Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids.

Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes.

Frataxin participates to the hypoxia-induced response in tumors.

Defective expression of frataxin is responsible for the degenerative disease Friedreich's ataxia. Frataxin is a protein required for cell survival since complete knockout is lethal. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. We therefore sought to investigate the pathways through which frataxin enhances stress resistance in tumor cells. We found that frataxin expression is upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, frataxin upregulation in response to hypoxia is dependent on hypoxia-inducible factors expression and modulates the activation of the tumor-suppressor p53. Importantly, we show for the first time that frataxin is in fact increased in human tumors in vivo. These results show that frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could have a critical role in tumor cell survival and/or progression.

Acid reflux into the oesophagus does not influence exercise-induced airway narrowing in bronchial asthma.

OBJECTIVES: A few studies on small patient series have investigated the relationship between gastroesophageal reflux and bronchial responsiveness as expressed by exercise-induced bronchoconstriction (EIB), with non-conclusive results. The aim of this study was to evaluate whether the presence of acid in the oesophagus may influence EIB. METHODS: 45 patients with bronchial asthma underwent spirometry, exercise challenge on bicycle ergometer and 24 h oesophageal pH monitoring. Subjects with EIB (Forced expiratory volume in the first second (FEV1)) percentage decrease after exercise (DeltaFEV1) > or =15%, n = 28) were retested after a 2 week treatment course with omeprazole 40 mg/daily. Exercise at baseline was performed at the same time as oesophageal pH monitoring. RESULTS: In basal condition, there was no difference in FEV1, acid exposure time or number of refluxes measured during 24 h pH monitoring between patients with and without EIB. There was no relationship between spirometry results and DeltaFEV1 on one hand, and parameters of gastroesophageal reflux on the other. Nine patients with EIB (31.0%) and six patients without EIB (37.5%) had one or more episodes of GER during exercise challenge, without significant differences between the two groups. After gastric acid inhibition by omeprazole, DeltaFEV1 did not change significantly. CONCLUSIONS: The results indicate that acid in the oesophagus, or its short-term inhibition by proton pump inhibitors, has no influence on exercise-induced bronchoconstriction.

Silencing of SH-PTP2 defines a crucial role in the inactivation of epidermal growth factor receptor by 5-aminosalicylic acid in colon cancer cells.

Recent studies have suggested that 5-aminosalicylic acid (5-ASA) inhibits colorectal cancer (CRC) development. However, the mechanism underlying the antineoplastic effect of 5-ASA remains unknown. We here examined the effect of 5-ASA on epidermal growth factor receptor (EGFR) activation, a pathway that triggers mitogenic signals in CRC cells. We show that 5-ASA inhibits EGFR activation, through a mechanism that does not rely on CRC cell death induction. 5-ASA enhances the activity, but not expression, of phosphorylated (p)-EGFR-targeting phosphatases (PTPs), and treatment of cells with PTP inhibitors abrogates the 5-ASA-mediated EGFR dephosphorylation. Both SH-PTP1 and SH-PTP2 interact with EGFR upon 5-ASA treatment. However, knockdown of SH-PTP2 but not SH-PTP1 by small interference RNAs prevents the 5-ASA-induced EGFR dephosphorylation. Finally, we show that 5-ASA attenuates p-EGFR in ex vivo organ cultures of CRC explants. Data indicate that 5-ASA disrupts EGFR signalling by enhancing SH-PTP2 activity, and suggest a mechanism by which 5-ASA interferes with CRC growth.

The clinical relevance of the expression of several multidrug-resistant-related genes in patients with primary acute myeloid leukemia.

Multidrug resistance (MDR) is a complex phenomenon that includes the expression of many different genes regulating drug transport or metabolism, cellular repair or detoxification mechanisms. The co-expression of several genes could be at the basis of the resistant phenotype in vivo. In order to test a possible prognostic role of the expression and co-expression of several MDR-related genes (MDR1, topoisomerase IIalpha, topoisomerase IIbeta, MRP, GSTpi, LRP), 35 patients affected by acute myeloid leukemia (AML) were tested by RT-PCR assays. In our series, topoisomerase IIbeta was significantly co-expressed with MRP (p = 0.05), GSTpi (p = 0.017) and LRP (p = 0.005). GSTpi was co-expressed with LRP (p = 0.03) and MRP (p = 0.007); on the other hand, 53.8% of patients were LRP and MRP-positive (p = 0.02). The PCR-positivity did not differ according to biological/clinical characteristics of patients, including age; this latter was the only parameter conditioning the response and overall survival. Neither the expression nor the co-expression of the tested genes was significantly correlated with the response to the induction treatment and long-term outcome.

Asthma prevalence and drug prescription in asthmatic children.

A cross-sectional study was conducted on among 28,856 children aged from birth to 14 years to determine the prevalence of asthma and assess its treatment in a sample of asthmatic children. Children diagnosed with asthma were identified by a sensitive algorithm applied to the information stored in the computerized medical records between 1997 and 1998. Pediatricians then reviewed and validated the diagnosis. Specific information was obtained, after age stratification under 5 yrs and over 6 ys, from the medical records and by interview regarding their personal details and treatment of asthmatic patients. In all, 1,263 cases of asthma were identified (64% males) with a prevalence of 6.3% among males and 4% among females in under 5 year-olds, and 3.9% for males and 2.1% for females in over 6 year-olds. The prevalence of asthma diagnosed directly by the pediatrician was consequently higher among under 5 year-olds, in both genders, than among the older children. Contrary to the international guidelines, pediatricians prescribed more oral corticosteroids and nebulized short-acting beta-2 agonists for children under 5 ys olds than for over 6 year-olds (13.3% Vs 4.8% and 25% Vs 10.9%, respectively, p < 0.001). For the > or = 6 year-olds, the most commonly prescribed treatments were oral antihistamines (13.9% Vs 12.6%), inhaled corticosteroids via metered-dose inhaler (30.8% Vs 28.7%) and sodium cromoglycate (12.1% Vs 4.8%, p < 0.001).

Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis.

Increased brain ganglioside levels are a hallmark of various neuroinflammatory pathologies. Here, we provide evidence that murine microglia can secrete disialoganglioside GD3 upon exposure to inflammatory stimuli. Comparison of different neural cell types revealed a particular and specific sensitivity of oligodendrocytes towards exogenous GD3. Oligodendrocyte death triggered by GD3 was preceded by degeneration of cellular processes, and associated with typical features of apoptosis, such as chromatin condensation, exposure of phosphatidylserine, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential, followed by the loss of plasma membrane integrity and detachment of disintegrated oligodendrocytes. Overexpression of bcl-2 partially protected oligodendrocytes from death. In contrast, treatment with the pan-caspase inhibitor zVAD-fmk did not prevent phosphatidylserine exposure, chromatin margination at the nuclear periphery, and death, although caspase-3 was blocked. Thus, GD3 produced by microglia under neuroinflammatory conditions may function as a novel mediator triggering mitochondria-mediated, but caspase-independent, apoptosis-like death of oligodendrocytes.

Novel anti-inflammatory effects of the inhaled corticosteroid fluticasone propionate during lung myofibroblastic differentiation.

Asthma is characterized by an irreversible subepithelial fibrosis with the appearance of myofibroblasts, which can be now considered important early participants in inflammatory responses as well as potential targets for anti-inflammatory drugs. In this study, we show that fluticasone propionate (FP), a powerful inhaled corticosteroid (ICS), displays novel anti-inflammatory effects on human lung fibroblasts during their myofibroblastic differentiation. Indeed, FP inhibits in lung myofibroblasts, at a very early stage of differentiation, the activation of Janus kinase/STAT pathways induced by IL-13 (tyrosine kinase 2, STAT1, STAT3, STAT6, mitogen-activated protein kinase). Contrarily, in mildly or fully differentiated myofibroblastic cultures, FP still displays a potential anti-inflammatory activity even if it only inhibits tyrosine kinase 2 phosphorylation. Moreover, FP inhibits constitutive and TGF-beta-induced expression of alpha-smooth muscle actin, the main marker of myofibroblastic differentiation, both in very early and in mild differentiated myofibroblasts. Finally, FP displays an additional powerful anti-inflammatory effect, decreasing nuclear translocation of NF-kappaB independent of the degree of myofibroblastic differentiation. These data 1) suggest that myofibroblasts are priority targets for ICS, which is able to revert them to a normal phenotype even if they appear to be already engaged in their differentiation, and 2) may help to explain why asthma is improved by an early ICS treatment, whereas advanced asthma is more resistant to these drugs.

UVB-induced apoptosis of human dendritic cells: contribution by caspase-dependent and caspase-independent pathways.

Dendritic cells (DCs) play a central role in the initiation and regulation of the immune response. The modalities by which DCs are committed to undergo apoptosis are poorly defined. Here it is shown that, unlike death receptor ligands, UVB radiation triggers apoptosis of human DCs very efficiently. UVB exposure is followed by the activation of caspases 8, 9, and 3, by the loss of mitochondrial transmembrane potential (deltaPsim), and by cellular and nuclear fragmentation. Caspase inhibitors substantially prevented the occurrence of cellular and nuclear fragmentation but had no effect on UVB-induced deltaPsim dissipation. Importantly, mature DCs (MDCs) displayed relative resistance to UVB; UVB-induced caspase activation and apoptosis were substantially delayed compared to immature DCs (IDCs). Resistance correlated with the strong up-regulation of cellular FLIP and bcl2 observed in MDCs compared to IDCs.

Unusual morphology in a case of large granular cell leukemia.

Large granular lymphocyte proliferative status represents a group of clonal and nonclonal lymphoproliferative disorders of natural killer (NK) or T-cell lineages with common morphological features. Cellular differences may sustain the clinical polymorphism observed in these disorders. Here we report a case of large granular lymphocyte disease unusually expressing CD4+CD8+ clonal T cells and atypical cell morphology in bone marrow.

Additive effects of salmeterol and fluticasone or theophylline in COPD.

BACKGROUND: ss(2)-Agonists and corticosteroids or theophylline can interact to produce beneficial effects on airway function in asthma, but this has not been established in COPD. METHODS: Eighty patients with well-controlled COPD were randomized to receive 3 months of treatment in one of four treatment groups: (1) salmeterol, 50 microg bid; (2) salmeterol, 50 microg, plus fluticasone propionate, 250 microg bid; (3) salmeterol, 50 microg, plus fluticasone propionate, 500 microg bid; and (4) salmeterol, 50 microg, plus titrated theophylline bid. At each visit, a dose-response curve to inhaled salbutamol was constructed using a total cumulative dose of 800 microg. RESULTS: A gradual increase in FEV(1) was observed with each of the four treatments. Maximum significant increases in FEV(1) over baseline values that were observed after 3 months of treatment were as follows: salmeterol, 50 microg bid, 0.163 L (95% confidence interval [CI], 0.080 to 0.245 L); salmeterol, 50 microg, plus fluticasone propionate, 250 microg bid, 0.188 L (95% CI, 0.089 to 0. 287 L); salmeterol, 50 microg, plus fluticasone propionate, 500 microg bid, 0.239 L (95% CI, 0.183 to 0.296 L); and salmeterol, 50 microg, plus titrated theophylline bid, 0.157 L (95% CI, 0.027 to 0. 288 L). Salbutamol always caused a significant dose-dependent increase in FEV(1) (p < 0.001), although the 800-microg dose never induced further significant benefit when compared with the 400-microg dose. The mean differences between the highest salbutamol FEV(1) after salmeterol, 50 microg, plus fluticasone propionate, 500 microg bid, and that after salmeterol, 50 microg, plus titrated theophylline bid or salmeterol, 50 microg bid, were statistically significant (p < 0.05). CONCLUSION: These data show that both long-acting ss(2)-agonists and inhaled corticosteroids have a role in COPD. The data also show that fluticasone propionate and salmeterol given together are more effective than salmeterol alone. Moreover, it suggests that the addition of fluticasone propionate to salmeterol allows a greater improvement in lung function after salbutamol, although regular salmeterol is able to improve lung function in COPD patients without development of a true subsensitivity to its bronchodilator effect. In any case, patients must be treated for at least 3 months before a real improvement in lung function is achieved.

Intracellular mediators of programmed cell death initiated at the cell surface receptor Fas.

Apoptosis is a programmed cell death process, which plays a pivotal role in development, in tissue homeostasis and in several human diseases. Fas (CD95/Apo-1) is a member of the "death receptors" family, a group of cell surface proteins that trigger apoptosis upon binding with their natural ligands. In the immune system, intracellular signal transduction triggered from Fas splits into two different pathways. The proteolytic pathway is mediated by a family of cysteine proteases, the caspases, responsible for the morphological changes occurring in the apoptotic process. To complete this death program, another series of events, involving a lipid pathway, is necessary. Upon Fas stimulation, a sequential activation of specific enzymes results in the accumulation of ceramides and GD3 ganglioside. GD3 directly induces mitochondrial damage and triggers the release of apoptogenic factors, allowing efficient execution of Fas-mediated apoptosis.

GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion.

Lipid and glycolipid diffusible mediators are involved in the intracellular progression and amplification of apoptotic signals. GD3 ganglioside is rapidly synthesized from accumulated ceramide after the clustering of death-inducing receptors and triggers apoptosis. Here we show that GD3 induces dissipation of DeltaPsim and swelling of isolated mitochondria, which results in the mitochondrial release of cytochrome c, apoptosis inducing factor, and caspase 9. Soluble factors released from GD3-treated mitochondria are sufficient to trigger DNA fragmentation in isolated nuclei. All these effects can be blocked by cyclosporin A, suggesting that GD3 is acting at the level of the permeability transition pore complex. We found that endogenous GD3 accumulates within mitochondria of cells undergoing apoptosis after ceramide exposure. Accordingly, suppression of GD3 synthase (ST8) expression in intact cells substantially prevents ceramide-induced DeltaPsim dissipation, indicating that endogenously synthesized GD3 induces mitochondrial changes in vivo. Finally, enforced expression of bcl-2 significantly prevents GD3-induced mitochondrial changes, caspase 9 activation, and apoptosis. These results show that mitochondria are a key destination for apoptogenic GD3 ganglioside along the lipid pathway to programmed cell death and indicate that relevant GD3 targets are under bcl-2 control.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes.

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

Lipopolysaccharide induces jun N-terminal kinase activation in macrophages by a novel Cdc42/Rac-independent pathway involving sequential activation of protein kinase C zeta and phosphatidylcholine-dependent phospholipase C.

The activation of kinases of the mitogen-activated protein kinase superfamily initiated by lipopolysaccharide (LPS) plays an important role in transducing inflammatory signals. The pathway leading to the induction of stress-activated protein kinases in macrophages stimulated with LPS was investigated. The activation of Jun N-terminal kinases (JNK) by LPS is herbimycin sensitive. Using specific inhibitors, it was shown that the pathway involves the activation of phosphoinositide 3-kinase (PI 3-K). However, in contrast to previous reports, the small GTPases Cdc42 and Rac are not required downstream of PI 3-K for JNK activation. Instead, the phosphoinositides produced by PI 3-K stimulate protein kinase C (PKC) zeta activation through PDK1. In turn, activation of this atypical PKC leads to the stimulation of phosphatidylcholine phospholipase C (PC-PLC) and acidic sphingomyelinase (ASMase). It is therefore proposed that PKCzeta regulates the PC-PLC/ASMase pathway, and it is hypothesized that the resultant ceramide accumulation mediates the activation of the SEK/JNK module by LPS.

A double-blind, placebo-controlled comparison of treatment with fluticasone propionate and levocabastine in patients with seasonal allergic rhinitis. FLNCO2 Italian Study Group.

Fluticasone propionate aqueous nasal spray (FPANS) is a topically active glucocorticoid which has been successfully used for the treatment of seasonal allergic rhinitis (SAR). Topical levocabastine is a highly selective H1 antagonist which has been proposed as an alternative treatment of SAR. The purpose of this study was to compare the clinical efficacy of two topical nasal treatments, FPANS and levocabastine, in the treatment of SAR. Additionally, the effect of treatments on nasal inflammation was examined during natural pollen exposure. A group of 288 adolescent and adult patients with at least a 2-year history of SAR to seasonal pollens participated in a multicenter, doubleblind, double-dummy, and placebo-controlled study. Patients were treated with either FPANS 200 microg, once daily (n = 97), or topical levocabastine, 200 microg, given twice daily (n = 96), or matched placebo (n = 95) for a period of 6 weeks, starting from the expected beginning of the pollen season. Clinically relevant pollens included Parietaria, olive, and grass. Assessment of efficacy was based on scores of daily nasal symptoms and on nasal cytology of nasal lavage. Nasal lavage was performed immediately before, during, and at the end of treatment in 39 patients. FPANS significantly increased the percentage of symptom-free days for nasal obstruction on waking and during the day, rhinorrhea, sneezing, and itching. FPANS provided a better control for night and day nasal obstruction (P<0.02 and P<0.01) and rhinorrhea (P<0.01) than levocabas tine. In addition, fewer patients treated with FPANS used rescue medication (P<0.025). The percentage of eosinophils in nasal lavage was reduced only during treatment with FPANS. The results of this study indicate that FPANS 200 microg, once daily, provides a better clinical effect than levocabastine 200 microg, twice daily, in patients with SAR. Unlike levocabastine, FPANS significantly attenuates nasal eosinophilia during pollen exposure, a feature which may explain its therapeutic efficacy.

Differential activity of glycosaminoglycans on colony-forming cells from cord blood. Preliminary results.

Heparin, heparan sulfate and chondroitin sulfate were evaluated for their possible role on proliferation and differentiation of hematological precursor cells from cord blood. For these purposes, different concentrations of glycosaminoglycans were added to methyl-cellulose in colony assay performed with human cord blood derived cells. A volume of 10 microg/ml heparin induces a significant increase of both granulocyte-monocyte and granulocyte colonies, and a decrease of erythroid-colonies, more evident in the presence of 100 microg/ml. Heparan sulfate-treatment induces a significant increase of all granulocyte-monocyte colonies derived from CFU-granulocyte-monocyte, CFU-granulocyte and CFU-monocyte precursors. A significant decrease of multipotent cells was also observed. On the other hand, chondroitin sulfate induces an increase of granulocyte-colonies and a decrease of erythroid-colonies. Glycosaminoglycans with different structure may be useful to increase the number of specific colonies. The selective and differential binding of glycosaminoglycans with several growth factors and the regulation of their activities is discussed.

Distinct mechanisms target stress and extracellular signal-activated kinase 1 and Jun N-terminal kinase during infection of macrophages with Salmonella.

The interaction between bacteria and macrophages is central to the outcome of Salmonella infections. Salmonella can escape killing by these phagocytes and survive and multiply within them, giving rise to chronic infections. Cytokines produced by infected macrophages are involved in the early gastrointestinal pathology of the infection as well as in the induction and maintenance of the immune response against the invaders. Jun N-terminal kinases (JNK) are activated by inflammatory stimuli and play a role in cytokine production. We have investigated the signaling routes leading to JNK activation in Salmonella-infected macrophages and have discovered that they differ radically from the mechanisms operating in epithelial cells. In particular, activation of the JNK kinase stress and extracellular-activated kinase 1 (SEK1) and of JNK in macrophages occurs independently of actin rearrangements and of the GTPases Cdc42 and Rac, essential mediators in other cells. Activation of JNK is effected by a novel pathway comprising tyrosine kinase(s), phosphoinositide 3-kinase and, likely, atypical protein kinase C zeta. SEK1 is stimulated by a distinct mechanism involving phosphatidylcholine-phospholipase C and acidic sphingomyelinase. Dominant-negative SEK1 can block JNK activation by LPS, but not by Salmonella. These data demonstrate that SEK1 and JNK are activated independently in Salmonella-infected macrophages and offer experimental support for the concept that incoming signals can direct the selective coupling of downstream pathways to elicit highly specific responses. Inhibitors of stress kinase pathways are receiving increasing attention as potential anti-inflammatory drugs. The precise reconstruction of stimulus-specific pathways will be instrumental in predicting/evaluating the effects of the inhibitors on a given pathological condition.