be a relatively frequent cause of autosomal dominant hearing loss.