Human Papillomavirus Genome Copy Number Is Maintained by S-Phase Amplification, Genome Loss to the Cytosol during Mitosis, and Degradation in G1 Phase.

The current model of human papillomavirus (HPV) replication is comprised of three modes of replication. Following infectious delivery, the viral genome is amplified during the establishment phase to reach up to some hundred copies per cell. The HPV genome copy number remains constant during the maintenance stage. The differentiation of infected cells induces HPV genome amplification. Using highly sensitive in situ hybridization (DNAscope) and freshly HPV16-infected as well as established HPV16-positive cell lines, we observed that the viral genome is amplified in each S phase of undifferentiated keratinocytes cultured as monolayers. The nuclear viral genome copy number is reset to pre-S-phase levels during mitosis. The majority of the viral genome fails to tether to host chromosomes and is lost to the cytosol. Cytosolic viral genomes gradually decrease during cell cycle progression. The loss of cytosolic genomes is blocked in the presence of NH4Cl or other drugs that interfere with lysosomal acidification, suggesting the involvement of autophagy in viral genome degradation. These observations were also made with HPV31 cell lines obtained from patient samples. Cytosolic viral genomes were not detected in UMSCC47 cells carrying integrated HPV16 DNA. Analyses of organotypic raft cultures derived from keratinocytes harboring episomal HPV16 revealed the presence of cytosolic viral genomes as well. We conclude that HPV maintains viral genome copy numbers by balancing viral genome amplification during S phase with the loss of viral genomes to the cytosol during mitosis. It seems plausible that restrictions to viral genome tethering to mitotic chromosomes reset genome copy numbers in each cell cycle. IMPORTANCE HPV genome maintenance is currently thought to be achieved by regulating the expression and activity of the viral replication factors E1 and E2. In addition, the E8^E2 repressor has been shown to be important for restricting genome copy numbers by competing with E1 and E2 for binding to the viral origin of replication and by recruiting repressor complexes. Here, we demonstrate that the HPV genome is amplified in each S phase. The nuclear genome copy number is reset during mitosis by a failure of the majority of the genomes to tether to mitotic chromosomes. Rather, HPV genomes accumulate in the cytoplasm of freshly divided cells. Cytosolic viral DNA is degraded in G1 in a lysosome-dependent manner, contributing to the genome copy reset. Our data imply that the mode of replication during establishment and maintenance is the same and further suggest that restrictions to genome tethering significantly contribute to viral genome maintenance.

Experimental Support for Human Papillomavirus Genome Amplification Early after Infectious Delivery.

Even though replication and transcription of human papillomavirus type 16 (HPV16) has been intensively studied, little is known about immediate-early events of the viral life cycle due to the lack of an efficient infection model allowing genetic dissection of viral factors. We employed the recently developed infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846) to investigate genome amplification and transcription immediately after infectious delivery of viral genome to nuclei of primary keratinocytes. Using 5-ethynyl-2'-deoxyuridine (EdU) pulse-labeling and highly sensitive fluorescence in situ hybridization, we observed that the HPV16 genome is replicated and amplified in an E1- and E2-dependent manner. Knockout of E1 resulted in failure of the viral genome to replicate and amplify. In contrast, knockout of the E8^E2 repressor led to increased viral genome copy number, confirming previous reports. Genome copy control by E8^E2 was confirmed for differentiation-induced genome amplification. Lack of functional E1 had no effect on transcription from the early promoter, suggesting that viral genome replication is not required for p97 promoter activity. However, infection with an HPV16 mutant virus defective for E2 transcriptional function revealed a requirement of E2 for efficient transcription from the early promoter. In the absence of the E8^E2 protein, early transcript levels are unaltered and even decreased when normalized to genome copy number. Surprisingly, a lack of functional E8^E2 repressor did not affect E8^E2 transcript levels when normalized to genome copy number. These data suggest that the main function of E8^E2 in the viral life cycle is to control genome copy number. IMPORTANCE It is being assumed that human papillomavirus (HPV) utilizes three different modes of replication during its life cycle: initial amplification during the establishment phase, genome maintenance, and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model. Using our recently established infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846), we demonstrate herein that viral genome is indeed amplified in an E1- and E2-dependent manner. Furthermore, we find that the main function of the viral repressor E8^E2 is to control viral genome copy number. We did not find evidence that it regulates its own promoter in a negative feedback loop. Our data also suggest that the E2 transactivator function is required for stimulation of early promoter activity, which has been debated in the literature. Overall, this report confirms the usefulness of the infection model for studying early events of the HPV life cycle using mutational approaches.

Genome-Wide Transcriptome Analysis of Human Papillomavirus 16-Infected Primary Keratinocytes Reveals Subtle Perturbations Mostly due to E7 Protein Expression.

It is established that the host cell transcriptomes of natural lesions, organotypic rafts, and human papillomavirus (HPV)-immortalized keratinocytes are altered in the presence of HPV genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at 7 days postinfection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107, and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to the E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus had perturbed transcriptional regulation of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.IMPORTANCE To establish infection and complete the viral life cycle, human papillomavirus (HPV) needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocyte-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an extracellular matrix (ECM)-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S-phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and, possibly, disease progression.

Novel Triazole Hybrids of Betulin: Synthesis and Biological Activity Profile.

Betulin derivatives containing a 1,2,3-triazole ring possess a wide spectrum of biological activities, including antiviral, anticancer, and antibacterial activity. A series of novel triazoles were prepared by the 1,3-dipolar cycloaddition reaction between the alkyne derivatives of betulin and organic azides. The chemical structures of the obtained compounds were defined by ¹H and 13C NMR, IR, and high-resolution mass spectrometry (HR-MS) analysis. The target triazoles were screened for their antiviral activity against DNA and RNA viruses. The cytotoxic activity of the obtained compounds 5a-k and 6a-h was determined using five human cancer cell lines (T47D, MCF-7, SNB-19, Colo-829, and C-32) by a WST-1 assay. The bistriazole 6b displayed a promising IC50 value (0.05 µM) against the human ductal carcinoma T47D (500-fold higher potency than cisplatin). The microdilution method was applied for an evaluation of the antimicrobial activity of all of the compounds. The triazole 5e containing a 3'-deoxythymidine-5'-yl moiety exhibited antibacterial activity against two gram-negative bacteria vz. Klebsiellapneumoniae and Escherichia coli (minimal inhibitory concentration (MIC) range of 0.95-1.95 µM).

The effects of killer cell immunoglobulin-like receptor (KIR) genes on susceptibility to HIV-1 infection in the Polish population.

Killer cell immunoglobulin-like receptors (KIR) are the most polymorphic receptors of natural killer (NK) cells. Their activity diversifies the functions of NK cells in the antiviral immune response, so the presence of certain KIR may affect transmission of HIV-1. The aim of the study was to evaluate the influence of KIR genes on the susceptibility to HIV-1 infection in the Polish population depending on the route of exposure. We determined the frequencies of activating (2DS1, 2DS2, 2DS3, 2DS4f, 2DS4del, 2DS5, 3DS1) and inhibitory (2DL1, 2DL2, 2DL3, 2DL5, 3DL1) KIRs in HIV-1-positive patients (n = 459), individuals exposed to HIV-1 but uninfected (EU, n = 118) and in uninfected, healthy blood donors (BD, n = 98). Analysis was performed using stepwise logistic regression. Apart from KIRs, CCR5-∆32, and CCR2-64I, alleles were also analyzed, as we knew or suspected that these features could affect susceptibility to HIV infection. The regression confirmed the protective effect of CCR5-∆32 (OR = 0.25, p = 0.006) and CCR2-64I (OR = 0.59, p = 0.032) against HIV infection. Among KIR genes, 2DL3 was found to be a protective factor (OR = 0.30, p = 0.015). A similar effect was seen for 3DS1 but only in intravenous drug users (IDUs) (OR = 0.30, p = 0.019), not in sexually exposed people. 2DL5 was found to be a factor facilitating HIV infection (OR = 2.13, p = 0.013). A similar effect was observed for 2DL2 but only in females (OR = 2.15, p = 0.040), and 2DS1 in IDUs (OR = 3.03, p = 0.022). Our results suggest a beneficial role of KIR3DS1 and 2DL3 supporting resistance to HIV infection and a harmful effect of 2DS1, 2DL5, and 2DL2 genes promoting HIV acquisition.

The role genes encoding of killer cell immunoglobulin-like receptors (KIRs) and their ligands in susceptibility to and progression of HIV infection.

NK cells are a part of the innate antiviral response. Their activity is regulated by signals from the surface receptors. Some of them, known as killer cell immunoglobulin-like receptors (KIRs), determine the quality and intensity of the immunological response, together with their ligands (HLA class I). KIR genes are very polymorphic, and this is reflected in the NK activity modulation. The stimulation of NK cells, especially in the early stages of the infection, can reduce the transmission of HIV or slow down the progression of infection. The varied KIR/HLA repertoire is a limiting factor for the risk of HIV infection and disease progression. Such diversity enables optimal regulation of NK cells and maintenance of the balance between activation to eliminate infected cells and inhibition. The control of NK cell activity via KIR3DL1/3DS1 and HLA-Bw4 (especially Bw4-80I) seems to be very important in the HIV context. With a few exceptions, it leads to a reduction of susceptibility to HIV infection and better viremia control, and slows down depletion of CD4+ T cells. Incompatibility of sexual partners for KIRs and HLA may oblige NK cells from the exposed partner to reject incoming cells from the HIV-positive partner. The presence of the inhibitory KIR, in the absence of its ligand, results in a lower threshold of NK cell activation, which reduces the chance of infection. The presence of an inhibitory receptor with a low affinity to the ligand (KIR2DL3+HLA-C1) is associated with lower susceptibility, and the effective NK cell inhibition (KIR2DL2+HLA-C1) results in increased susceptibility to HIV infection. The advantage of activating KIRs, especially in the presence of their ligands, is associated with higher cytolytic abilities, and thus reduced risk of HIV infection. If the virus is not eliminated in an early stage of infection, massive activation of NK is unfavorable due to the excessive stimulation of the immune system.

Comparison of microbiological and physicochemical methods for enumeration of microorganisms.

Determination of the number of cultured bacteria is essential for scientific and industrial practice. A spread plate technique is the most common and accurate method for counting of microorganisms. However, time consuming incubation does not allow for a quick estimation of the number of bacteria in a growing culture. In the present study, the results of photometric measurements: direct optical density method (OD at 585 nm), UV absorbance at 260 and/or 280 nm of separated and lysed bacteria by sodium hydroxide and surfactant with the spread plate technique were compared. The linear regression model for bacterial strains Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli was used to compare these three methods. The UV measurement method enabled determination of the number of bacteria with similar precision. The procedure for solubilized bacteria UV measurement is robust, and is not influenced by dispersions in the original culture medium.

An Exonuclease V-qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues.

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC. Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues Alternate Protocol: Determining HPV integration type or integrity of HPV episome.

[Host genetic factors associated with susceptibility to HIV infection and progression of infection]. / Czynniki genetyczne zwiazane z podatnoscia na zakazenie HIV oraz z progresja zakazenia.

HIV infection causes progressive immune system deficiency, the development of AIDS and eventual death. Genetic factors play a very important role in the susceptibility to HIV infection and disease progression. Estimation of their effects is realized by comparing different patients groups. Four group of HIV-infected patients are taken into account: RP - rapid progressors, SP - slow progressors, LTNP - long term non-progressors and EU - exposed but uninfected. Genetic factors influencing the course of disease can be divided in groups, for example genes coding proteins connected with viral entry into cells (chemokine receptors, chemokines, lectins receptors), proteins engaged in the innate response to viral infections (cytokines, MBL, cyclophilin A, TRIM-5 alpha, APOBEC3G), human leukocyte antigens (HLAs) and killer cell immunoglobulin-like receptors (KIRs). It is suggested that some sequences of human endogenous retroviruses (HERV) and microRNA (miRNA) can also interact with the course of HIV infection. Knowledge of the roles of genetic determinants in HIV infection is very important and useful for their cognitive significance as well as in the prognosis of AIDS progression and the selection of specific therapies for individual patients. It is also a basis for the development of new antiviral drugs and vaccines.

Hampering Herpesviruses HHV-1 and HHV-2 Infection by Extract of Ginkgo biloba (EGb) and Its Phytochemical Constituents.

Despite the availability of several anti-herpesviral agents, it should be emphasized that the need for new inhibitors is highly encouraged due to the increasing resistant viral strains as well as complications linked with periods of recurring viral replication and reactivation of latent herpes infection. Extract of Ginkgo biloba (EGb) is a common phytotherapeutics around the world with health benefits. Limited studies, however, have addressed the potential antiviral activities of EGb, including herpesviruses such as Human alphaherpesvirus 1 (HHV-1) and Human alphaherpesvirus 2 (HHV-2). We evaluated the antiviral activity of EGb and its phytochemical constituents: flavonoids and terpenes against HHV-1 and HHV-2. Pretreatment of the herpesviruses with EGb prior to infection of cells produced a remarkable anti-HHV-1 and anti-HHV-2 activity. The extract affected the viruses before adsorption to cell surface at non-cytotoxic concentrations. In this work, through a comprehensive anti-HHV-1 and anti-HHV-2 activity study, it was revealed that flavonoids, especially isorhamnetin, are responsible for the antiviral activity of EGb. Such activity was absent in quercetin and kaempferol. However, EGb showed the most potent antiviral potency compared to isorhamnetin. EGb could augment current therapies for herpes labialis and genital herpes. Moreover, the potential use of EGb in multidrug therapy with synthetic anti-herpes compounds might be considered.

The Infectious Etiology of Alzheimer's Disease.

BACKGROUND: Inflammation is a part of the first line of defense of the body against invasive pathogens, and plays a crucial role in tissue regeneration and repair. A proper inflammatory response ensures the suitable resolution of inflammation and elimination of harmful stimuli, but when the inflammatory reactions are inappropriate it can lead to damage of the surrounding normal cells. The relationship between infections and Alzheimer's Disease (AD) etiology, especially lateonset AD (LOAD) has been continuously debated over the past three decades. METHODS: This review discusses whether infections could be a causative factor that promotes the progression of AD and summarizes recent investigations associating infectious agents and chronic inflammation with AD. Preventive and therapeutic approaches to AD in the context of an infectious etiology of the disease are also discussed. RESULTS: Emerging evidence supports the hypothesis of the role of neurotropic viruses from the Herpesviridae family, especially Human herpesvirus 1 (HHV-1), Cytomegalovirus (CMV), and Human herpesvirus 2 (HHV-2), in AD neuropathology. Recent investigations also indicate the association between Hepatitis C virus (HCV) infection and dementia. Among bacteria special attention is focused on spirochetes family and on periodontal pathogens such as Porphyromonas gingivalis or Treponema denticola that could cause chronic periodontitis and possibly contribute to the clinical onset of AD. CONCLUSION: Chronic viral, bacterial and fungal infections might be causative factors for the inflammatory pathway in AD.

VSV based virotherapy in ovarian cancer: the past, the present and future?

The standard approach to treating patients with advanced epithelial ovarian cancer (EOC) after primary debulking surgery remains taxane and platinum-based chemotherapy. Despite treatment with this strategy, the vast majority of patients relapse and develop drug-resistant metastatic disease that may be driven by cancer stem cells (CSCs) or cancer initiating cells (CICs). Oncolytic viruses circumvent typical drug-resistance mechanisms, therefore they may provide a safe and effective alternative treatment for chemotherapy-resistant CSCs/CICs. Among oncolytic viruses vesicular stomatitis virus (VSV) has demonstrated oncolysis and preferential replication in cancer cells. In this review, we summarize the recent findings regarding existing knowledge on biology of the ovarian cancer and the role of ovarian CSCs (OCSCs) in tumor dissemination and chemoresistance. In addition we also present an overview of recent advances in ovarian cancer therapies with oncolytic viruses (OV). We focus particularly on key genetic or immune response pathways involved in tumorigenesis in ovarian cancer which facilitate oncolytic activity of vesicular stomatitis virus (VSV). We highlight the prospects of targeting OCSCs with VSV. The importance of testing an emerging ovarian cancer animal models and ovarian cancer cell culture conditions influencing oncolytic efficacy of VSV is also addressed.

Association between Periodontal Health Status and Cognitive Abilities. The Role of Cytokine Profile and Systemic Inflammation.

BACKGROUND: Contemporary neurobiology, periodontal medicine, and immunology are now focusing on the relationship between chronic periodontitis and systemic diseases, which also include Alzheimer's disease (AD). However a causative relationship between dementia and periodontitis has yet to be confirmed. OBJECTIVE: The aim of the study was to determine whether periodontal health status and cognitive abilities are correlated with the relative changes in systemic measures of pro- and anti-inflammatory cytokines as a reflection of systemic inflammation. We hypothesized that poor periodontal health status may be associated with cognitive impairment and dementia via the exacerbation of systemic inflammation. METHODS: Based on the periodontal and psychiatric examinations and the cytokine levels produced by unstimulated and LPS-stimulated PBL isolated from 128 participants, we have examined if the coexisting of these two clinically described conditions may have influence on the systemic inflammation. Mini- Mental State Examination (MMSE) and Bleeding on Probing (BoP) test results were combined into the one mathematical function U, which determines the severity of specific condition, called Cognitive and periodontal impairment state. Similarly, the levels of cytokines were combined into the one mathematical function V, whose value determines the level of Inflammatory state. The correlation between U and V was determined. RESULTS: These results confirm that the presence of cognitive decline and the additional source of proinflammatory mediators, like periodontal health problems, aggravate the systemic inflammation. CONCLUSION: It is most likely that the comorbidity of these two disorders may deepen the cognitive impairment, and neurodegenerative lesions and advance to dementia and AD.

Graves' disease as an immune reconstitution syndrome in an HIV-1-positive patient commencing effective antiretroviral therapy: case report and literature review.

Combination antiretroviral therapy (cART) reduces morbidity and mortality in human immunodeficiency virus (HIV) infection, but it may also alter the clinical course of subclinical opportunistic infections and can even induce autoimmune disease. These atypical presentations are known as immune restoration disease (IRD), immune reconstitution syndrome/immune recovery syndrome (IRS), or immune restoration inflammatory syndrome (IRIS). We report the case of a 27-year-old, HIV-1-positive woman who developed hyperthyroidism attributable to Graves' disease (GD) after commencing potent cART. At the initiation of cART, her CD4 T cell count was 15 cells/microL and plasma HIV RNA 35 000 copies/mL. Her commencement of cART resulted in complete viral suppression and subsequent improvement of the CD4 T-cell count. Three years later, the diagnosis of GD was established based on a typical clinical picture and the results of hormonal and immunological analyses. It coincided with a 58-fold rise of the CD4 T cells. Retrospective analysis of serum samples revealed normal thyroid function and lack of anti-thyroid peroxidase (anti-TPO), anti- thyroid-stimulating hormone receptor (anti-TSHR), and anti-thyroglobulin (anti-TG) autoantibodies at the beginning of cART. HLA class II gene examination did not reveal susceptibility for the GD development in this patient. We suggest that GD in our patient was an IRD, and advise this as a possible differential diagnosis in patients presenting with hyperthyroidism on cART. To provide further details relevant to this case, we also review the literature concerning IRD-GD.

[Retroviruses-derived sequences in the human genome. Human endogenous retroviruses (HERVs)]. / Sekwencje pochodzenia retrowirusowego w genomie czlowieka. Ludzkie endogenne retrowirusy (HERV).

Retroviruses-derived elements in the human genome constitute 90% of non-coding mobile sequences. Reverse transcriptase (RT) plays an essential role in their transposition as do long terminal repeats (LTRs), which contain promotors, enhancers, and regulatory sequences. Some retroelements (pseudogens and retrogenes, e.g. SINE) are non-autonomic and do not possess their own RT. These elements are dependent on autonomic elements (retroposons, e.g. LINE, retrotransposons, exo- and endogenous retroviruses). The genome of retroviruses is composed of gag, pol, and env genes flanked by long terminal repeats. Endogenous retroviruses are probably the remnants of ancient germ cell infection by exogenous retroviruses and are transmissible to the next generation in a Mendelian way. Most of them are defective (because of mutation accumulation), but some are still active and their expression is regulated by different factors (UV radiation, inflammatory cytokines, steroid hormones, and exogenous virus products). Retroelements as well as their gene products exert influence on the organism's functions. They influence the plasticity and evolution of genomes, are a source of promotors and regulatory sequences, but they also supply additional signals of transcription initiation, mRNA splicing, and STOP codons. One of the positive aspects of human endogenous retroviruses (HERVs) is the participation of their products in normal syncytiotrophoblast formation. They also block exogenous retrovirus replication by receptor interference or antisense mRNA. Their presence is considered to be connected with a number of autoimmunological diseases (multiple sclerosis, insulin-dependent diabetes mellitus, systemic lupus erythematosus), cancer, or even psychiatric disorders (schizophrenia). There are also other problems connected with the potential role of ERVs in genomic therapy (with retroviruses vectors) and transplantology (xenotransplantation).

Genetic (KIR, HLA-C) and Some Clinical Parameters Influencing the Level of Liver Enzymes and Early Virologic Response in Patients with Chronic Hepatitis C.

Natural killer cells play an important role as effectors of innate immunity and regulators of adaptive immunity. They are important elements of the innate response to viral infections, which they detect using human leukocyte antigen (HLA) class I-binding receptors. Most polymorphic of these are killer cell immunoglobulin-like receptors (KIRs) which exist as two basic isotypes, activating or inhibitory receptors and are encoded by genes distributed differently in unrelated individuals. We searched for links between selected clinical data (including HCV viremia, liver enzymes level and liver histology parameters) and the presence of genes encoding these receptors and their ligands in hepatitis C virus-infected individuals subjected to pegylated interferon-α and ribavirin therapy. Genomic DNA samples from two hundred and ninety-two chronically infected patients were typed by polymerase chain reaction for the presence or absence of genes for KIRs and their ligands, class I HLA molecules, and clinical data of the patients were collected. Our results suggest an importance of clinical parameters and the contribution of KIR and HLA genes to the course of hepatitis C virus infection and the response to therapy. The study revealed that levels of liver enzymes before therapy were about 30% higher in patients who possessed a variant KIR2DS4 gene with 22-base pair deletion. Decrease of ALT activity after treatment was higher in HLA-C C2-positive than negative individuals. Beside it, patients demonstrated early virologic response to the therapy if the time lag before treatment was short, particularly in women.

Contribution of genes for killer cell immunoglobulin-like receptors (KIR) to the susceptibility to chronic hepatitis C virus infection and to viremia.

BACKGROUND: Natural killer (NK) cells are an important element of innate immunity against viruses, although their numbers decrease in the liver during chronic HCV infection. NK cells express a large panel of inhibitory and activating receptors. The most polymorphic of these are killer cell immunoglobulin-like receptors (KIRs) which are encoded by multiple genes that may be present or absent in given individuals depending on their genotype. This variability results in differential susceptibility to viral infections and diseases, including HCV infection and its consequences. AIMS AND METHODS: The aim of this study was to test whether chronical infection with HCV and the viremia levels are associated with any KIR gene in the Polish population. We typed 301 chronically HCV-infected patients and 425 non-infected healthy individuals for the presence or absence of KIR genes and their ligands, HLA-C C1 and C2 groups as well as HLA-B and HLA-A Bw4-positive alleles. RESULTS: We found that males, but not females, possessing KIR2DS2 and KIR2DL2 genes had a 1.7 higher probability to become chronically HCV-infected than males negative for these genes (p=0.0213). In accord with this, centromeric B region, containing KIR2DS2 and KIR2DL2 genes, was also associated with chronic HCV infection in males. In addition, patients of both genders possessing KIR2DS3 but not KIR2DS5 gene exhibited, on average, 2.6 lower level of viremia than HCV-infected individuals with other genotypes (p=0.00282). This was evident in those infected at a young age. KIR2DS3-positive patients also had lower mean levels of bilirubin than KIR2DS3-negative ones (p=0.02862). CONCLUSION: Our results suggest a contribution of the KIR2DS2 and KIR2DL2 genes (cenB haplotype) to the susceptibility to chronic HCV infection, and an association of the KIR2DS3 gene in the absence of KIR2DS5 with low viremia levels.

Frequency of human endogenous retroviral sequences (HERV) K113 and K115 in the Polish population, and their effect on HIV infection.

BACKGROUND: The human genome contains about 8% of endogenous retroviral sequences originated from germ cell infections by exogenous retroviruses during evolution. Most of those sequences are inactive because of accumulation of mutations but some of them are still capable to be transcribed and translated. The latter are insertionally polymorphic HERV-K113 and HERV-K115. It has been suggested that their presence and expression was connected with several human diseases. It is also believed that they could interfere with the replication cycle of exogenous retroviruses, including HIV. RESULTS: Prevalence of endogenous retroviral sequences HERV-K113 and HERV-K115 was determined in the Polish population. The frequencies were found as 11.8% for HERV-K113 and 7.92% for HERV-K115. To verify the hypothesis that the presence of these HERVs sequences could affect susceptibility to HIV infection, comparison of a control group (HIV-negative, not exposed to HIV; n = 303) with HIV-positive patients (n = 470) and exposed but uninfected (EU) individuals (n = 121) was performed. Prevalence of HERV-K113 and HERV-K115 in the EU group was 8.26% and 5.71%, respectively. In the HIV(+) group we detected HERV-K113 sequences in 12.98% of the individuals and HERV-K115 sequences in 7.23% of the individuals. There were no statistically significant differences between groups studied. CONCLUSION: The frequency of HERV-K113 and HERV-K115 sequences in Poland were found to be higher than usually shown for European populations. No relation between presence of the HERVs and HIV infection was detected.

Protective effect of CCR5-Δ32 against HIV infection by the heterosexual mode of transmission in a Polish population.

Effects of chemokine receptor alleles (CCR5-Δ32 and CCR2-64I) on susceptibility to human immunodeficiency virus (HIV) infection were studied in a Polish population. The CCR5 and CCR2 genotypes were determined for 311 healthy, HIV-negative individuals (control group), 121 exposed to HIV infection but uninfected (EU group), and 470 HIV-positive patients. The frequency of the alleles in the control group was calculated as 0.12 for both CCR5-Δ32 and CCR2-64I. The logistic regression method was used to analyze the effects of the described factors. A protective effect was observed for the CCR5-Δ32 allele but only in the case of heterosexual exposure. Prevalence of the CCR5-Δ32/+ genotype in HIV(+) patients infected via the heterosexual route (n=61; 8.2%) was much lower than in the control group (n=311; 21.5%); in the heterosexually exposed uninfected group it was slightly higher (n=28; 25%). This suggested that in this mode of infection, the native CCR5 expression level was crucial for establishment of infection. Individuals with the CCR5-Δ32 allele have more than three times less chance of infection in the case of HIV heterosexual exposure (odds ratio, 3.37; 95% confidence interval, 1.055-10.76). However, a protective effect of the CCR5-Δ32/+ genotype was not observed in the case of intravenous drug users (IDUs). The rates of the genotype were similar in HIV-infected IDU individuals (n=356; 17.7%) and in exposed uninfected patients (n=84; 15.5%), not significantly different from control group. No effect of the CCR2 genotype was observed. The analysis revealed that the important factor increasing infection risk was, in particular, hepatitis C virus (HCV) infection (odds ratio, 12.9). Moreover, the effect of HCV infection was found to be age dependent. Susceptibility to HIV infection resulting from HCV positivity became weaker (6% per year) with increasing age.