High Intrapatient Variability of Tacrolimus Concentrations Predicts Accelerated Progression of Chronic Histologic Lesions in Renal Recipients.

High intrapatient variability (IPV) of tacrolimus concentrations is increasingly recognized as a predictor of poor outcome in solid organ recipients. How it relates to evolution of histology has not been explored. We analyzed tacrolimus IPV using the coefficient of variability (CV) from months 6-12 after transplantation in a cohort of 220 renal recipients for whom paired protocol biopsies at 3 mo and 2 years were available. Recipients in the highest CV tertile had an increased risk of moderate to severe fibrosis and tubular atrophy by 2 years compared with the low-IPV tertile (odds ratio [OR] 2.47, 95% confidence interval [CI] 1.09-5.60, p = 0.031; and OR 2.40, 95% CI 1.03-5.60, p = 0.043, respectively). Other predictors were donor age, severity of chronic lesions at 3 mo, and presence of borderline or subclinical rejection at 3 mo. Chronicity score increased significantly more in the high CV tertile group than in the middle and low tertiles (mean increase 1.97 ± 2.03 vs. 1.18 ± 2.44 and 1.12 ± 1.80, respectively; p < 0.05). CV did not predict evolution of renal function, which did not deteriorate within the 2-year follow-up period. These results indicate that high IPV is related to accelerated progression of chronic histologic lesions before any evidence of renal dysfunction.

Endarterectomy combined with retrograde stenting for tandem lesions of the carotid artery.

OBJECTIVES: Due to its location in the chest wall, surgical treatment of lesions at the origin of the brachiocephalic trunk or common carotid artery (CCA) is unattractive. Complete endovascular treatment of lesions at the origin of the common carotid artery or brachiochephalic trunk combined with high-grade lesions at the carotid bifurcation carries a high risk for distal emboli before cerebral protection is installed. Therefore, the combination of open carotid endarterectomy with retrograde stenting of the proximal lesion through one stage is most attractive. METHODS: Eleven patients were treated with a combined procedure for tandem lesions at the origin of the brachiocephalic trunk or common carotid artery (CCA) and the carotid bifurcation. Endpoint of this evaluation was the 30-day MACE (Major Adverse Cardiovascular Events). RESULTS: All procedures were finished as planned and no conversion was necessary. Thirty-day mortality was 0%. One patient developed a restenosis after only 4 days for which he underwent a re-PTA procedure. The 30-day MACE was 0%. None of the patients needed additional treatment during follow-up (mean follow-up 33 months; range: 11 to 60) although one patient developed a non-significant stenosis during follow-up. CONCLUSIONS: Combined treatment of tandem lesions of the carotid artery is safe and effective in the long-term.

Severe stomatitis complicating immune-suppressive switch after cardiac transplantation.

Everolimus is a recently developed immunosuppressive drug for patients following solid organ transplantation. Its mechanism of action, independent of calcineurin, is different from that of ciclosporin and tacrolimus and because of its lack of nephrotoxicity, it is a good alternative for calcineurin inhibitors in patients with renal dysfunction. In this paper we describe the case report of a 66-year-old caucasian female who underwent heart transplantation in December 2006. After induction with rabbit anti-thymocytic globulin, her immunosuppressive therapy comprised the combination of tacrolimus, mycophenolate mofetil (MMF) and steroids. Because of renal dysfunction, tacrolimus was changed for everolimus after 6 months. Unfortunately our patient developed severe stomatitis with aphthous ulcerations, shortly after the switch. Despite oral therapy (local anaesthetics), severe pain and malnourishment prompted interruption of everolimus and MMF and therapy was changed to ciclosporin and azathioprine. In addition, thalidomide was added. During the following weeks, there was progressive healing of the ulcerations. MMF was re-introduced and thalidomide was stopped after 6 weeks, without recurrent lesions after 4 months of follow-up.

Highly variable clinical phenotype of carbamylphosphate synthetase 1 deficiency in one family: an effect of allelic variation in gene expression?

Deficiency of the urea cycle enzyme carbamylphosphate synthetase 1 (CPS1) causes hyperammonemia with a vast range of clinical severity from neonatal onset with early lethality to onset after age 40 with rare episodes of hyperammonemic confusion. The cause for this variability is not understood. We report two patients from one family with highly divergent clinical course, one presenting neonatally with a fatal form and the other at age 45 with benign diet-responsive disease. The patients are compound heterozygous for two mutations of the CPS1 gene, c.3558 + 1G > C and c.4101 + 2T > C. The haplotypes containing each mutation are identical between the two patients, as are the sequences of CPS1 exons and flanking introns. Transcriptional experiments show that the abnormal CPS1 transcripts generated by both mutations are identical in these two patients. We characterize promoter and enhancer sequences of the CPS1 gene and find also in these regions no sequence differences between patients. Finally, we perform cloning experiments and find that in the neonatal-onset case, clones of messenger RNA (mRNA) expressed from the allele carrying the c.4101 + 2T > C mutation are threefold more than clones of mRNA from the allele with the c.3558 + 1G > C mutation, whereas in the adult-onset case the two types of clones are equal, indicating skewed expression towards the c.4101 + 2T > C allele in the neonatal case. Although we are yet to understand the mechanism of this differential expression, our work suggests that allelic imbalance may explain clinical variability in CPS1 deficiency in some families.

Pseudodeficiency of glutamine in infant liver disease.

Gamma-glutamyltransferase (gamma-GT) is an early marker for cholestasis and has the capability of glutamine-deamidation. Two infants with elevated serum gamma-GT had a decreased serum glutamine. A time course of glutamine and glutamate concentration changes was performed. This revealed a time dependent decrease of glutamine far below the normal lower limit while glutamate increased above the normal upper limit. In conclusion, increased in vitro gamma-GT can cause pseudodeficiency of glutamine. To avoid pitfalls, physicians should inform the laboratory on accompanying pathologies.

Glutamine synthetase is essential for proliferation of fetal skin fibroblasts.

Background. Glutamine synthetase (GS) is ubiquitously expressed in the human and plays a major role for many metabolic pathways. However, little is known about its role during the fetal period. Methods. Cultured skin fibroblasts derived from an aborted fetus deficient in GS activity due to a R324C exchange as well as fetal and mature controls were used to determine the level of GS-expression, apoptosis, and proliferation in presence or absence of exogenous glutamine. Results. Glutamine synthetase can be found at early gestational stages. Loss of GS activity either inherited or induced through l-methionine sulfoximine leads to an upregulation of the GS protein but not of the GS mRNA and results in a significant drop in the proliferation rate but has no effect on apoptosis. Exogenous glutamine does not influence the rate of apoptosis but increases proliferation rates of the fetal but not the mature fibroblasts. Conclusion. GS can be found during early human fetal stages when it displays a significant effect on cell proliferation.

Influence of age on pulsatile luteinizing hormone release and responsiveness of the gonadotrophs to sex hormone feedback in men.

The influence of aging on serum LH and testosterone (T) pulse frequency and gonadotroph sensitivity to androgen and estrogen feedback was studied in young (less than 55 yr old) and elderly (greater than 65 yr) Trappist monks. LH pulse frequency (sampling interval, 20 min) was significantly lower [0.25 +/- 0.03 (+/- SEM) vs. 0.38 +/- 0.02 pulses/h; P less than 0.01] in elderly (n = 21) than in young monks (n = 27); the pulse amplitudes were similar. Similarly, T pulse frequency was lower in the elderly than in the young monks (0.13 +/- 0.04 vs. 0.23 +/- 0.02 pulses/h; P less than 0.01). In elderly men, the hypothalamo-pituitary complex was more sensitive to 5 alpha-androstan-17 beta-ol-3-one feedback, as determined by the decrease in serum LH and T levels. Moreover, during 5 alpha-androstan-17 beta-ol-3-one (125 mg/day, percutaneously, for 10 days) administration, the LH response to LHRH (100 micrograms, iv) was significantly higher in the elderly men compared to the pretreatment response. During estradiol (1.5 mg/day, percutaneously for 10 days) administration, the LH response to LHRH was decreased in the elderly men, but unchanged in the young men, suggesting greater responsiveness to estradiol in the elderly men. We conclude that in aged men, decreased testicular androgen secretion is not exclusively the consequence of a primary testicular alteration, but that important changes occur in hypothalamo-pituitary function, specifically decreased LH pulse frequency and increased LH responsiveness to sex hormone feedback.

A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs.

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.

Distribution of paroxetine in three postmortem cases.

Paroxetine (Paxil) is a selective serotonin reuptake inhibitor, one of a new class of antidepressants used in the treatment of obsessive-compulsive disorder, panic disorder, and depression. Paroxetine potentiates serotonergic activity through the selective inhibition of serotonin reuptake in the central nervous system. There are few reported overdoses in the literature, and of these, three were fatal. Three coroner's cases in which paroxetine was directly associated with the cause of death are reported. In case #1, paroxetine was the only drug detected in significant concentrations. The heart blood paroxetine concentration was 4.0 mg/L. Case #2 was a known suicide in which the decedent herself admitted taking pills and alcohol. The hospital blood sample drawn at admission was analyzed and contained a 0.25% ethanol level and no paroxetine. Death occurred 10 h later. The postmortem heart blood contained ethanol at 0.06%, paroxetine at 3.7 mg/L, fluoxetine at 0.86 mg/L, and norfluoxetine at 0.65 mg/L. In case #3, death was attributed to an apparent adverse drug interaction between paroxetine and imipramine/desipramine. The postmortem heart blood contained paroxetine at 1.4 mg/L, imipramine at 3.0 mg/L, and desipramine at 9.6 mg/L.

The ABC transporter BcatrB from Botrytis cinerea is a determinant of the activity of the phenylpyrrole fungicide fludioxonil.

This study demonstrates that the ATP-binding cassette (ABC) transporter BcatrB from Botrytis cinerea influences the activity of phenlpyrrole fungicides against the pathogen. This conclusion is based on toxicity assays and northern analysis experiments which show that BcatrB replacement mutants, which do not express the BcatrB gene, show an increased sensitivity to the phenylpyrrole fungicides fludioxonil and fenpiclonil. Mutants overexpressing BcatrB exhibit a decreased sensitivity to these fungicides. In addition, accumulation of fludioxonil by BcatrB replacement mutants was higher than by wild-type isolates. For mutants overexpressing BcatrB the reverse was observed. Additional ABC and major facilitator superfamily (MFS) transporter genes were identified in an expressed sequence tag (EST) database, suggesting that B cinerea has gene families of ABC and MFS transporters. Corresponding fragments of ten ABC (BcatrC-BcatrN) and three MFS transporter genes (Bcmfs1-4) were cloned and characterised. Fludioxonil affected the transcript level of some members of these gene families in germlings during a short treatment with the fungicide at sub-lethal concentrations. Hence, other ABC and MFS transporters may affect the activity of phenylpyrrole fungicides as well. Other fungicides such as the anilinopyrimidine fungicide cyprodinil, the azole fungicide tebuconazole, the dicarboximide fungicide iprodione and the strobilurin fungicide trifloxystrobin also induced transcription of some of the ABC and MFS transporter genes identified. Therefore, we propose that various ABC and MFS transporters function in protection of the fungus against fungicides and are involved in multi-drug resistance development.

Cardiac surgery in octogenarians.

OBJECTIVE: The elderly segment of the Western population is increasing rapidly, and cardiac surgeons are being asked to consider the very elderly for cardiac surgery. Our objective was to obtain data on the outcome of cardiac surgery in octogenarians in order to improve the indication for surgery and to give more accurate information to patients, family and general practitioners. METHODS AND RESULTS: From January 1990 through December 1998, 127 octo- and nonagenarians (age 80-94, mean 82.2 years) underwent cardiac surgery (coronary artery bypass grafting, valve surgery and aortic surgery) at the University Hospital of Antwerp. A retrospective review of the patients' medical records was performed. Follow-up information was obtained by mail or telephone from each patient's primary physician or cardiologist. Hospital mortality was 7.1% (9/127) and late mortality (mean follow-up 2 years) was 23% (30/127). Actuarial survival at 108 months was 70% (90/127). Eighty-three percent of the patients were having class III or IV anginal symptoms before operation. At follow-up 76% of the survivors were in NYHA class I or II. CONCLUSION: Cardiac surgery in the very old often permits survival with improved symptoms. Therefore surgery should not be refused solely because of old age.

[Acquired coronary-cameral fistula complicated by a ventricular pseudoaneurysm]. / Fistule coronaro-camérale acquise compliquée d'un pseudo-anévrysme ventriculaire.

Coronary-cameral fistulas are usually congenital, rarely acquired; the complication of this anomaly with ventricular pseudoaneurysm is exceptional. We report a new case of acquired coronary-cameral fistula, occurred in a patient who had received a bypass graft and who had suffered from angina 1 year after the surgery. On computed tomography coronary angiography, the fistula seems to communicate the first diagonal to a left ventricle pseudoaneurysm. Embolization of the fistula and filling of the pseudoaneurysm by neurocoil were successfully performed. The clinical and angiographic control after 3 months showed symptoms improvement and absence of recanalization of the fistula.

Effects of fixation on RNA integrity in a liquid-based cervical cytology setting.

AIMS: Despite many improvements, cervical cancer screening is still subject to shortcomings. Diagnostic accuracy may improve by using molecular biological techniques, requiring RNA of superior quality. This study determined the effect of SurePath fixation on RNA integrity to assess the suitability of clinical samples collected in this medium for RNA-based molecular assays. METHODS: RNA isolation was performed on fresh and fixed HeLa cells and exfoliated cervical cells fixed in SurePath. The RNA integrity was evaluated by analysis of ribosomal RNA as an indicator of quality. The effect of SurePath preservation on PCR amplification was evaluated by real-time reverse transcriptase (RT)-PCR. RESULTS: In contrast to unfixed cells, SurePath-fixed cells yielded less and severely degraded RNA, as shown by the absence of ribosomal RNA bands. RNA derived from SurePath-fixed cells showed poor real-time RT-PCR amplification characteristics, as evidenced by the absent correlation between threshold values and log cDNA concentration. CONCLUSIONS: Implementation of molecular biology in a clinical context is on the rise and may alleviate shortcomings in current screening and diagnostics. This study shows that SurePath fixation gives rise to highly fragmented RNA with insufficient quality for further reliable analysis by standard real-time RT-PCR applications. The increasing prominence of molecular screening stresses the importance of this finding, which must be considered in relation to choice of an appropriate liquid-based cytology system.

Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis.

The three Apx toxins of Actinobacillus pleuropneumoniae have potential value for use in vaccines and diagnostic tests which will be species specific instead of serotype specific, provided that the Apx toxins are species specific and all field strains produce these toxins. We examined 114 A. pleuropneumoniae field strains and found that they secreted either ApxI, ApxII, ApxI and ApxII, or ApxII and ApxIII and secreted no other cytolytic activities. However, proteins similar to ApxI and ApxII were also produced by Actinobacillus suis.

Transmission of Actinobacillus pleuropneumoniae in pigs is characterized by variation in infectivity.

Ten transmission trials with Actinobacillus pleuropneumoniae were carried out. The observed transmission was highly variable, which was surprising since the design of the trials was very similar. We investigated whether the variable transmission could be explained by variation in infectivity of A. pleuropneumoniae infected pigs. We looked for measurable characteristics, which could be indicative for infectious pigs or for the level of infectivity. The characteristic that appeared to be most indicative for a pig being infectious was an A. pleuropneumoniae positive tonsil at necropsy. The characteristic that was correlated to the level of infectivity was the number of A. pleuropneumoniae colonies isolated from the nasal swab, i.e. the probability for an infectious pig to infect a susceptible pig was tenfold higher on days where at least ten colonies were isolated. In this study it is shown that it is possible to measure the bacterial transmission of A. pleuropneumoniae under controlled circumstances if variation in infectivity is taken into account.