Genetic identification and subsequent LC-QTOF MS analysis of plant remains (Oenanthe spp.) could prove the cause of an undetermined sudden death.

In recent years, recorded cases related to forensic botany and, in particular, of plant poisoning have become rare. We report on the medicolegal characteristics of an undetermined sudden death (USD) of a woman in which scene there were remnants of a vegetal peeling. After the autopsy, macroscopic findings reported multiorgan failure and requested the investigation of the cause of death. Postmortem blood was firstly investigated on cyanide toxicity presumptively coming from a yucca-like root; however, found cyanide levels were under normality. Because of the lack of morphological features of the encountered plant remains, a genetic nrDNA ITS2 sequence investigation was followed. The resulting DNA sequence could identify the evidence as the water dropwort (Oenanthe spp.) which contains oenanthotoxin, a potent toxin that may be fatal, similar to the more commonly found in hemlock Conium or cowbane Cicuta species. A liquid chromatography-tandem high resolution mass spectrometry (LC-QTOF MS) was later applied to analyse the vegetal extract and stomach content and successfully confirmed the toxin existence. Medicolegal and analytical findings at the forensic laboratory were described, where both biological and chemical techniques could successfully conjugate, as an interdisciplinary research, and explain premortem symptoms and postmortem findings. Present data can be helpful in future investigation on poisoning cases by conjugated polyacetylenes . The present work tries to emphasize the often undervalued plant evidence in legal medicine diagnosis in the context of an unexplained death.

The NOS3 (27-bp repeat, intron 4) polymorphism is associated with susceptibility to osteomyelitis.

Cytokines generate nitric oxide (NO) in osteoblasts and neutrophils through the induction of NO synthase isoforms, endothelial (NOS3) and inducible (NOS2), thereby producing bone loss. In osteomyelitis (OM), a chronic infection of the bone, homozygosity for the NOS3 (27-bp repeat, intron 4 polymorphism) 4 allele was significantly more frequent among the 80 patients than in 300 healthy controls (p=0.044). No significant differences were found for other polymorphisms of the NOS genes such as NOS3, the promoter (-786T/C), and the missense change (E298D) in exon 7, and for NOS2, the G/A substitution at position 37498 in exon 22, the (CCTTT)(n), and (TAAA)(n) micro-satellites and the -954G/C in the promoter. Serum NO levels were significantly higher only in the OM patients homozygous for the NOS3 (27-bp repeat, intron 4 polymorphism) 4 allele, compared to controls. In the presence of bacteria or bacterial products, the neutrophils of these patients produced more NO. However, immunolabelling of osteoblasts for NOS3 in biopsy tissues did not correlate with the carriage of a determined NOS polymorphism but with the presence of bone inflammation. This is the first report of an association between a NOS3 polymorphism and the risk of developing OM.

The Toll-like receptor 4 (Asp299Gly) polymorphism is a risk factor for Gram-negative and haematogenous osteomyelitis.

Osteomyelitis is a bone infection caused mostly by Staphylococcus aureus but also by Gram-negative bacteria. Toll-like receptors (TLRs), after recognizing microbial products, induce a signal in neutrophils, leading to NF-kappaB activation and transcription of pro-inflammatory genes. Polymorphisms in TLR2 (Arg753Gln) and TLR4 (Asp299Gly, Thr399Ile) genes are associated with bacterial infections, we therefore studied these polymorphisms in osteomyelitis patients. Homozygotes for the TLR4 (Asp299Gly) polymorphism were significantly more frequent among the 80 osteomyelitis patients than in the 155 healthy controls (3/80, 3.8%versus 0/155, 0%; P = 0.038). Carriers of one or two G alleles of this tlr4 polymorphism were more likely to have Gram-negative, haematogenous and/or chronic osteomyelitis than those without this mutation (P < 0.031). Patients with the TLR4 (Thr399Ile) mutant, which cosegregates with the TLR4 (Asp299Gly), were also carriers of this second polymorphism. No differences for the TLR2 (Arg753Gln) genotypes were found between patients and controls. Neutrophils of patients homozygous for the TLR4 (Asp299Gly) polymorphism showed lower LPS-induced apoptosis reduction, phosphorylation of the inhibitor of NF-kappaB, and lower IL-6 and TNF-alpha levels (P < 0.05). We report here for the first time an association between this TLR4 polymorphism and susceptibility to Gram-negative bacteria and haematogenous osteomyelitis.

Characterization of Salmonella-induced cell death in human macrophage-like THP-1 cells.

Salmonella strains are facultative intracellular pathogens that produce marked cytopathology during infection of host cells. Different forms of cytopathic effects have been associated with the virulence systems encoded by the two Salmonella pathogenicity islands (SPI-1 and SPI-2) and the spv locus. We used Salmonella enterica serovar Dublin to investigate the induction of cytopathology during infection of the human macrophage-like cell line THP-1. Analysis of host cells by flow cytometry using a fluorescent terminal deoxynucleotidyltransferase dUTP-biotin nick end labeling (TUNEL) assay revealed that 70% of THP-1 cells showed DNA fragmentation after 4 h of infection, increasing to greater than 90% by 5.5 h. Moreover, the results showed that gentamicin-killed or chloramphenicol-treated bacteria did not induce DNA fragmentation. Serovar Dublin strains with mutations in SPI-1, SPI-2, or spvB induced these cytopathic effects similar to wild-type bacteria. In contrast, a mutation in the phoP regulatory gene abolished DNA fragmentation in the TUNEL assay. Caspase-3 activation was detected during Salmonella infection of THP-1 cells, but caspase-8 and caspase-9 activities were not found. However, inhibition of caspase-3 did not block Salmonella-induced DNA fragmentation. These results identify a previously undetected apoptotic effect in Salmonella-infected cells that is dependent on phoP gene function.

In vivo interleukin-6 protects neutrophils from apoptosis in osteomyelitis.

Polymorphonuclear neutrophils are critical for resolution of bacterial infections. In tissues, most of the neutrophils quickly die through apoptosis. Using propidium iodide DNA staining and DNA gel electrophoresis, we found that spontaneous apoptosis of neutrophils from patients suffering osteomyelitis (n = 52) was significantly decreased in relation to control neutrophils (n = 20) (40.2% +/- 25.2% versus 54.5% +/- 23.5%; P < 0.03). Incubation of neutrophils from normal volunteers with sera from patients with osteomyelitis reduced apoptosis from 79.1% +/- 14.8% in control sera to 62.2% +/- 18.7% in osteomyelitis sera. A significant increase of serum interleukin-6 (IL-6) and IL-1alpha was found in osteomyelitis (IL-6, 8.8 +/- 11.9 pg/ml versus 1.8 +/- 1.2 pg/ml in controls [P < 0.004]; IL-1alpha, 3.8 +/- 6.4 pg/ml versus 1.0 +/- 2.2 pg/ml in controls [P < 0.02]). No differences in the levels of other cytokines, such as tumor necrosis factor alpha, were found. There was an inverse correlation between IL-6 levels and neutrophil apoptosis (r = -0.855; P < 0.007), but this was not the case for other cytokines. The antiapoptotic effect of the osteomyelitis sera was reversed with anti-IL-6 antibodies (P < 0.03) and was reproduced with recombinant human IL-6 (P < 0.001). The longer life span of neutrophils in osteomyelitis induced by IL-6 could contribute to the tissue damage that occurs in these chronic bone infections.

IL-1 alpha (-889) promoter polymorphism is a risk factor for osteomyelitis.

As osteomyelitis (OM) induces the synthesis of inflammatory cytokines and IL-1 mediates bone resorption by osteoclasts we determined if there is an association between certain common polymorphisms of the genes encoding proinflammatory cytokines (IL-1 alpha and beta, IL-6, TNF-alpha) and OM in adults. The IL-1 alpha (-889) TT genotype was significantly more frequent among 52 OM patients than in 109 healthy controls (13/52, [25.0%] vs. 9/109, [8.3%], P = 0.0081, chi(2) = 7.01, OR = 3.7, 95% CI, 1.35-10.34). Patients who were homozygous for the T allele were younger than the rest of the OM patients (mean age 35.7 +/- 11.5 vs. 58.1 +/- 18.6 years, P = 0.001). IL-1 beta TT (+3953) polymorphism was also more frequent in OM patients (P = 0.014, chi(2) = 5.12, OR = 5.1, 95% CI, 1.21-52.14), but IL-1 beta is in linkage disequilibrium with the IL-1 alpha *T (P < 0.001). Route of infection, chronicity of the infection, type of microorganism isolated, and frequency of relapses were similar in patients with and without the IL-1 alpha TT genotype. There were no associations between OM and polymorphisms of other cytokines genes. IL-1 alpha serum levels were significantly increased in all the OM patients independently of their IL-1 genotype compared to the controls (P = 0.021). Although IL-1 alpha serum levels were not significantly higher in patients with the IL-1 alpha (-889) polymorphism, this does not exclude a difference in production of IL-1 alpha by osteoclasts or other inflammatory cells at the site of infection.