Identification of SH2 Domain-Containing Protein 3C as a Novel, Putative Interactor of Dipeptidyl Peptidase 3.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent exopeptidase with broad specificity for four to eight amino acid residue substrates. It has a role in the regulation of oxidative stress response NRF2-KEAP1 pathway through the interaction with KEAP1. We have conducted stable isotope labeling by amino acids in a cell culture coupled to mass spectrometry (SILAC-MS) interactome analysis of TRex HEK293T cells using DPP3 as bait and identified SH2 Domain-Containing Protein 3C (SH2D3C) as prey. SH2D3C is one of three members of a family of proteins that contain both the SH2 domain and a domain similar to guanine nucleotide exchange factor domains of Ras family GTPases (Ras GEF-like domain), named novel SH2-containing proteins (NSP). NSPs, including SH2D3C (NSP3), are adaptor proteins involved in the regulation of adhesion, migration, tissue organization, and immune response. We have shown that SH2D3C binds to DPP3 through its C-terminal Ras GEF-like domain, detected the colocalization of the proteins in living cells, and confirmed direct interaction in the cytosol and membrane ruffles. Computational analysis also confirmed the binding of the C-terminal domain of SH2D3C to DPP3, but the exact model could not be discerned. This is the first indication that DPP3 and SH2D3C are interacting partners, and further studies to elucidate the physiological significance of this interaction are on the way.

Interdisciplinary Study of the Effects of Dipeptidyl-Peptidase III Cancer Mutations on the KEAP1-NRF2 Signaling Pathway.

Dipeptidyl peptidase III (DPP III) is associated with cancer progression via interaction with KEAP1, leading to upregulation of the KEAP1-NRF2 oxidative stress pathway. Numerous DPP III mutations have been found in human tumor genomes, and it is suggested that some of them may alter affinity for KEAP1. One such example is the DPP III-R623W variant, which in our previous study showed much higher affinity for the Kelch domain of KEAP1 than the wild-type protein. In this work, we have investigated the effects of this mutation in cultured cells and the effects of several other DPP III mutations on the stability of KEAP1-DPP III complex using an interdisciplinary approach combining biochemical, biophysical and molecular biology methods with computational studies. We determined the affinity of the DPP III variants for the Kelch domain experimentally and by molecular modeling, as well as the effects of the R623W on the expression of several NRF2-controlled genes. We confirmed that the R623W variant upregulates NQO1 expression at the transcriptional level. This supports the hypothesis from our previous study that the increased affinity of the R623W variant for KEAP1 leads to upregulation of the KEAP1-NRF2 pathway. These results provide a new perspective on the involvement of DPP III in cancer progression and prognosis.

An outbreak of ertapenem-resistant, carbapenemase-negative and porin-deficient ESBL-producing Klebsiella pneumoniae complex.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae is an emerging healthcare-associated pathogen with dynamic molecular epidemiology. This study presents a retrospective analysis of the distribution and antibiotic resistance patterns of ertapenem-resistant ESBL-producing K. pneumoniae strains recovered during an outbreak from 2012 to 2014 in a Croatian University hospital. METHODS: We aimed to estimate genetic relatedness of clinical isolates and underlying mechanisms that conferred the ertapenem-resistant phenotype. RESULTS: Expression analysis of genes involved in the antibiotic resistance showed reduced expression of major non-selective porin channel OmpK35. Reduced expression of OmpK36 porin channel in isolates resistant to at least one more carbapenem, apart from the ertapenem, was found to a lesser degree. Pulsed-field gel electrophoresis analysis of genomic DNA revealed that almost all isolates belonged to the same genetic clone. CONCLUSIONS: Caution regarding ertapenem-resistant, carbapenemase-negative porin-deficient mutants of K. pneumoniae is required as they are widespread, and under selective pressure this could result in a local clonal outbreak.

Binding of dipeptidyl peptidase III to the oxidative stress cell sensor Kelch-like ECH-associated protein 1 is a two-step process.

This work is about synergy of theory and experiment in revealing mechanism of binding of dipeptidyl peptidase III (DPP III) and Kelch-like ECH-associated protein 1 (KEAP1), the main cellular sensor of oxidative stress. The NRF2 ̶ KEAP1 signaling pathway is important for cell protection, but it is also impaired in many cancer cells where NRF2 target gene expression leads to resistance to chemotherapeutic drugs. DPP III competitively binds to KEAP1 in the conditions of oxidative stress and induces release of NRF2 and its translocation into nucleus. The binding is established mainly through the ETGE motif of DPP III and the Kelch domain of KEAP1. However, although part of a flexible loop, ETGE itself is firmly attached to the DPP III surface by strong hydrogen bonds. Using combined computational and experimental study, we found that DPP III ̶ Kelch binding is a two-step process comprising the endergonic loop detachment and exergonic DPP III ̶ Kelch interaction. Substitution of arginines, which keep the ETGE motif attached, decreases the work needed for its release and increases DPP III ̶ Kelch binding affinity. Interestingly, mutations of one of these arginine residues have been reported in cBioPortal for cancer genomics, implicating its possible involvement in cancer development. Communicated by Ramaswamy H. Sarma.

An in vitro and in silico evaluation of bioactive potential of cornelian cherry (Cornus mas L.) extracts rich in polyphenols and iridoids.

In the present research, seven different cornelian cherry (Cornus mas L.) cultivars and selections were examined. In vitro and in silico methods were applied for determining and correlating phytochemical constituents and biological potential. Loganic acid, cornuside, cyanidin3-galactoside, and pelargonidin 3-galactoside were determined as the most dominant compounds, presenting ≥90% of the all detected iridoid and phenolic constituents in the extracts. Cornelian cherry fruits were characterized by high antioxidant capacity and antiproliferative activity on human colon cancer cells (HT29). It was observed the strong inhibitory potential of α-amylase, α-glucosidase, and dipeptidyl peptidase III (DPP III) enzyme activities. Principal component analysis (PCA) was used as a very helpful tool to discriminate the constituents with the highest contribution to tested bioactivities and to highlight the most potent genotypes. PCA, together with binding energies measurements and docking analysis, pointed out pelargonidin 3-robinobioside as the strongest inhibitor of α-glucosidase.

The first dipeptidyl peptidase III from a thermophile: Structural basis for thermal stability and reduced activity.

Dipeptidyl peptidase III (DPP III) isolated from the thermophilic bacteria Caldithrix abyssi (Ca) is a two-domain zinc exopeptidase, a member of the M49 family. Like other DPPs III, it cleaves dipeptides from the N-terminus of its substrates but differently from human, yeast and Bacteroides thetaiotaomicron (mesophile) orthologs, it has the pentapeptide zinc binding motif (HEISH) in the active site instead of the hexapeptide (HEXXGH). The aim of our study was to investigate structure, dynamics and activity of CaDPP III, as well as to find possible differences with already characterized DPPs III from mesophiles, especially B. thetaiotaomicron. The enzyme structure was determined by X-ray diffraction, while stability and flexibility were investigated using MD simulations. Using molecular modeling approach we determined the way of ligands binding into the enzyme active site and identified the possible reasons for the decreased substrate specificity compared to other DPPs III. The obtained results gave us possible explanation for higher stability, as well as higher temperature optimum of CaDPP III. The structural features explaining its altered substrate specificity are also given. The possible structural and catalytic significance of the HEISH motive, unique to CaDPP III, was studied computationally, comparing the results of long MD simulations of the wild type enzyme with those obtained for the HEISGH mutant. This study presents the first structural and biochemical characterization of DPP III from a thermophile.

Cervical HPV type-specific pre-vaccination prevalence and age distribution in Croatia.

The main etiological factor of precancerous lesion and invasive cervical cancer are oncogenic human papillomaviruses types (HPVs). The objective of this study was to establish the distribution of the most common HPVs in different cervical lesions and cancer prior to the implementation of organized population-based cervical screening and HPV vaccination in Croatia. In this study, 4,432 cervical specimens, collected through a 16-year period, were tested for the presence of HPV-DNA by polymerase chain reaction (PCR) with three sets of broad-spectrum primers and type-specific primers for most common low-risk (LR) types (HPV-6, 11) and the most common high-risk (HR) types (HPV-16, 18, 31, 33, 45, 52, 58). Additional 35 archival formalin-fixed, paraffin embedded tissue of cervical cancer specimens were analyzed using LiPA25 assay. The highest age-specific HPV-prevalence was in the group 18-24 years, which decreased continuously with age (P<0.0001) regardless of the cytological diagnosis. The prevalence of HR-HPV types significantly increased (P<0.0001) with the severity of cervical lesions. HPV-16 was the most common type found with a prevalence (with or without another HPV-type) of 6.9% in normal cytology, 15.5% in atypical squamous cells of undetermined significance, 14.4% in low-grade squamous intraepithelial lesions, 33.3% in high-grade squamous intraepithelial lesions, and 60.9% in cervical cancer specimens (P<0.0001). This study provides comprehensive and extensive data on the distribution of the most common HPV types among Croatian women, which will enable to predict and to monitor the impact of HPV-vaccination and to design effective screening strategies in Croatia.

Prominent role of exopeptidase DPP III in estrogen-mediated protection against hyperoxia in vivo.

A number of age-related diseases have a low incidence in females, which is attributed to a protective effect of sex hormones. For instance, the female sex hormone estrogen (E2) has a well established cytoprotective effect against oxidative stress, which strongly contributes to ageing. However, the mechanism by which E2 exerts its protective activity remains elusive. In this study we address the question whether the E2-induced protective effect against hyperoxia is mediated by the Nrf-2/Keap-1 signaling pathway. In particular, we investigate the E2-induced expression and cellular distribution of DPP III monozinc exopeptidase, a member of the Nrf-2/Keap-1 pathway, upon hyperoxia treatment. We find that DPP III accumulates in the nucleus in response to hyperoxia. Further, we show that combined induction of hyperoxia and E2 administration have an additive effect on the nuclear accumulation of DPP III. The level of nuclear accumulation of DPP III is comparable to nuclear accumulation of Nrf-2 in healthy female mice exposed to hyperoxia. In ovariectomized females exposed to hyperoxia, supplementation of E2 induced upregulation of DPP III, Ho-1, Sirt-1 and downregulation of Ppar-γ. While other cytoprotective mechanisms cannot be excluded, these findings demonstrate a prominent role of DPP III, along with Sirt-1, in the E2-mediated protection against hyperoxia.

Mechanisms of Carbapenem Resistance in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from a Croatian Hospital.

Pseudomonas aeruginosa is an opportunistic pathogen, one of the leading causes of nosocomial infections such as pneumonia, urinary tract infections, and bacteraemia. The bacterial resistance to structurally unrelated antibiotics and its spread within hospitals limits the efficient antimicrobial options and patients' outcome. Carbapenems are important agents for the therapy of infections due to multidrug-resistant (MDR) P. aeruginosa; hence, the development of carbapenem resistance severely hampers effective therapeutic options. The aim of this investigation was to examine mechanisms of carbapenem resistance and genomic diversity in carbapenem-resistant MDR strains of P. aeruginosa, which caused an outbreak among patients in Clinical Hospital Rijeka. Most of the isolates showed decreased expression of porin that is important for the entry of carbapenems (oprD). Overexpression of MexAB-OprM, MexCD-OprJ, and MexEF-OprN efflux systems was observed in many of the isolates. Production of metallo-ß-lactamases was not detected. Typing by pulsed-field gel electrophoresis discriminated the isolates into five clusters. The clonal distribution of the strains was related to the location of hospital departments where the isolates were collected, which implies that most of the infections were caused by spread of the epidemic strains within the hospital.

Characterization and whole genome analysis of human papillomavirus type 16 e1-137463nt variants.

BACKGROUND: The variation of the most common Human papillomavirus (HPV) type found in cervical cancer, the HPV16, has been extensively investigated in almost all viral genes. The E1 gene variation, however, has been rarely studied. The main objective of the present investigation was to analyze the variability of the E6 and E1 genes, focusing on the recently identified E1-1374^63nt variant. METHODOLOGY/PRINCIPAL FINDINGS: Variation within the E6 of 786 HPV16 positive cervical samples was analyzed using high-resolution melting, while the E1-1374^63nt duplication was assayed by PCR. Both techniques were supplemented with sequencing. The E1-1374^63nt duplication was linked with the E-G350 and the E-C109/G350 variants. In comparison to the referent HPV16, the E1-1374^63nt E-G350 variant was significantly associated with lower grade cervical lesions (p = 0.029), while the E1-1374^63nt E-C109/G350 variant was equally distributed between high and low grade lesions. The E1-1374^63nt variants were phylogenetically closest to E-G350 variant lineage (A2 sub-lineage based on full genome classification). The major differences between E1-1374^63nt variants were within the LCR and the E6 region. On the other hand, changes within the E1 region were the major differences from the A2 sub-lineage, which has been historically but inconclusively associated with high grade cervical disease. Thus, the shared variations cannot explain the particular association of the E1-1374^63nt variant with lower grade cervical lesions. CONCLUSIONS/SIGNIFICANCE: The E1 region has been thus far considered to be well conserved among all HPVs and therefore uninteresting for variability studies. However, this study shows that the variations within the E1 region could possibly affect cervical disease, since the E1-1374^63nt E-G350 variant is significantly associated with lower grade cervical lesions, in comparison to the A1 and A2 sub-lineage variants. Furthermore, it appears that the silent variation 109T>C of the E-C109/G350 variant might have a significant role in the viral life cycle and warrants further study.

Effect of ferric ions on reactive oxygen species formation, cervical cancer cell lines growth and E6/E7 oncogene expression.

As iron ions may participate in the pathogenesis of cancer and viral infections, the aim of this study was to monitor their influence on proliferation, E6 and E7 oncogene expression and reactive oxygen species (ROS) production in two human papilloma virus (HPV) positive cervical carcinoma cell lines (HeLa and SiHa) and one HPV negative vulvar cell line (A431). The anti-anaemic drug, ferric-sorbitol-citric acid complex (FSC) as a source of Fe(III) ions was used. Cells were treated with FSC at the concentrations between 0.001 and 1 mM Fe(III) for different time periods. Fe(III) ions inhibited the viability of HeLa and A431 cells while it had no influence on SiHa cells. Furthermore, Fe(III) treatment showed a time-dependent and a higher stimulatory effect on E6/E7 expression in SiHa cells than in HeLa cells. Fe(III) ion treatment with concentrations lower than 0.1mM showed a time and a concentration dependent intracellular ROS production in all tested cell lines, while the treatment with 1mM concentration decreased ROS production in all tested cell lines. In conclusion, Fe(III) ion treatment apart from having an anti-tumour effect, as we previously described, enhances survival of HPV 16-positive cells and might be associated with HPV oncogenesis.

Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates.

Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.

Advances in cervical cancer control and future perspectives.

The knowledge that the persistent infection with high-risk (HR) human papillomavirus (HPV) is the etiological factor in the development of cervical cancer has led to the development of the HPV DNA detection methods as well as the prophylactic vaccine against the most common HR-HPV types, HPV 16 and 18. Despite HPV vaccination, cervical cancer screening will remain the main preventive measure for both vaccinated and non-vaccinated women, but the nature of screening and management of women with cervical disease is being adapted to the new technologies. Although, HPV DNA detection is more sensitive that cytology, its specificity is lower, since most HPV infections are transient. Therefore, other methods are considered to improve the management of women with cervical disease. Typing of HPV DNA and viral load measurements are still used for research purposes only. Detection of viral oncogene E6/E7 transcripts, which is the marker of the productive infection, is a promising tool for follow-up of HPV DNA-positive women. The detection of p16INK4a over-expression, as an indirect test of E6/E7 expression, is used for confirmation of cervical neoplasia. Despite the lack of standardization, the detection of p16INK4a is useful in clinical settings, however its reproducibility in the management of low-grade and borderline cases is low. Future perspectives include the determination of the methylation status of several cellular genes that could predict the progression of the disease.

A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis.

Studies on the variability of human papillomavirus (HPV) type 16 are based mostly on DNA sequencing of the viral oncogenes E6 and E7. In order to simplify variant identification, high resolution melting (HRM) analysis, which has been shown to distinguish amplicons differing in a single nucleotide, was employed. Optimised HRM analysis was applied to 255 anogenital samples positive for HPV 16. The E6/E7 region of the HPV 16 genome was amplified using nested PCR with subsequent melting of the amplicons. Samples giving ambiguous melting profiles were melted again in the presence of reference HPV 16 DNA to define and confirm the novel melting profiles. Out of 219 samples of Croatian origin, 65 reference variants, 119 E6-360G variants and 35 novel melting profiles were found. Samples containing unusual profiles were sequenced for identification. In addition, a subset of samples with two common variants, 23 reference and 34 E6-350G variants, was also sequenced to confirm the findings of high resolution melting. Concordance between the melting analysis and sequencing was 93.9%, while HRM sensitivity and specificity were 92.9% and 94.7%, respectively. This study showed that HRM analysis can be useful for the identification of HPV 16 variants. The HRM method will be useful in low resource settings as it saves considerable time and resources compared to sequencing.

Identification of human papillomavirus type 16 integration sites in high-grade precancerous cervical lesions.

OBJECTIVES: Infection with oncogenic human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. In many cases of cervical cancer and all cervical cancer derived cell lines oncogenic HPV DNA is found to be integrated, indicating the importance of integration in disease development. In this study, 176 HPV 16 positive precancerous cervical lesions were analyzed for the physical state of viral genome to determine the sites of integration into a host cell DNA and to evaluate the incidence of the integration in different stages of cervical lesions. METHODS: The detection of integrated papillomavirus sequences (DIPS) method in combination with the amplification by polymerase chain reaction (PCR) of E1/E2 region was used to identify the physical state of HPV 16 genome. The site of integration within a host cell genome was determined by sequencing of unusual sized DIPS amplicons. RESULTS: The combined results of DIPS and E1/E2 PCR revealed the integration of HPV 16 DNA in 7.4% samples. The integration was found only in high grade cervical lesions indicating that it is a late event in disease progression. Sequencing of 11 DIPS amplicons revealed HPV DNA from 6 samples (54.5%) to be integrated in cellular genes (VMP1, PVRL1, CHERP, CEACAM5, AHR, MRF-2) and also 6 (54.5%) within the common fragile sites (CFS). CONCLUSIONS: Although, the HPV integration is known to be a random event, this study indicates that HPV 16 integrates more than by chance within or close to CFSs. As most of the genes affected by HPV 16 integration can be linked with some aspects of tumor formation, this indicates that the site of HPV DNA integration might play a role in the rate and the nature of tumor development.

Identification of a novel human papillomavirus type 16 E1 gene variant with potentially reduced oncogenicity.

The human papillomavirus (HPV) 16 genome has been studied extensively, although no study has focused on the E1 gene that is implicated in viral DNA replication. After analyzing the E1 region of HPV 16 genomes in 429 cervical samples, 11.2% were found to contain a 63 nucleotides duplication in this region. Sequence analysis of the E6 and the E7 regions has shown that all samples containing this duplication were related to E6-G350 variant of the HPV 16 (Chi square test, P = 0.0012). A comparison of cervical lesion severity of the examinees having regular or variant E1 genes has shown that the variant group had a significantly (Fischer's exact test, P = 0.0401) lower percentage of high grade disease cases, suggesting that this particular duplication might reduce the oncogenic potential of HPV 16, and also might clarify the differences of E6-G350 variant oncogenicity observed in European populations. Albeit, a decreased incidence of high grade cervical lesions can be linked to the prevalence of multiple HPV infection, the additional decrease of those cases with the variant E1 gene versus those without (10.5% and 18.6%, respectively) can only be ascribed to the effect of this particular HPV variant. Further research is needed to clarify the biology of these HPV 16 E1 variants.

Detection and typing of human papillomaviruses combining different methods: polymerase chain reaction, restriction fragment length polymorphism, line probe assay and sequencing.

The identification of the etiological factor of many cervical precancerous lesions and cervical cancer, the human papillomavirus (HPV) is widely used. In this study, we evaluated the consensus and type-specific (TS) polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), line probe assay (LiPA, Innogenetics) and sequencing to determine the HPV types in cervical specimens. Out of 690 High-grade Squamous Intraepithelial Lesion (HSIL) samples, 86.7% were HPV positive and 13.3% HPV negative by consensus primers (MY09/MY11, L1C1/L1C2-1/L1C2-2 and/or GP5/6) directed PCR. Out of 598 HPV positive samples, 85.3% were typed by TS-PCR being HPV 6/11, 16, 18, 31 and/or 33, while 14.7% remained untyped. The most prevalent HPV type in the study group was HPV 16, identified in 35.5% cases, while HPV 31 was the second most frequent HPV type with a prevalence of 10.5%. They were followed by HPV types 6/11, 33 and 18 with a prevalence of 7.4%, 6.2% and 4.9%, respectively. Multiple HPV infections with two or more HPV types (6/11, 16, 18, 31 and/or 33) were found in 9.4% cases. A subset of 88 samples was further typed by RFLP and LiPA to determine the rare HPV types in HSIL samples. The most frequent low abundant HPV types in single infections in decreasing order were HPV 53, 58, 66, 56 and 52, while HPV 51 was the most frequent low abundant HPV type found in multiple HPV infections. Multiple HPV infections were mostly found by LiPA in 27.3% cases versus 14.8% cases found by RFLP. The perfect agreement between RFLP and LiPA assay pair was observed only for HPV types 16, 18, 34 and 59 (kappa value of 1). For other HPV types, the inter-assay agreement ranged from very good to no agreement indicating that neither assay is perfect. Sequencing was performed for 33 samples in cases where both RFLP and LiPA were inconclusive. Sequencing was shown to be a very good method in case of single HPV infection but not applicable in case of multiple HPV infections. Both RFLP and LiPA are good assays for epidemiological studies, although RFLP being cumbersome and time-consuming is less applicable than LiPA. Careful consideration has to be made before the implementation of either HPV typing methods in clinical laboratories.

Retrospective study of the prevalence of high-risk human papillomaviruses among Croatian women.

The infection with Human papillomavirus (HPV) is the necessary cause for cervical cancer. There are at least 15 High-Risk (HR) HPV types that are significantly associated with progression of cervical intraepithelial neoplasia to cervical cancer. Since previous studies showed that the prevalence of HPV in cervical cancers varies among different geographic regions, we wanted to investigate the prevalence of HPV types in Croatia, especially low abundant HR HPV types. By means of consensus primers directed polymerase chain reaction (PCR), we analysed cervical DNA samples of 2,136 Croatian women, mostly with abnormal cervical smears, in order to detect the presence of HPV Type-specific primers were then used to determine Low-Risk (LR) HPV types 6/11 and HR HPV types 16, 18, 31, 33, 45, 52 and 58. Out of 2,136 specimens, 1,255 (58.8%) were positive for HPV More than half of positive samples were typed (64.5%) and 35.5% still remained untyped. Multiple HPV infections were found in 10.3% of the cases. The most prevalent type, including both single and multiple infections, was HPV16 with the prevalence of 15.9%, followed by HPV types 31, 6/11, 33, 18, 52, 45 and 58 with 8.7%, 7.1%, 4.5%, 3.8%, 2.3%, 1.2% and 1.1%, respectively. The significant increase of frequency from Low-grade Squamous Intraepithelial Lesions (LSIL) to High-grade Squamous Intraepithelial Lesions (HSIL) was observed for HR HPV types 16, 18, 31 and 33 but not 45, 52 and 58. The frequency of unknown HPV types was almost the same in cervical specimens of women with LSIL and those with HSIL, 19.8% and 21.1%, respectively. The prevalence of HPV infection rate decreased significantly with patient age from 68.5% (age group 12 to 24 years) to 38.8% (age group 45 to 54 years). But, in women aged 55 or older the overall prevalence increased to 56.6%. Our results indicate that prevalence of HR HPV types in Croatia is similar to other countries. We suggest that HPV positive women in Croatia should be closely monitored by typing for HR HPV types: 16, 18, 31, 33, 45, 52 and 58.

Possible role of bacterial and viral infections in miscarriages.

OBJECTIVE: To determine the role of infections in miscarriages. Chorionic villi from aborted material were subjected to cytogenetic evaluation and analyzed for the presence of Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, human cytomegalovirus (HCMV), adeno-associated virus (AAV), and human papillomaviruses (HPV). DESIGN: Retrospective study. SETTING: University hospital and academic research institution. MAIN OUTCOME MEASURE(S): Karyotyping and detection of bacterial and viral DNA by means of polymerase chain reaction (PCR) in placenta specimens. RESULT(S): In 54 (50%) of 108 samples the karyotype was normal, in 38 (35%) samples it was abnormal, and in 16 (15%) samples karyotype was undetermined. No U. urealyticum, M. hominis, HCMV, or AAV-2 DNA was detected, while C. trachomatis DNA was detected in one (1%) and HPV DNA in eight (7%) samples. No significant correlation of HPV-positive findings with karyotype status was established. CONCLUSION(S): Our findings do not support a role of C. trachomatis, U. urealyticum, M. hominis, HCMV, or AAV infections in miscarriages during the first trimester of pregnancy. However, further investigation should be made to determine a possible involvement of HPVs in the development of genetic abnormalities of the fetus and in miscarriages.

[Detection of human papillomaviruses and other agents causing sexually transmitted diseases with molecular diagnosis methods]. / Otkrivanje papilomavirusa covjeka i drugih uzrocnika spolno prenosivih bolesti metodama molekularne dijagnostike.

Causative agents of sexually transmitted diseases (STD) are different types of bacteria, viruses, fungi and protozoa. The last two decades of the twentieth century were marked with a sudden rise in the number of cases of STDs. Human immunodeficiency virus (HIV), which emerged in the 1980s, is the most prominent STD agent because of its fast spread and severity of the disease it causes, acquired immunodeficiency syndrome (AIDS). Beside HIV, human papillomaviruses (HPVs), herpes simplex viruses (HSVs) and Chlamydia trachomatis are nowadays among most health-threatening STD pathogens. In order to stop the spread of infection, apart from education about precautions, early detection of the disease is essential. Although most STD pathogens can be detected by classical methods of cultivation, biochemical and/or serologic methods, molecular diagnosis of infectious diseases has largely simplified and accelerated their detection. For instance, HPVs that cause benign and malignant tumors of genital skin and mucosa cannot be routinely detected on cell culture, whereas serologic analysis is not sensitive and informative enough. Moreover, cytologic (Pap smear) and histologic analyses can indicate changes associated with HPV infection, but neither of these methods can prove the presence of HPV. That is why the molecular methods are essential to demonstrate the presence of the infection and, even more important, to determine the type of the virus, which is associated either with low-grade or high-grade genital lesions. There are numerous methods based on hybridization with DNA or RNA probes, some of them are suitable for detecting wide range of types and screening of large collection of samples. However, the most sensitive and informative methods are based on polymerase chain reaction (PCR), and they have the advantage of being able to determine the type of the virus and distinguishing between multiple infections. Herein, we present when and why molecular analysis is useful and necessary for the detection of STD agents.