Upregulation of pro-inflammatory cytokines in the intercostal muscles of COPD patients.

Muscle dysfunction is a characteristic feature of chronic obstructive pulmonary disease (COPD). Recent studies suggest that cytokines may operate as local regulators of both muscle function and regeneration. The aim of the present study was to characterise the expression of different cytokines in the external intercostal muscle of COPD. Muscle biopsies were obtained from 25 stable COPD patients and eight healthy controls. Local tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, -6 and -10 expressions (real-time PCR and ELISA), sarcolemmal damage (immunohistochemistry), and the transcript levels of CD18 were assessed. Muscle TNF-alpha and IL-6 transcripts were significantly higher in COPD patients compared with controls, and IL-1beta and sarcolemmal damage showed a strong tendency in the same direction. Similar results were observed at protein level. The CD18 panleukocyte marker was similar in COPD and controls. Respiratory muscle function was impaired in COPD patients and it correlated to both the severity of lung function impairment and TNF-alpha muscle expression. Chronic obstructive pulmonary disease is associated with the upregulation of pro-inflammatory cytokines in the intercostal muscles. This phenomenon might be involved in respiratory muscle dysfunction.

Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers.

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4 degrees C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period.

Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure.

The potential to recover genetic profiles from evidence samples has substantially increased since robust and sensitive amplification kits are commercially available. Nevertheless, even the best amplification kits cannot succeed when the extracted DNA is of poor quality. In this study we compared the efficiency of silica (QIAamp DNA Mini Kit), Chelex and Phenol-Chloroform (PC) based protocols to recover DNA from different categories of samples (blood and saliva on cotton swabs, muscles, cigarette butts, saliva on foods and epidermal cells on clothes). The efficiency of the QIAamp system was improved when samples were treated with QIAshredder homogenizing columns. Overall, conventional Chelex or PC protocols allowed to recover conclusive SGM Plus profiles for 61% of the samples considered in this study. Contrastingly, 82% of them were successfully genotyped after being treated with a combination of QIAshredder and QIAamp systems. Our results further suggested that the QIAshredder/QIAamp protocol was particularly helpful to analyze evidence samples with few DNA and/or that were collected on substrates containing PCR inhibitors.

Characterisation of three Amerindian populations from Hidalgo State (Mexico) by 15 STR-PCR polymorphisms.

The purpose of this study is to report allele frequency data of three ethnic Amerindian population samples: the Otomi (Hna-hnu) from eastern Sierra Madre and Ixmiquilpan valley and the Huasteco from La Huasteca. These groups were characterised by 15 STR-PCR polymorphisms (HumTH01, HumvWA, D18S51, HumTPOX, D19S433, D16S539, D13S317, D8S1179, D7S820, D5S818, HumFGA, CSF1PO, D2S1338, D3S1358 and D21S11). No significant deviations in observed allelic frequencies from Hardy-Weinberg equilibrium were found for all the studied systems. From the forensic point of view, the heterozygosity value, power of discrimination and the a priori chance of exclusion were calculated.

[Analysis of respiratory muscle structure and tumor necrosis and insulin-like growth factor expression in chronic obstructive pulmonary disease: are samples valid if obtained during thoracotomy performed because of localized pulmonary neoplasia?]. / Análisis estructual y expresión de los factores de necrosis tumoral y crecimiento insulina-like en los músculos respiratorios de pacientes con EPOS. Son válidas las muestras obtenidas en el curso de una toracotomía por neoplasia pulmonar localizada?

OBJECTIVE: Various methods have been used to obtain samples to study the structure of human respiratory muscles and the expression of diverse substances in them. Samples are most often obtained from autopsies, from muscle biopsies during thoracotomy performed because of a localized pulmonary lesion (TLL), and from ambulatory thoracoscopic biopsy in patients free of comorbidity (AT). The disadvantage of the first 2 of these methods lies in the possibility of interference from factors related to the patient's death in the first case or from the disease that necessitated surgery in the second. Although AT is free from the disadvantages of the other 2 methods, it is impossible to obtain samples of the diaphragm the principal respiratory muscle with this procedure. The objective of this study was to analyze the fibrous structure of the external intercostal muscle of patients with chronic obstructive pulmonary disease and to quantify the expression of the principal inflammatory cytokine tumor necrosis factor alpha (TNF-alpha)- and of insulin-like growth factor (IGF-1) in the same muscle, comparing the results obtained with TLL and AT samples. METHODS: Prospective and consecutive samples were taken of the external intercostal muscle (fifth space, anterior axillary line) in 15 patients with chronic obstructive pulmonary disease (mean [SD] age 66 [6] years; forced expiratory volume in 1 second 49% [9%] of predicted; PaO2 75 [9] mm Hg). Samples were taken during TLL (8 patients, all with pulmonary neoplasms but carefully selected in order to rule out systemic effects) or TA (7 patients). Patients with serious comorbidity were excluded from the second group. Samples were processed for structural analysis of fibers (immunohistochemical and enzymatic histochemical) and genetic expression of TNF-alpha and IGF-1 (real-time polymerase chain reaction). RESULTS: No differences in the structure of fibers were found between the 2 groups. No differences were observed in the expression of TNF-alpha or IGF-1. CONCLUSIONS: Using rigorous criteria, the TLL method appears to be suitable for studying the structural characteristics and expression of inflammatory cytokines and growth factors in the external intercostal muscle. Moreover, it can also be inferred that TLL is probably also useful for obtaining samples of the diaphragm, a muscle which cannot currently be sampled by any alternative method.

Tunisian population data on 10 Y-chromosomal loci.

Alleles and haplotypes frequencies for 10 Y-chromosome STR loci (DYS19, DYS385 I/II, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438 and DYS439), included in the Y-Plex6 and Y-Plex5 kits were determined for a Tunisian population sample of 100 male individuals.

A case of suicide disguised as natural death.

A case report describing the suicide of a physician classified initially as a natural death. Faced with the vehement protest of the family of the deceased the magistrate decided to request a 'preventive' forensic autopsy. Forensic investigations revealed the cause of death as being a pentobarbital intoxication and the circumstances favoured the hypothesis of a genuine suicide. This case illustrates that the tendency of magistrates to request or not an autopsy is related to their experience or intuition.

Tunisian population data on 15 PCR-based loci.

Genotype and allele frequencies distribution for 15 PCR-based loci included in the Promega PowerPlex 16 kit were determined for a Tunisian population sample of 196 unrelated individuals.

[Interleukin 10 and tumor necrosis factor alpha gene expression in respiratory and peripheral muscles. Relation to sarcolemmal damage]. / Expresión de los genes de la interleucina 10 (IL-10) y del factor de necrosis tumoral alfa (TNF-alpha) en músculos respiratorios y periféricos. Relación con el daño sarcolémico.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) has been implicated in loss of muscle mass in chronic obstructive pulmonary disease and other consumptive processes. TNF-alpha production would be related to inflammation arising from pulmonary disease itself or, alternatively, from smoking, and would be carried to the muscle through the blood stream. However, it has also been suggested that TNF-alpha may be expressed directly in muscle tissue. Regardless the site of production of TNF-alpha, its relation to subsequent muscle damage is unclear. OBJECTIVE: We studied the expression of TNF-alpha and an interleukin inhibitor of its production (IL-10) in the main respiratory muscles and a peripheral muscle in the dog. METHOD: Nine young, male Beagle dogs were included. From all animals we obtained a biopsy of the diaphragm (Dph) and external intercostal (ExtI) muscles and a leg muscle (internal vastus of the quadriceps, IntV). TNF-alpha and IL-10 gene expressions were measured through the analysis of messenger RNA levels, using reverse transcription and polymerase chain reaction. We also assessed sarcolemmal damage using intracellular fibronectin detection (immunohistochemistry). RESULTS: The expression of both cytokines showed wide interindividual variability. On the one hand, TNF-alpha (was very low in Dph and ExtI (0.02 0.03 and 0.05 0.06 a.u., respectively), but relatively high in the IntV (0.14 0.08 a.u.). IL-10 expression, on the other hand was low in the Dph (0.06 0.05 a.u.) and slightly higher in the ExtI (2.7 1.9 a.u., p < 0.01) and IntV (1.6 1.7 a.u.). Sarcolemmal damage was minimal in all three muscles and was related to TNF-alpha expression in the peripheral muscle (r = 0.682, p < 0.05). CONCLUSIONS: 1) TNF-alpha and IL-10 appear to be constitutively expressed within the skeletal muscle in dogs. 2) Basal TNF-alpha expression is lower in respiratory muscles than in peripheral muscles. 3) The expression in the latter is related to membrane damage.

DNA hypermethylation at the D17S5 locus is not a frequent event in human urothelial cancer.

OBJECTIVE: To analyse the DNA methylation status and the loss of heterozygosity (LOH) at the D17S5 locus (17p13.3) in urothelial cancer. MATERIALS AND METHODS: DNA methylation was assayed and LOH analysed by Southern blotting in a series of 33 transitional cell carcinomas of the bladder and renal pelvis. RESULTS: DNA hypermethylation and LOH at the D17S5 locus were detected in six (18%) and 17 (52%) of the tumours, respectively. The six cases with DNA hypermethylation were of the papillary type, and four also had LOH at this locus. CONCLUSION: In contrast to other epithelial tumours, DNA hypermethylation at the D17S5 locus is not a frequent event in human urothelial cancer.

Expresión de los genes de la interleucina 10 (IL-10) y del factor de necrosis tumoral alfa (TNF- ) en músculos respiratorios y periféricos. Relación con el daño sarcolémico / Interleukin 10 and tumor necrosis factor alpha gene expression in respiratory and peripheral muscles. Relation to sarcolemmal damage

ANTECEDENTES: El factor de necrosis tumoral alfa (TNF) se ha implicado en la pérdida de masa muscular en la enfermedad pulmonar obstructiva crónica (EPOC) y otras enfermedades consumtivas. Su producción estaría en relación con la inflamación derivada de la propia enfermedad pulmonar o el hábito tabáquico, llegando al músculo por el torrente sanguíneo. Sin embargo, también se ha sugerido que el TNF- podría expresarse directamente en el tejido muscular. Sea cual fuere el lugar de producción, la relación del TNF con el daño muscular subsiguiente no es clara. OBJETIVO: Hemos estudiado la expresión del TNF y una interleucina inhibidora de su producción (IL-10) en los músculos respiratorios principales y en un músculo periférico del perro. MÉTODOS: Se han incluido nueve animales machos, adultos jóvenes de raza Beagle. En todos ellos se tomaron biopsias del diafragma (Df), del intercostal externo (IExt) y de un músculo de las extremidades (vasto interno del cuádriceps o VInt). Se analizó la expresión de los genes de TNF e IL-10, a través de la determinación de sus ARNm correspondientes, utilizando la transcripción reversa y reacción en cadena de la polimerasa (RT-PCR). También se evaluó el daño sarcolémico mediante detección de fibronectina intracelular (inmunohistoquímica). RESULTADOS: Los valores de expresión de ambas citocinas evidenciaron una amplia dispersión interindividual. Por un lado, la expresión de TNF fue mínima en Df y IExt (respectivamente, 0,02 ñ 0,03 y 0,05 ñ 0,06 u.a.), con valores relativamente elevados en VInt (0,14 ñ 0,08 u.a.). A su vez, la IL-10 evidenciaba valores bajos en DF (0,6 ñ 0,5 u.a.), que eran algo superiores en IExt (2,7 ñ 1,9 u.a.; p < 0,01) y VInt (1,6 ñ 1,7 u.a.). El daño sarcolémico era mínimo en los tres músculos y se relacionaba con la expresión de TNF en el músculo periférico (r = 0,682; p < 0,05). CONCLUSIONES: a) TNF e IL-10 parecen expresarse constitutivamente en el músculo esquelético canino; b) los valores basales de expresión del TNF son menores en los músculos repiratorios que en los de las extremidades mostrando relación en estos últimos con el daño de membrana.. (AU)

Sudden death related to small coronary artery disease.

Two cases of sudden death of young people in apparently good health are reported. The only pathologic change found was a fibromuscular dysplasia of the artery supplying the conduction system of the heart with an important narrowing of the lumen and strong thickening of the arterial wall. The first case was of a 12-year-old girl who died suddenly while skiing; the second was of a 32-year-old man who died while talking to his wife. No other pathologic changes were found at autopsy, and the results of toxicologic analysis were negative. There was no individual or family history of cardiac diseases. These cases illustrate the importance of an analysis of the conduction system, including examination of the intramural coronary arteries supplying the conduction system.

Comparative genomic hybridization analysis of transitional cell carcinomas of the renal pelvis.

We used comparative genomic hybridization to analyze 10 primary tumor samples from patients with transitional cell carcinoma of the renal pelvis. The most frequent loss was located at 9q, that is, in 50% of the tumors. Gains of DNA sequences were most frequently observed in chromosome regions 1q21 approximately q23, 2p23 approximately p25, 8q21.1 approximately q22 and in the whole chromosome 20. High level amplifications at 1q21 approximately q25, 6p22 approximately p23, 8q21 approximately q22, 8q22 approximately q24.1, 11q13, and 12q14 approximately q21 were detected. Most of these regions have previously been reported to be involved in transitional cell carcinoma of the bladder, thus confirming the importance of an increasing number of chromosome imbalances in the development and progression of this type of tumors.

The reconstruction: a useful tool in forensic sciences.

The intervention of a forensic pathologist may be required at any stage of a judicial investigation. Clearly, he or she plays an important role at a crime scene and later on during the examination of the body. But the forensic pathologist's contribution is equally important during reconstruction, which is most frequently called for in cases of violent deaths, homicides in particular. However, reconstruction may also be very useful in cases of less importance in terms of criminal law. It requires little investment and can provide precious evidence. In the two cases presented here we were able to exclude, respectively, the presumption of the adulteration of a blood sample taken for alcohol dosage and the accidental origin of an attempted murder. In fact, reconstruction may be a valuable procedure in any event requiring the participation of a forensic expert.

The challenge of identification following the tragedy of the Solar Temple (Cheiry/Salvan, Switzerland).

On October 5, 1994, 48 members of the Sect of the Solar Temple were found dead at two different locations in Switzerland: 23 victims in Cheiry and 25 victims in Salvan. Our Institute was commissioned to solve the forensic problems presented by this tragedy. Our goals were to establish the time of death, determine its causes, help elucidate the surrounding circumstances and identify the victims. This work presented us with the following challenges. This catastrophe was of an 'open' type: there was no list of 'passengers'; the victims were of five different nationalities and many had just arrived in Switzerland to participate in this event; family ties were very complex within this group; half of the victims were burned and sometimes charred; the exceptionally intense media converage of the story put a lot of pressure on the investigators and our Institute. In spite of these difficulties, all the victims were positively identified within 1 month. In the present report, we describe the steps realized to progress in our work. A special section describes our relationship with the journalists and their invaluable help in our investigations. The importance of being prepared for such an event is discussed.

Forensic evaluation of mtDNA in a population from south west Switzerland.

The polymorphism of the two hypervariable segments (HVI and HVII) of the control region of mtDNA was analyzed in a population of 154 unrelated individuals from south west Switzerland using a fluorescent based capillary electrophoresis sequencing method. In our population data of 154 random individuals, 137 mtDNA types were observed. Of these, 124 sequences were observed only in one individual whereas 10 sequences were observed in 2 individuals, 2 sequences in 3 individuals and 1 sequence in 4 individuals. The probability of two unrelated individuals having the same sequence was 0.84%. The results were compared with four other Caucasian populations. Furthermore, the usefulness of the mtDNA sequencing was tested, for exclusion and inclusion, in 18 forensic cases including 69 evidence samples and 44 reference samples. Despite the fact that 55% of the evidence samples yielded a negative result for the nuclear DNA with the human dot quantitation system, the success rate of the mtDNA sequencing was 71.0%. This validation study proves the great usefulness and sensitivity of the mtDNA sequencing technique using nested PCR and fluorescent capillary electrophoresis.

Study of allelic losses on 3p, 6q, and 17p in human urothelial cancer.

Forty-eight transitional cell carcinomas of the bladder and three transitional cell carcinomas of the renal pelvis were examined for loss of heterozygosity (LOH) on chromosomes 3p, 6q, and 17p. The most frequent allelic loss was seen on 17p (18/36, 50%) followed by 6q (6/22, 27%), and 3p (5/22, 23%). In cases with LOH at more than one locus, the same DNA sample often varied in degree of signal reduction for missing alleles. This observation indicates that LOH studies can serve to detect intratumor heterogeneity. No correlation was found between allelic losses at these chromosome arms and tumor grade and stage. Allelic losses on 6q were associated with tumors having a solid growth pattern; in this kind of tumors, allelic losses on 3p were associated with invasion.

Analysis of 3p allelic losses in renal cell carcinomas: comparison with cytogenetic results.

We performed a study of loss of heterozygosity (LOH) at 3p by restriction fragment length polymorphism analysis in a series of 22 renal tumors. In 11 cases, molecular results could be compared with those of cytogenetic studies. The highest frequency of allelic losses at 3p was seen in clear cell non-papillary renal tumors, whereas none of the papillary renal cell carcinomas showed LOH at 3p. Allelic losses on 3p were found to be independent of tumor grade or stage or both. One case analyzed showed a discrepancy between cytogenetic and LOH studies. This tumor displayed rearrangements of chromosome 3 and no LOH at the c-RAF-1 (close to the Von Hippel Lindau gene) locus.

Forensic validation of the short tandem repeat HUMACTBP2 using capillary electrophoresis.

Experiments were performed to evaluate the forensic identification of the short tandem repeat (STR) HUMACTBP2 (human beta-actin-related pseudogene) using automated fluorescence-based capillary electrophoresis. The HUMACTBP2 is a complex tetranucleotide STR locus with more than 32 alleles in the range of 202-323 bp. The reproducibility of genetic typing using a fluorescent labeled allelic ladder was determined by comparison of the calculated fragment size after consecutive (within-day) and nonconsecutive (day to day) injection. The maximum variation in size (window) observed for any allele was 0.23 bp for the within-day and 0.8 bp for the day-to-day precision. Furthermore, it is possible to achieve a 1 bp resolution, the precision of the reproducibility assays being about 99.95%. Sixty blood samples and twenty stains were typed with both automated fluorescent sequencer ABI 373A and ABI 310. Identical genotypes were obtained with both techniques and the ABI 310 seemed to be more sensitive than the ABI 373A. A population sample of 197 unrelated individuals from southwest Switzerland was analyzed and the genotype frequencies observed were similar to those reported by others. Thirty-one alleles and 126 genotypes were found. The observed heterozygosity was 0.934. Mixtures from two different blood samples varying in their ratio were typed and the minor fraction was detectable to about 1:10. The practical usefulness of the HUMACTBP2 is illustrated by analyzing casework samples. This validation study proves the usefulness of the HUMACTBP2 locus in forensics and the detection efficiency using fluorescent capillary electrophoresis.