Co-expression of cancer testis antigens and topoisomerase 2-alpha in triple negative breast carcinomas

Triple negative breast cancers (TNBC) are characterized by aggressive tumor biology, lack of targeted treatments and poor prognosis. Anthracyclins were shown to induce immunogenic death in target cells, potentially leading to “endogenous” vaccination. We comparatively assessed expression of cancer testis antigens (CTA) and topoisomerase 2-alpha (TOPO2A), a well defined molecular target of anthracyclins, in TNBC fully characterized for basal-like (BL) immunophenotype, BL morphology and conventional clinicopathological factors. The study included 83 patients undergoing surgery between January 2003 and December 2009. Tissue sections were stained with CK5/6, CK14, EGFR, Ki-67, TOPO2A, MAGE-A1, MAGE-A10, NY-ESO and multi-MAGE-A specific reagents. Of the 83 TNBC, >66.3% had BL immunophenotype and 48.2% had BL morphology. MAGE-A1 specific staining was most frequently detectable (69.2%), followed by multi-MAGE-A (58%), NY-ESO (27.1%) and MAGE-A10 (16%) specific staining. MAGE-A10 expression significantly correlated with tumor size (p = 0.026). Furthermore, MAGE-A1, MAGE-A10 and multi-MAGE-A specific stainings significantly correlated with advanced clinical stage (p = 0.024, p = 0.041, p = 0.031, respectively). We found no significant association between CTA expression and disease free (DFS) or overall survival (OS). Most interestingly, a significant correlation was observed between expression of MAGE-A10 and NY-ESO and expression of TOPO2A (p = 0.005, p = 0.013). Expression of defined CTA and TOPO2A are significantly correlated in TNBC. Considering the limited therapeutic options for TNBC, these findings might suggest novel forms of combination therapies that should be further explored.     

Evaluation of Primary Stability of Self‐Tapping and Non‐Self‐Tapping Dental Implants. A 12‐Week Clinical Study

Purpose: The aim of this study was to investigate the relationship between surgical techniques and implant macro‐design (self‐tapping/non‐self‐tapping) for the optimization of implant stability in the low‐density bone present in the posterior maxilla using resonance frequency analysis (RFA). Materials and Methods: A total of 102 implants were studied. Fifty‐six self‐tapping BlueSkyBredent® (Bredent GmbH&Co.Kg®, Senden, Germany) and 56 non‐self‐tapping Standard Plus Straumann® (Institut Straumann AG®, Waldenburg, Switzerland) were placed in the posterior segment of the maxilla. Implants of both types were placed in sites prepared with either lateral bone‐condensing or with bone‐drilling techniques. Implant stability measurements were performed using RFA immediately after implant placement and weekly during a 12‐week follow‐up period. Results: Both types of implants placed after bone condensing achieved significantly higher stability immediately after surgery, as well as during the entire 12‐week observation period compared with those placed following bone drilling. After bone condensation, there were no significant differences in primary stability or in implant stability after the first week between both implant types. From 2 to 12 postoperative weeks, significantly higher stability was shown by self‐tapping implants. After bone drilling, self‐tapping implants achieved significantly higher stability than non‐self‐tapping implants during the entire follow‐up period. Conclusions: The outcomes of the present study indicate that bone drilling is not an effective technique for improving implant stability and, following this technique, the use of self‐tapping implants is highly recommended. Implant stability optimization in the soft bone can be achieved by lateral bone‐condensing technique, regardless of implant macro‐design.     

Comparison of Pulmonary Inflammatory and Antioxidant Responses to Intranasal Live and Heat-Killed Streptococcus pneumoniae in Mice

Inflammatory and antioxidant responses, in male C57Bl6J mice, to single intranasal inoculations with live or heat-killed Streptococcus pneumoniae were studied in order to tease out differences in responses. Heat-killed bacteria elicited weak lung neutrophil infiltration and raised concentrations (peak 6–8 h), in serum or lung tissue, of CXCL1 and 2, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and granulocyte-macrophage-colony stimulating factor, with later increases in CCL2 and IL-1β. Live bacteria induced profound pulmonary neutrophil infiltration and acute chemokine/cytokine elevations. After 72–96 h, live S. pneumoniae induced a delayed rise in chemokines CXCL2 and CCL2, preceded by increases in TNFα, IL-1β, and IL-6 and mononuclear infiltration of lungs. With both live and heat-killed bacteria, alveolar epithelial type II cells and alveolar macrophages were the main sources of TNFα and IL-1β. Only live bacteria caused an acute decrease in lung glutathione peroxidase, an increase in superoxide dismutase, and a sustained increase in serum amyloid protein A. Acute innate immune responses to live and heat-killed S. pneumoniae are similar. In response to live bacteria, inflammation is greater, accompanied by changes in antioxidant enzymes and has an additional, later mononuclear component.     

Human Papillomavirus Genotype Specificity of Hybrid Capture 2 Low-Risk Probe Cocktail

A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.Low-risk alpha-human papillomaviruses (HPV) have never been at the fore in HPV research, due to their connection with benign neoplasm only. However, interest in these genotypes has increased substantially in recent years, due to the fact that quadrivalent HPV vaccine contains, in addition to a “cervical cancer component” (against HPV16 and HPV18), virus-like particles of the two most important low-risk alpha-HPV genotypes: HPV6 and HPV11. These two closely related HPV genotypes are the etiological agents of at least 90% of genital warts and laryngeal papillomas (1, 8, 10, 20) and at least 12.1% of cervical low-risk squamous intraepithelial lesions (5). In phase III clinical trials, this quadrivalent prophylactic HPV vaccine was shown to be highly effective against genital warts, e.g., reducing the burden of genital warts by 99% (95% confidence interval = 93.7 to 100%) among HPV-naïve vaccinated women aged 16 to 26 years (11). The quadrivalent HPV vaccine is currently licensed in more than 105 countries and has already been included in national vaccination programs in several countries. The widespread use of this vaccine has created an immediate need for a very specific detection tool for low-risk alpha-HPV.The Hybrid Capture 2 HPV DNA test (hc2), originally developed by Digene Corporation (Gaithersburg, MD) and currently marketed by Qiagen (Hilden, Germany), is the most widely used molecular method for the detection of a subset of clinically important HPV genotypes (14-16, 18). In this assay, exfoliated cells are first treated with alkali-denaturing reagent, and the processed samples are hybridized under high-stringency conditions with two mixtures of unlabeled full-genomic-length RNA probes, one specific for 13 high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68) and one for 5 low-risk HPV genotypes (HPV6, HPV11, HPV42, HPV43, and HPV44). Positive specimens are detected by binding the hybridization complexes onto the surface of a microplate well coated with monoclonal antibodies specific to RNA-DNA hybrids. Immobilized hybrids are detected by the addition of an alkaline phosphatase-conjugated antibody to RNA-DNA hybrids, followed by the addition of a chemiluminiscent substrate. The emission of light is measured as relative light units (RLU) in a luminometer. Thus, hc2 does not allow the exact determination of HPV genotype(s) present in a clinical specimen but rather expresses the results of tested high-risk or low-risk HPV genotypes as positive or negative. The hc2 high-risk cocktail is very reliable for the routine detection of clinically important HPV infection and is, at present, the only commercially available HPV DNA assay with sufficient scientific data to support its performance in a clinical setting. However, several studies have shown significant analytical inaccuracy of the high-risk cocktail, mainly due to cross-reactivity with several untargeted HPV genotypes (2-4, 6, 9, 16, 19, 21-26, 27). This phenomenon certainly reduces the analytical specificity of the high-risk cocktail, but cross-reactivity with some HPV genotypes has proven to be clinically beneficial (4, 19).The U.S. version of hc2, containing the high-risk probe cocktail only, is approved by the U.S. Food and Drug Administration (FDA) for triage (in cases of equivocal cytology results showing the presence of atypical squamous cells of undetermined significance) to determine which patients should be referred to physicians for a colposcopy and as a screening test for use in addition to cytology screening for women 30 years of age and older (15). Although the use of the hc2 low-risk probe cocktail is not recommended in the U.S. due to lack of FDA approval, the “Conformité Européene” (CE)-certified version of hc2, containing both high-risk and low-risk probe cocktails, is currently used in at least 40% of laboratories outside the U.S., mainly for individuals with clinically suspected low-risk HPV infection or as a reflex test for women with atypical squamous cells of undetermined significance who tested negative for high-risk HPVs. In contrast to the established cross-reactivity of the high-risk probe cocktail with several untargeted HPV genotypes, the specificity of the hc2 low-risk cocktail has never been studied in detail. According to the data presented in the hc2 package insert, the only recognized cross-reactivity of the hc2 low-risk cocktail is with HPV13, a genotype commonly detected in lip lesions of certain ethnic groups but never in anogenital lesions (17). In the present study, therefore, we have for the first time systematically examined the analytical specificity of the hc2 low-risk cocktail by determining the exact HPV genotype(s) present in 285 consecutive samples recognized using the hc2 low-risk probe cocktail as HPV DNA positive.To determine the specificity and accuracy of hc2 in the detection of the five HPV genotypes (HPV6, HPV11, HPV42, HPV43, and HPV44) included in the low-risk probe cocktail, 285 consecutive cervical specimens obtained from the same number of women and recognized as HPV positive using the hc2 low-risk probe cocktail were included in the study. Fifty-six out of 285 samples were positive using both low-risk and high-risk probe cocktails. The specimens included in the study were collected between June 2007 and May 2008, using a DNAPaP cervical sampler and specimen transport medium (STM; Digene Corporation, Gaithersburg, MD), from Croatian and Slovenian women undergoing routine gynecological examination. hc2 testing was performed not later than 5 days after the collection of specimens, strictly following the manufacturer''s instructions. According to the manufacturer''s interpretation criteria, the specimens with a RLU-per-cutoff (RLU/CO) value higher than 1.0 were considered positive for one or more low-risk HPV genotypes included in the cocktail, and the specimens with a RLU/CO value of <1.0 were considered negative for the five low-risk HPV genotypes tested.The presence of the five targeted low-risk genotypes, as well as other, untargeted low-risk and high-risk HPV genotypes, in the hc2 low-risk cocktail-positive samples was determined by using seven different PCR-based genotyping methods. To keep the number of tests to a minimum, the seven methods were used consecutively, e.g., if the first genotyping method reliably detected at least one of the five targeted HPV genotypes in the particular hc2 low-risk cocktail-positive sample, this sample was excluded from further testing. All seven genotyping tests were performed on the same sample used for hc2 testing, i.e., 500 μl of STM was removed before the addition of hc2 denaturing reagent solution (first step of hc2 testing) and kept at −70°C until genotyping. After the isolation of DNA from 200 μl of nondenatured STM using a QIAamp DNA mini kit (Qiagen, Hilden, Germany), all 285 samples were first tested using a recently developed real-time PCR (RT-PCR) assay that allows very specific detection and reliable differentiation of HPV6 and HPV11 (13). The sensitivity of the test, assessed by probit analysis at a 95% detection level, is 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and nonprototypic HPV6 variants and HPV11, respectively (13). Sixty-six samples were found to be positive for HPV6, and 6 samples contained HPV11 (Table ​(Table1).1). The remaining 213 samples were then tested using HPV42 genotype-specific RT-PCR, and 105 were found to contain this low-risk HPV. The remaining 108 samples were then tested using HPV43 genotype-specific RT-PCR, followed by testing of all negative samples using HPV44/55 genotype-specific RT-PCR. By using the two RT-PCR tests, the presence of HPV43 and HPV44/55 was identified in 8 and 17 samples, respectively (Table ​(Table1).1). The HPV42, HPV43, and HPV44/55 genotype-specific primers and probes were designed according to the L1 long control region genomic sequences of the prototype isolates (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"M73236","term_id":"333211","term_text":"M73236"}}M73236, {"type":"entrez-nucleotide","attrs":{"text":"AJ620205","term_id":"40804474","term_text":"AJ620205"}}AJ620205, {"type":"entrez-nucleotide","attrs":{"text":"U31788","term_id":"1020242","term_text":"U31788"}}U31788, and {"type":"entrez-nucleotide","attrs":{"text":"U31791","term_id":"1020266","term_text":"U31791"}}U31791, respectively). As shown in Table ​Table2,2, two primers and two hybridization (fluorescent resonance energy transfer) probes were selected for each HPV genotype studied. A common probe was designed for HPV44 and HPV55, which has recently been recognized as a subtype of HPV44 and is no longer considered a separate genotype (7). A BLAST search of the GenBank nr database was performed for each sequence in order to verify HPV genotype specificity. The assays were set up on a LightCycler 2.0 real-time system (Roche Diagnostics GmbH, Mannheim, Germany) and performed using a QuantiTect probe PCR kit (Qiagen, Hilden, Germany) according to the manufacturer''s instructions.

TABLE 1.

Summary of genotyping results of 285 consecutive samples recognized as HPV DNA positive using the hc2 low-risk probe cocktail

Spatial and temporal distribution of Ki-67 proliferation marker, Bcl-2 and Bax proteins in the developing human tooth

Objective

To investigate the spatial and temporal expression of proliferation Ki-67 marker, pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins during early development of the human tooth.

Materials and methods

Histological sections of eight human conceptuses, 5–10 postovulatory weeks old, were used for immunolocalization for Ki-67, Bax and Bcl-2 markers. Quantification was performed by calculating the fraction of Ki-67 positive cells, expressed as a mean ± SD, and analysed by Mann–Whitney test, Kruskal–Wallis and Dunn's post hoc test.

Results

In 6th–7th developmental weeks, the tooth germ and dental crest contained 37% of proliferating cells, which increased to 40% in the 8th week, and then decreased to 15% in the 10th week, whilst the proliferation in the ectomesenchyme subsequently dropped from 37% to 23%. Epithelial parts of the enamel organ displayed similar proliferation activity (31–36%), dental crest 10%, whilst enamel knot showed no proliferating activity. The tooth ectomesenchyme contained more proliferating cells (50%) than the jaw ectomesenchyme (35%), and both dropped to 28% in the 10th week. Ectomesenchyme between the tooth germs contained 23%, whilst the jaw ectomesenchyme contained 15% of proliferating cells. Bcl-2 expression had following pattern: strong in proliferating cells, moderate in tooth germs and dental crest, and weak in the ectomesenchyme. Bax co-expressed with Bcl-2 in the tooth germ and dental crest. In the reticulum and inner enamel epithelium Bcl-2 had prevalent expression, whilst Bax prevailed in the outer enamel epithelium and tooth ectomesenchyme.

Conclusions

Proliferating cells most likely influence growth of the tooth germ, Bcl-2 affects proliferation and differentiation of specific cell lineages, whilst Bax influences process of cell death.     

Levels of Organochlorine Compounds in the Mediterranean Blue Mussel from the Adriatic Sea

The distribution of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) was investigated in Mediterranean blue mussel collected at four locations in Mali Ston Bay few times a year in 2005–2007. OCPs were found in all samples and levels ranged between 0.07 and 7.58 ng g⁻¹ dry wt. Levels of PCBs ranged between 0 (below detection limit) and 21.55 ng g⁻¹ dry wt. For most analyzed compounds there were no significant level changes between the 3 years. Exceptions are decreased levels of β-HCH, DDD, and PCB-138 and increased levels of γ-HCH and DDT in 2007. However, mussels from this area are applicable for human diet.     

Refractive Changes Associated with Diabetes Mellitus

Background and methods: Diabetes mellitus is the most prevalent endocrine disease in developed countries. In people with diabetes in addition to vision loss caused by diabetic retinopathy transient visual disturbances may occur frequently caused by refractive changes. These changes in refraction are associated with variations in blood glucose levels but the underlying mechanism is still not fully understood. A systematic review with a comprehensive literature search was performed in order to clarify the underlying mechanisms regarding the connection of glycaemic control and refractive shift.

Results: Some studies have shown that increased blood sugar leads to a myopic shift whilst others demonstrated that this change is in a hyperopic direction. Changes in visual acuity in patients with diabetes could be an indicator of inadequate metabolic control or even the first sign of diabetes mellitus.

Conclusion: This review gives a brief overview of current research regarding potential mechanisms of glycemic control influence on refractive error. The aim is to emphasize the importance of understanding the relationship of blood glucose concentration and refractive changes as one of the common but overlooked diabetic complications.     

Transplacental genotoxicity of antiepileptic drugs: animal model and pilot study on mother/newborn cohort

Antiepileptic drugs (AED) as transplacental agents are known to have adverse effects on fetal development. Genotoxicity of AEDs is still not fully understood. The aim of present study was to investigate the transplacental genotoxicity of valproate on animal model and in 21 mothers and their newborns receiving AED. In both studies, in vivo micronucleus (MN) assay was used. Pregnant dams were exposed to Na-valproate (100mg/kg) on gestational days 12-14. Dams and pups receiving Na-valproate showed a significantly increased MN frequency (5.17 ± 1.17/1000; 5.20 ± 1.48/1000) compared to the control (1.0 ± 0.58/1000; 1.67 ± 1.03/1000). In mother/newborn study a significant increase of MN frequency was detected in newborns of mothers taking AEDs (3.09 ± 0.49/10,000) compared to the referent newborns (1.56 ± 0.22/10,000). The results of this study suggest that AEDs may act as transplacental genotoxins. Launching the mother/newborn cohorts for genotoxicological monitoring may give a significant new insight in health effects of AEDs.     

Topical Azithromycin and Clarithromycin Inhibit Acute and Chronic Skin Inflammation in Sensitized Mice, with Apparent Selectivity for Th2-Mediated Processes in Delayed-Type Hypersensitivity

Macrolide antibiotics inhibit the secretion of Th1 cytokines while their effects on the release of Th2 cytokines are variable. We investigated molecular and cellular markers of Th1- and Th2-mediated inflammatory mechanisms and the anti-inflammatory activity of azithromycin and clarithromycin in phorbol 12-myristate 13-acetate (PMA) and oxazolone (OXA)-induced skin inflammation. Dexamethasone (50 μg/ear), azithromycin, and clarithromycin (500 μg/ear) reduced TNF-α and interleukin (IL)-1β concentration in ear tissue by inhibiting inflammatory cell accumulation in PMA-induced inflammation. In OXA-induced early delayed-type hypersensitivity (DTH), the macrolides (2 mg/ear) and dexamethasone (25 μg/ear) reduced ear tissue inflammatory cell infiltration and secretion of IL-4 while clarithromycin also decreased IFN-γ concentration. Macrolides showed better activity when administered after the challenge. In OXA-induced chronic DTH, azithromycin (1 mg/ear) reduced the number of ear tissue mast cells and decreased the concentration of IL-4 in ear tissue and of immunoglobulin (Ig)E in serum. Clarithromycin (1 mg/ear) reduced serum IgE concentration, possibly by a mechanism independent of IL-4, while both macrolides attenuated mast cell degranulation. In conclusion, azithromycin and clarithromycin attenuate pro-inflammatory cytokine production and leukocyte infiltration during innate immune reactions, while selectively affecting Th2 rather than Th1 immunity in DTH reactions.     

Chemical methods for determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in biological material

This review presents methods for the analysis of hydroxylated metabolites of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in humans and animals. These metabolites serve as biomarkers of human and animal exposure to the mentioned pollutants, but some metabolites also have toxic properties. Most are analysed in urine, which is the most accessible sample, but they can also be analysed in human and animal liver, bile, and adipose tissue.Their analysis is important for assessing bioavailability of aromatic pollutants and their toxicity in human organism, but also the toxicity of metabolites themselves. Advancements in analytical methods have made it possible to analyse multiple metabolites in a sample at the same time. New extraction techniques and more precise and selective qualitative and quantitative analyses can now detect very low metabolite concentrations. An extra advantage is that these simple techniques require less chemicals and time.