Molecular Biomarkers & Diagnosis

Lead (Pb) is one of the most important environmental pollutant metals accumulating in the atmosphere, water, foods, and in organisms living in contaminated areas. Skin is one of the main targets of Pb toxicity based on its ability of direct penetration upon exposure. The underlying cell damaging pathomechanisms have not been revealed in detail. Herein, we focus on Pb-induced oxidative and nitrosative stress that has not been previously thoroughly investigated. We investigated these effects in order to elucidate the pathomechanisms and as well to identify potential biological markers that may indicate Pb toxicity. Human immortalized keratinocytes (HaCaT cells) were exposed to Pb (100 μM) either for 5 minutes or 6 hours. Pb-induced cellular damage was evaluated by immunocytochemistry analysis of multiple signalling cascades, e.g. apoptosis, Akt, MAPK, NOS, nitrotyrosine and 8-isoprostane formation, detection of nitrosative stress using Diaminofluorescein (DAF-FM) and oxidative stress using 3'-(p aminophenyl) fluorescein (APF). We found that Pb exposure resulted in significantly enhanced NO and ROS production in HaCaT cells. Pb led to enhanced eNOS-phosphorylation at Ser1177, and Ser116 residues but not Thr495. AKT phosphorylation but not MAP kinases were enhanced by Pb In addition, Pb induced apoptosis as shown by Caspase-3 activation and PARP cleavage. Our results suggest that Pb mediates its toxic effect in keratinocytes through oxidative and nitrosative stress which is accompanied by differential changes of eNOS phosphorylation and apoptosis. These data significantly contribute to understanding of underlying mechanisms of Pb-induced cellular damage.

Background: The development of senile cataract is a multifactorial process with oxidative stress and its sequelae are clearly involved in its etiology. Cataract is also one of the earliest secondary complications of diabetes mellitus. Paraoxonase (PON) enzyme is an antioxidant High-Density Lipoprotein (HDL)-associated enzyme. In mammals, three genes of paraoxonase, PON1, PON2, and PON3, have been identified.

Aim: To assess the contribution of PON1-55 and PON1-192 gene polymorphisms as risk factors for senile cataract formation among diabeticand non-diabetic Egyptian patients.

Methods: 132 Egyptian cataract patients (66 without diabetes and 66 with diabetes) and 106 subjects with matched age and sex free of cataract and diabetes control subjects were included in the present study using multiplex PCR for PON1-55 and PON1-192 gene polymorphisms followed by restriction fragment length polymorphism analysis.

Results: The study revealed that there was a significant difference in PON1-55 genotypes; LL, LM and MM (p=0.0001) and in PON1-192 genotypes; QQ, QR and RR (p=0.0001) genotypes distribution among cataract patients with and without diabetes and controls. Also there was a significant difference in L and M (p=0.003) and in Q and R allele frequencies (p=0.005) among cataract patients with and without diabetes and controls. In addition there was a significant difference in the distribution of 55 LM/192 RR combined genotypes with the highest frequency in cataract diabetic subgroup (75%), while 55LL/192RR, 55LL/192QR and 55LM/192QR combined genotypes showed the highest frequencies among the control group (52.4%, 59.1% and 66.7% respectively).

Conclusion: For the first time, we provide evidence that functional polymorphisms in the PON1 gene may influence the risk of cataract in both non-diabetic and diabetic subgroups in Egyptian populations, suggesting new clues that help to clarify the pathogenesis of cataract.

Langerhans Cell Sarcoma (LCS) is extremely rare, with only 37 cases reported in English literature. We present the case of a 38 year old woman with a 2 month history of left neck swelling and pain. A diagnosis of LCS was made based on pathological findings of the biopsy of the tonsil. IHC on the neoplastic cells was positive for LCA, S100, CD 68 and CD1a (with membranous and paranuclear dot like patterns). A literature review is also presented to enhance the understanding of LCS and the importance of early diagnosis and treatment of this unusual lesion.

Background: In large epidemiological studies it is important to test the stability of biomarkers as a function of both temperature and duration of storage. In this study the stability of seven lipid parameters have been tested in human serum samples after storage at three different temperatures up to 1 year.

Methods: Serum samples of 16 human individuals were used in this study. The concentration of all parameters have been determined at T=0 and at several time points up to 1 year after storage at –20°C, –70°C and –196°C.

Results: Most of the lipid biomarkers, cholesterol, triglycerides HDL- and LDL cholesterol, apolipoprotein-A1 and –B are stable on long-term storage for one year at the three temperatures tested. The levels of both HDL- and LDL cholesterol showed a small decrease for samples stored at -20°C only. The free fatty acids, however, are not stable and showed a decrease to about 80% of the starting value. The rank order between the samples, however, remained very good.

Conclusions: This study shows that free fatty acids are not stable in human serum samples probably due to thawing and freezing effects, although the rank order and correlation between the samples from different time points remained the same. HDL- and LDL-cholesterol showed a very small deviation on storage at -20°C. The other lipid parameters remained perfectly stable in this study. No differences were observed between storage at -70°C or -196°C, Therefore it is advised to store serum samples at -70°C for longer periods.

To investigate the effect of blocking agent of heme oxygenase-1 zinc protoporphyrin (Znpp) on lipopolysaccharide (LPS)-induced autophagy in acute lung dysfunction, the rats were divided into control (C), LPS (L), LPS +Hemin (Hemin) and LPS+ZnPP (ZnPP) groups. Treatment with ZnPP induced autophagy and accelerated oxidative damages during lipopolysaccharide treatment in rat lung, LPS+ZnPP increased pathological alterations in lung tissues, the number of ballooned pulmonarycytes, serum tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels, and myeloperoxidase (MPO) and malondialdehyde (MDA) levels in lung tissues (P < 0.05) but attenuated by LPS +Hemin treatment. Thus, ZnPP may aggravate LPS-induced acute lung dysfunction in rats, possibly by increasing inflammation and accelerating oxidative damages. LPS+Hemin group prolonged the median survival time and reduced lung dysfunction. Moreover, HO-1 may significantly contribute to lung protection.

Over 15,000 women die from ovarian cancer and there are approximately 23,000 new cases diagnosed each year. Platinum-based chemotherapy is still the primary treatment for ovarian cancer. Most patients with the disease are initially responsive to chemotherapeutic treatment. However, a majority of ovarian cancer patients eventually relapse and become refractory to additional treatment. This drug-resistance is a major impediment to the successful treatment of ovarian cancer. To date the mechanisms of drug-resistance remain poorly understood. Previous studies have suggested that many proteins, such as BRCA1, BRCA2, MDR1, MRP1, MDM2, hMLH1, HSP27, and HSP70, are differentially expressed in drug-resistant ovarian tumor cells by mRNA differential display analysis. However, biomarkers that can be used to differentiate chemotherapy responders from non-responders have not yet been developed. With recent developments in proteomic technologies, differential protein expression in complex biological samples can be analyzed. In this cell model based study, we applied a label-free protein quantification technology to discover potential protein biomarker candidates that can differentiate chemo-drug responders from non-responders. This experimental approach could also serve as a model tool for further clinical validation and biomarker development for other diseases.