Human identification and analysis of DNA in bones

The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall

OBJECTIVE: The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. METHODS: Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65±6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. RESULTS: Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. CONCLUSION: DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

A utilização de retalho composto de pele e tecido mamário na reparação de área cruenta resultante da deiscência de esternotomia em cirurgia cardíaca

OBJETIVO: Descrever nova técnica para a reparação de áreas cruentas resultantes de infecção e deiscência de esternotomia na cirurgia cardíaca em seis mulheres que, além de área cruenta extensa na região esternal, haviam sido submetidas a uma primeira reintervenção com recidiva. MÉTODOS: É descrita a técnica cirúrgica utilizada baseada em retalho triangular composto de pele e tecido mamário com base no sulco inframamário inferior, transposto para a área cruenta a fim fornecer uma cobertura de tecido bem vascularizado. RESULTADOS: São discutidos os diversos tratamentos da fase aguda e também de reconstrução nas deiscências de região esternal. CONCLUSÃO: Este retalho preenche as necessidades em relação às dimensões da perda de tecido da área cruenta além de fornecer uma maior proteção contra infecção a uma área que, pela deiscência ocorrida, permaneceu por certo tempo exposta.

Reparação de defeitos ósseos em fêmures de ratos machos e fêmeas espontaneamente hipertensos: estudo histológico e histométrico

OBJETIVO: O objetivo deste estudo foi avaliar a reparação óssea em ratos espontaneamente hipertensos (SHR) e compará-la com a de ratos normotensos, avaliando machos e fêmeas. MÉTODOS: Um defeito ósseo foi criado no fêmur esquerdo de 24 SHR (12 machos e 12 fêmeas) e 24 ratos normotensos (12 machos e 12 fêmeas). Os animais foram divididos em dois grupos com diferentes períodos de sacrifício: sete e 21 dias após o procedimento cirúrgico. Após processamento laboratorial de rotina, as análises histológica e histométrica foram realizadas e os dados submetidos à análise de variância (ANOVA) e ao teste de Tukey (5%). RESULTADOS: Machos e fêmeas do mesmo grupo apresentaram características histológicas similares. Após sete dias, todos os animais apresentaram trabéculas ósseas irregulares, entretanto, os osteoblastos periosteais eram achatados nos SHR, enquanto nos normotensos, essas células apresentavam formato cúbico. Após 21 dias, todos os espécimes mostraram fechamento linear em toda a extensão superficial do defeito ósseo e os SHR apresentaram osteoblastos achatados, enquanto os normotensos apresentaram células cúbicas circundando as trabéculas ósseas. A análise estatística dos dados histométricos indicou médias similares entre machos e fêmeas, exceto para ratos normotensos, aos sete dias. Além disso, aos 21 dias, foi observada maior neoformação óssea nos ratos hipertensos quando comparados aos normotensos, tanto machos quanto fêmeas. CONCLUSÃO: Concluiu-se que os animais SHR apresentaram maior neoformação e maturidade óssea nos defeitos que animais normotensos, no período de 21 dias após a cirurgia.

Ação local do alendronato sódico na reparação óssea de ratos espontaneamente hipertensos (SHR)

FUNDAMENTO: A hipertensão arterial é uma desordem caracterizada por alterações relevantes no tecido ósseo. O alendronato sódico tem indicação no tratamento de doenças ósseas, por causa de sua afinidade pela hidroxiapatita, inibindo as reabsorções ósseas. OBJETIVO: Analisar a ação local do alendronato sódico na reparação óssea de ratos espontaneamente hipertensos (SHR). MÉTODOS: Um defeito ósseo foi criado no fêmur esquerdo de 80 ratos. De acordo com o material utilizado no local, criaram-se quatro grupos: controle (C), amido (Am), alendronato 1 mol (A1) e alendronato 2 mol (A2). Após 7 e 21 dias, os animais foram sacrificados. Foram realizadas análises histológicas e histomorfométricas e os dados foram submetidos a análise de variância (ANOVA) e teste de Tukey (5%). RESULTADOS: Aos 7 dias, observou-se, na área do defeito, tecido conjuntivo com hemorragia e inflamação em todos os grupos. Alguns apresentavam matriz osteóide. Os grupos A1 e A2 apresentaram, ainda, uma rede de fibrina. Aos 21 dias, as trabéculas ósseas fechavam praticamente a extensão do defeito nos grupos C e Am. No grupo A1 de animais machos, observaram-se trabéculas que se irradiavam do canal medular até a área do defeito. Nos grupos A1 e A2, constatou-se apenas a presença de tecido conjuntivo com mínima deposição de osteóide. Um achado histológico marcante foi a formação de tecido ósseo extracortical subperiosteal nos animais dos grupos A1 e A2. CONCLUSÃO: Concluiu-se que a administração do alendronato sódico não contribuiu para o reparo ósseo nos ratos SHR, mas possivelmente tenha sido responsável pelas formações ósseas extracorticais observadas.

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera: Psychodidae)

Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probles for positively separting individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. welcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, buth this is based on circumstantial evidence because of the difficulties of identifying female sandflies wothin the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligationg short fragments of Sau 3A-resistricted, genomic DNA into the plasmid vector PUC 18. Differential screening of 1316 library clones with total genomic DNA of Ps. Wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (=ca. 0.5 namograms) was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.

Development of nuclear DNA probes for the typing of Trypanososma cruzi

We have developed and tested a new way of typing Trypanosoma cruzi, mamely the use of cloned nuclear DNA fragments as genetic markers. Restriction fragment length polymorphisms were verified on Soutern blots hybridized to random probes. Fragment patterns were analyzed and dendrograms constructed. Our results on well characterized laboratory strains correlate well to published isoenzyme studies. Some of the probes were also hybridized to chromosomes separated by pulse field gel electrophoresis a higher degree of heterogeneity was observed at this level.

Stability of isoenzyme and kinetoplast DNA (k-DNA) patterns in successively cloned Trypanosoma cruzi populations

Four Trypanosoma cruzi strains from zymodermes A, B, C and D were successively clonedon BHI-LIT-agar-blood BLAB). Twenty clones from the first generation (F1), 10 from The second (F2) and 4 from the third (F3) from the strains A138, B147 and C23 were isolated. The D150 strain provied 29 F1 and F2 clones. The strains and clones had their isoenzyme and K-DNA patterns determined. The clones from A138, Bl47 and C231 strains presented isoemzyme and K-DNA patterns identical between thewmselves and their respective parental strains. Therefore showing the homogenety and stability of isoenzyme and K-DNA patterns after successive cloning. The Dl50 strain from zymodeme D (ZD) showed heterogeneity. Twenty-eight out of 29 clones of the first generation were of zymodeme A and only one was of zymodeme C, confirming previous reports that ZD strains consisted of ZA and ZC parasite populations. The only D150 strain clone of zymodeme C showed a K-DNA pattern identical to its parental strain. The remining clones although similar among themselves were different from the parental strain. Thus the T. cruzi strains had either homonogeneus or heterogeneous populations. The clones produced by successive cloning provided genetically homonogeous populations. Their experimental use will make future results more reliable and reproducible.

A new repetitive DNA sequence from Trypanosoma cruzi

Tandemly repeated DNA sequences are found in the genome of higher eukaryotes, and have also been demonstrated in Trypanosoma cruzi. Repeated DNA sequences are potentially useful for the diagnostic detection of T. cruzi (A. Gonzales et al., 1984, Proc. Natl. Acad. Sci. USA, 81: 3356-3360). We have isoleted two clones from a genomic library of T. cruzi (Y strain) that contain, in one clone a family of at least seven copies of a repetitive sequence of approximately 600 base pairs, and in the other an independent copy of the same sequence. One copy of the repetition (HSP) and the independent clone (HCR) were sequenced by the Sanger procedure (Fig.). This sequence hybridized to four strains of T. cruzi tested and did not hybridize to eleven species of trypanosotids from five different Genera, being a good candidate for diagnostic assays. GenBank accession numbers: HSP#m31919, HCR#31920.

Detection of cytomegalovirus in urine of HIV-infected patients by DNA-DNA hybridization comparison with virus isolation, immunofluorescence and immunoperoxidase

Immunofluorescence and immunoperoxidase test directed against early viral antigens, and DNA-DNA hybridization were compared with viral isolation for their abilities to detect Cytomegalovirus (CVM) in the urine of 89 HIV infected patients. From the 100 urine samples collected, 70 were found positive by at least one method. Considering viral isolation as the "gold standard" technique, immunofluorescence and immunoperoxidase had a sensitivity of 92.3% and88% respectively, with a specificity in both cases of 95%. DNA-DNA hybridization showed a sensitivity of 90% but with lower (60%) specificity. All of the three assays were effective in detecting CVM from urine and the technical advantage of each is discussed.