Real-Time PCR Method as Diagnostic Tool for Detection of Periodontal Pathogens in Patients with Periodontitis.

The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent® semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for A. actinomycetemcomitans was 97.5%, for P. gingivalis 96.88%, and for P. intermedia 95.24%. The sensitivity for Tannerella forsythia and T. denticola was 100%. The Spearman correlation factor of two different measurements was 0.976 for A. actinomycetemcomitans, 0.967 for P. gingivalis, 0.949 for P. intermedia, 0.966 for Tannerella forsythia, and 0.917 for T. denticola. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.

Matrix metalloproteinases MMP-1, MMP-2, and MMP-13 are overexpressed in primary nodular melanoma.

BACKGROUND: The spread and invasion of malignant melanoma cells involve degradation and reorganization of the extracellular matrix by the activation of several matrix metalloproteinases (MMPs). This study analyzed the expression of MMP-1, MMP-2, and MMP-13 proteins in primary nodular melanoma (NM) and dysplastic nevi (DN) as a significant risk factor for melanoma development. The secondary goal was to analyze the correlation of MMPs protein expression in NM with tumor invasion, BRAF V600 mutation status, and overall survival. METHODS: Immunohistochemistry for MMP-1, MMP-2, and MMP-13 was performed on nodular melanoma (n = 52) and dysplastic nevi (n = 28) on tissue microarray (TMA). BRAF V600 mutation analysis on NM samples was performed by the Sanger sequencing method. RESULTS: A high level of MMPs expression in NM samples (>30%) compared with DN (<8%) was statistically significant (P < 0.001). BRAF V600 mutations were detected in 15 of 39 (38.5%) NM samples. This study revealed an interesting finding that MMP-1 and MMP-13 protein expression in the BRAF V600 mutated melanomas were significantly lower than in the BRAF V600 wild type (P < 0.05). CONCLUSION: Cox analysis revealed that Clark categories, Breslow thickness, and MMP-1 high protein expression are predictive factors for shorter overall survival (P < 0.05).

Genetic heritage of Croatians in the Southeastern European gene pool-Y chromosome analysis of the Croatian continental and Island population.

OBJECTIVES: The research objective of this study is to enlarge and deepen the Y chromosome research on the Croatian population and enable additional insights into the population diversity and historic events that shaped the current genetic landscape of Croatia and Southeastern Europe (SEE). MATERIALS AND METHODS: A high-resolution phylogenetic and phylogeographic analysis of 66 biallelic (SNPs) and 17 microsatellite (STRs) markers of the Y chromosome was performed using 720 Croatian samples. The obtained results were placed in a wider European context by comparison with ∼4450 samples from a number of other European populations. RESULTS: A high diversity of haplogroups was observed in the overall Croatian sample, and all typical European Y chromosome haplogroups with corresponding clinal patterns were observed. Three distinct genetic signals were identifiable in the Croatian paternal gene pool - I2a1b-M423, R1a1a1b1a*-M558, and E1b1b1a1b1a-V13 haplogroups. DISCUSSION: The analyses of the dominant and autochthonous I2a1b-M423 lineage (>30%) suggest that SEE had a significant role in the Upper Paleolithic, the R1a1a1b1a*-M558 lineage (19%) represents a signal from present day Slavic populations of Central Europe in the Croatian population, and the phylogeography of the E1b1b1a1b1a-V13 clade (around 9%) implies cultural diffusion of agriculture into Europe via the Balkan Peninsula. Am. J. Hum. Biol., 2016. © 2016 Wiley Periodicals, Inc. Am. J. Hum. Biol. 28:837-845, 2016. © 2016Wiley Periodicals, Inc.

Osteopontin expression is an independent factor for poor survival in oral squamous cell carcinoma: a computer-assisted analysis on TMA sections.

INTRODUCTION: Osteopontin (OPN) is non-collagenous extracellular matrix protein involved in various physiological and pathological events, including tumor progression. The aim of this study was to analyze the expression of OPN in normal oral mucosa and oral squamous cell carcinoma (OSCC) and to assess its prognostic significance. METHODS: The expression of OPN was immunohistochemicaly analyzed in 86 OSCC and compared with clinicopathological variable such as tumor size, nodal stage, WHO clinical stage, Ki-67 proliferation index, and patients' outcome. OPN mRNA was analyzed using quantitative real-time PCR and compared with protein OPN expression and clinical outcome in 18 OSCC samples. RESULTS: The expression of OPN protein was found in OSCC tumor cells (t-OPN) and various stromal cells (s-OPN). High level of t-OPN expression was associated with higher nodal stage (P = 0.045), higher WHO clinical stage (P = 0.033), and poor clinical outcome (P = 0.022). In multivariate analysis, t-OPN emerged as an adverse independent factor for survival (P = 0.049). Although correlated with t-OPN (P = 0.005), s-OPN was not significantly associated with clinical parameters, including patients' outcome. Also, there was no association between OPN and clinical parameters at the mRNA level. CONCLUSION: OPN is upregulated in tumor and stromal OSCC cells. Tumor cell-derived OPN is involved in tumor progression and can independently predict the clinical outcome. Stromal-derived OPN probably has a different function compared with OPN secreted from tumor cells.

Inherited Thrombophilia and Risk of Thrombosis in Children with Cancer: a Single-center Experience.

OBJECTIVES: Thrombosis is an increasingly recognized complication of childhood malignancy and its treatment. The incidence and etiology of pediatric cancer-related thrombosis is still not well understood. The aim of this study was to evaluate the prevalence of common prothrombotic genetic conditions in children with cancer, the frequency of thrombosis, and the role of inherited thrombophilia in the development of thrombosis in a pediatric oncology population. PATIENTS AND METHODS: Forty-seven children (36 treated for hematological malignancies and 11 for solid tumors) with a median age of 8.8. years (range 0.4 - 19.3 years) were included in the study. Genetic polymorphisms of Factor V Leiden (G1691A), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were determined by real-time polymerase chain reaction-based DNA analysis. RESULTS: Four (8.5%) patients were heterozygous for Factor V Leiden, 3 (6.4%) were heterozygous for prothrombin G20210A mutation, and 3 (6.4%) were homozygous for MTHFR C677T mutation. All patients had implanted central venous catheters. Four (8.5%) children had documented thrombosis, three of which were in the upper venous system. Two of the four patients with thrombosis had Factor V Leiden heterozygosity. CONCLUSIONS: Thrombosis is an important complication of childhood cancer. The risk of thrombosis may be increased in patients with Factor V Leiden. In the absence of consensus guidelines, our results support the recommendation for thrombophilia screening in children with cancer.

Refining the Global Phylogeny of Mitochondrial N1a, X, and HV2 Haplogroups Based on Rare Mitogenomes from Croatian Isolates.

Mitochondrial DNA (mtDNA) has been used for decades as a predominant tool in population genetics and as a valuable addition to forensic genetic research, owing to its unique maternal inheritance pattern that enables the tracing of individuals along the maternal lineage across numerous generations. The dynamic interplay between evolutionary forces, primarily genetic drift, bottlenecks, and the founder effect, can exert significant influence on genetic profiles. Consequently, the Adriatic islands have accumulated a subset of lineages that exhibits remarkable absence or rarity within other European populations. This distinctive genetic composition underscores the islands' potential as a significant resource in phylogenetic research, with implications reaching beyond regional boundaries to contribute to a global understanding. In the initial attempt to expand the mitochondrial forensic database of the Croatian population with haplotypes from small isolated communities, we sequenced mitogenomes of rare haplogroups from different Croatian island and mainland populations using next-generation sequencing (NGS). In the next step and based on the obtained results, we refined the global phylogeny of haplogroup N1a, HV2, and X by analyzing rare haplotypes, which are absent from the current phylogenetic tree. The trees were based on 16 novel and 52 previously published samples, revealing completely novel branches in the X and HV2 haplogroups and a new European cluster in the ancestral N1a variant, previously believed to be an exclusively African-Asian haplogroup. The research emphasizes the importance of investigating geographically isolated populations and their unique characteristics within a global context.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

EGFR protein overexpression correlates with chromosome 7 polysomy and poor prognostic parameters in clear cell renal cell carcinoma.

BACKGROUND: The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival. METHODS: The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining. RESULTS: Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient's survival, while there was no connection with pathological stage. CONCLUSION: In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification.

Gene amplification of epidermal growth factor receptor in atypical glottic hyperplasia.

The study searched for epidermal growth factor receptor (EGFR) gene amplification in hyperplastic glottis lesions. After classical pathohistological findings of hematoxylin-eosin (HE) slides and quantitative immunohistochemical (IHC) analysis, fluorescent in situ hybridization (FISH) was used on tissue microarrays of laryngeal hyperplastic tissue ranging from normal mucosa to abnormal and atypical hyperplastic lesions. FISH analysis of two atypical hyperplastic lesions discovered the amplification of EGFR gene while it was not found in simple and abnormal hyperplastic lesions. The results may indicate that EGFR gene amplifications could possibly correlate with the histopathologic picture. Tissue samples burdened with specific oncogen signatures like EGFR gene amplification could be detected in precancerous lesion. This might improve follow-up and treatment protocols of glottic lesions which are an everyday problem for ENT practitioners. Further research is mandatory to confirm our findings.

JAK2-v617F mutation is associated with clinical and laboratory features of myeloproliferative neoplasms.

The aim of this study is to investigate the differences of clinical and laboratory parameters between patients with JAK2-V617F positive myeloproliferative neoplasms (MPNs) and JAK2 wild type MPNs. DNA was isolated from peripheral blood granulocytes of 106 patients treated at Rijeka University Hospital Center: 41 with polycythemia vera (PV), 43 with essential thrombocythemia (ET), 9 with primary myelofibrosis (PMF) and 13 with myeloproliferative neoplasm--unclassifiable (MPN-u). The JAK2-V617F mutation was detected using allele specific PCR. Laboratory and clinical parameters were obtained from patient's medical records. The JAK2-V617F mutation was detected in 69% (73/106) patients with MPNs. The results revealed significantly different prevalence of JAK2-V617F mutation, between MPNs entities: 88% in PV 58% in ET, 56% in PMF and 54% in MPNs-unclassified disorders. The JAK2-V617F mutation significantly correlated with higher leukocyte count and alkaline phosphatase co re in ET group and with higher platelets count, leukocyte alkaline phosphatase score and serum lactate dehydrogenase in PV group. Vascular events were associated with elevated platelets count in whole MPNs group, with higher platelets and leukocyte count in ET and with splenomegaly in PVpatients. Clinical and laboratory data revealed significant contribution ofJAK2-V617F mutation to the development of clinical phenotype in patients with distinct subgroups of MPNs.

PD-L1 expression is regulated by microphthalmia-associated transcription factor (MITF) in nodular melanoma.

Malignant melanoma (MM) is known to avoid the host's immune response. Studies on in vitro melanoma cell lines link the microphthalmia-associated transcription factor (MITF) with the regulation of the PD-L1 expression. It seems that MITF affects the activation of the gene responsible for PD-L1 protein expression. Several proteins, including Bcl-2 and Cyclin D1, play major roles in malignant melanoma cell cycle regulation and survival. Our study aims to assess the relationship between MITF, Bcl-2, and cyclin D1 protein expression and the expression of the PD-L1 molecule. Additionally, we examined the association of BRAF mutation, MITF, and CCND1 gene amplification with PD-L1 protein expression. We performed immunohistochemical staining on fifty-two tumour samples from patients diagnosed with nodular melanoma (NM). BRAF V600 mutation, MITF, and CCND1 gene amplification analyses were analyzed by the Sanger sequencing and QRT-PCR methods, respectively. Statistical analyses confirmed the significant inverse correlation between cyclin D1 and PD-L1 expression (p = 0.001) and correlation between PD-L1 and MITF protein expression (p = 0.023). We found a statistically significant inverse correlation between the present MITF gene amplification and PD-L1 (p = 0.007) and MITF protein expression (p = 3.4 ×10-6), respectively. Our study, performed on clinical NM materials, supports the in vitro study findings providing a rationale for the potential MITF-dependent regulation of PD-L1 expression in malignant melanoma.

mRNA expression of bone morphogenetic proteins and their receptors in human renal cell carcinoma.

INTRODUCTION: Bone morphogenetic proteins (BMPs) have been studied in several cancers, but only limited information is available about renal cell carcinomas (RCCs). We determined the expression of mRNA of several BMP ligands and BMP receptors (BMPRs) in healthy kidney tissue and RCCs, and data were compared to clinicopathological parameters. MATERIAL AND METHODS: Sixty-four samples of RCCs and healthy renal tissues were prospectively examined. The expression of BMP2, BMP4, BMP6, BMP7, BMPRIA, BMPRIB and BMPRII mRNA was determined using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The expression levels of different BMP ligands and BMPRs were considerably higher in RCCs than in normal kidney tissue. BMP ligands showed elevated expression in clear-cell RCCs, whereas all three BMPRs showed higher expression levels in non-clear-cell RCCs. In clear-cell RCCs, the expression levels of BMP2 progressively increased and expression levels of BMP6, BMP7 and BMPRIB were lost with higher tumor stage. CONCLUSIONS: All BMPs and their receptors have stronger expression levels in RCC. The expression level of BMP2 is strongly elevated in kidney cancer.

Genetic polymorphisms of 15 STR loci in the population of the island of Cres (Croatia).

BACKGROUND: The population of the island of Cres presents one of the few persisting Eastern Adriatic isolates and is thereby suitable for human population differentiation analyses. AIM: The aim of this study was to analyse the genetic structure of the island of Cres with respect to its eight sub-populations and to compare the genetic variation of the island of Cres with other Eastern Adriatic islands and the Croatian mainland. SUBJECTS AND METHODS: Fifteen AmpFlSTR identifiler loci were analysed in a sample group of 122 unrelated autochthonous individuals from the island of Cres, Croatia. RESULTS: Analysis of STR polymorphisms revealed genetic homogeneity among sub-populations of the island of Cres and small but significant levels of genetic heterogeneity among geographically distant Eastern Adriatic islands. CONCLUSION: Despite a considerable degree of genetic homogeneity among the studied Eastern Adriatic islands, small but significant differentiation between distant islands indicates geographic sub-structuring which follows the isolation by distance model. This study is supportive of the notion that STR markers are useful for genetic differentiation between larger and geographically more distant regions.

Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis.

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).

Epidermal growth factor receptor protein expression and gene amplification in normal, hyperplastic, and cancerous glottic tissue: immunohistochemical and fluorescent in situ hybridization study on tissue microarrays.

AIM: To evaluate the importance of epidermal growth factor receptor (EGFR) protein overexpression and gene amplification in carcinogenesis of glottic cancer. METHOD: In order to evaluate EGFR expression at protein and gene level, immunohistochemical (IHC) analysis and fluorescent in situ hybridization (FISH) were performed on tissue microarrays of laryngeal tissue (145 samples) -- 38 samples of normal mucosa, 46 samples of hyperplastic lesions, and 61 samples of cancerous lesions. RESULTS: Membranous (mEGFR) and cytoplasmic (cEGFR) EGFR expression was significantly different between the analyzed groups. The differences were most striking in the suprabasal-transforming zone. IHC evaluation showed that high and low mEGFR staining contributed to the differentiation of dysplastic lesions, simple hyperplasia, and cancerous tissue, as well as between different degrees of atypia in hyperplastic lesions (P<0.050). EGFR gene amplification was not found in simple and abnormal hyperplastic lesions, but it was confirmed in 2/21 atypical hyperplasias, indicating that gene amplification can facilitate identification of malignant potential in hyperplastic lesions. In cancerous tissue, EGFR gene amplification was found in 8/50 samples. EGFR gene amplification was found in preinvasive cancer in one patient. In invasive carcinomas, gene amplification was not associated with stage or grade. Carcinomas with gene amplification showed significantly higher cEGFR expression (basal layer P=0.003; suprabasal layer P=0.002). CONCLUSIONS: This study confirmed an increase in EGFR protein expression and gene amplification with the increase in biological aggressiveness of glottic lesions. A correlation between EGFR gene amplification and protein expression was established. Gene amplification proved to be an early event in glottic carcinogenesis, indicating its importance for glottic cancer prevention, early detection, and protocol selection.

Forensic efficiency parameters for the 15 STR loci in the population of the island of Cres (Croatia).

Forensic parameters based on 15 AmpFISTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818 and FGA) were evaluated in the sample of 122 unrelated, autochthonous, adult individuals from the Island of Cres (Croatia). PCR amplification was performed with the AmpFISTR Identifiler PCR Amplification Kit and the amplified products were separated and detected using the ABI 3130 DNA genetic analyzer. The agreement with Hardy Weinberg Equilibrium (HWE) was confirmed for all loci (p > 0.05). The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 15 tested STR loci were 0.99999999999999997988728679 and 0.999997397, respectively. According to the presented data, D18S51 proved to be the most informative marker followed by markers D2S1338 and D21S11. Interpopulation comparisons in allele frequencies with other East Adriatic Islands revealed significant differences for all analyzed population pairs ranging from 4 loci (Cres vs. Hvar) to 1 locus (Cres vs. Krk). Furthermore, allele frequencies comparisons of Cres and Croatian mainland revealed the lack of statistically significant differences at all studied loci. The results of the current study indicate that the examined fifteen STR loci are useful genetic markers for individual identification and paternity testing in Croatian population from the Island of Cres.

Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation.

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.

Comparison of molecular assays for detection and typing of human papillomavirus.

OBJECTIVE: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA). STUDY DESIGN: A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. RESULTS: In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. CONCLUSION: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

Relationship between vascular endothelial growth factor and nuclear factor-kappaB in renal cell tumors.

AIM: To assess the relationship between protein and messenger RNA (mRNA) levels of vascular endothelial growth factor (VEGF) and subcellular localization of nuclear factor-kappa B (NF-kappaB), proliferation rate of tumor cells, and clinicopathological characteristics of renal cell tumors. METHODS: We analyzed 31 one renal cell tumors - 22 clear cell renal cell carcinomas (CCRCC) and 9 other histologic types (non-CCRCC). VEGF expression and subcellular localization of p65 member of NF-kappaB and Ki67 were immunohistochemically evaluated for the proliferation rate of tumor cells. Expression of VEGF mRNA was assessed using quantitative real-time polymerase chain reaction after total RNA extraction from snap-frozen tumor tissue samples. RESULTS: Cytoplasmic localization of VEGF protein in renal cell tumors showed a perimembranous and diffuse pattern, the former being more evident in CCRCC (27.1 -/+ 18.9 vs 3.3 -/+ 10 % tumors, P<0.001) and the latter in non-CCRCC type (71.7 -/+ 23.2 vs 31.1 -/+ 22.1 % tumors, P<0.001). Heterogeneity in VEGF gene expression was more pronounced in CCRCC type than in non-CCRCC type (P=0.004). In addition, perimembranous VEGF pattern was associated with higher VEGF mRNA levels (P=0.006) and diffuse VEGF pattern with lower VEGF mRNA levels (P<0.001). Nuclear and cytoplasmic staining of NF-kappaB/p65 was observed in the majority of tumor cells. A significant association was recorded between cytoplasmic NK-kappaB/65 staining and VEGF staining of diffuse pattern (P=0.026). Association between NF-kappaB/65 and proliferation rate of tumor cells was significant for cytoplasmic staining (P=0.039) but not for nuclear NFkB/p65 staining (P=0.099). CONCLUSION: Higher but inhomogeneous expression of VEGF in tumor cells, especially in CCRCCs, is associated with NF-kappaB/65 activity. This indicates that both VEGF and NF-kappaB/65 may be important in renal carcinogenesis, representing a possible molecular target in the treatment of renal cell carcinoma.

[Prenatal genotyping of the RhD locus by polymerase chain reaction in fetus at risk of hemolytic disease]. / Prenatalna genotipizacija RhD-lokusa s pomocu lancane reakcije polimerazom u fetusa s rizikom od hemoliticke bolesti.

Modern management of Rh alloimmunization includes early diagnosis of fetal RhD genotype, precise assessment of the severity of fetal anemia and the use of minimal number of invasive diagnostic and therapeutic procedures. The severity of fetal anemia can be assessed by Doppler ultrasound, while fetal RhD genotype is determined from the amniotic cells or fetal DNA extracted from the mother's serum by polymerase chain reaction (PCR). Although prenatal RhD genotype diagnostic techniques have been used in developed countries more than 10 years, they have not been available in Croatia until recently. As a consequence of unavailability of these techniques in Croatia there has been inadequate approach to the patient, in whom inappropriate and unnecessary visits, amniocentesis, cordocentesis and laboratory tests were performed. Therefore, we describe the first case of successful prenatal diagnosis of fetal RhD genotype by PCR analysis of DNA extracted from amniotic fluid cells.