Hepatitis C virus (HCV) genotype 1 NS5A resistance-associated variants are associated with advanced liver fibrosis independently of HCV-transmission clusters.

OBJECTIVES: The aim of the study was to characterize the differences in the frequencies of NS3 and NS5A resistance-associated variants (RAVs) among Polish therapy-naive genotype 1 (G1) hepatitis C virus (HCV)-monoinfected and human immunodeficiency virus (HIV)/HCV-coinfected patients including clustering patterns and association of RAV frequency with liver fibrosis. METHODS: NS3/NS5A RAVs were identified by population sequencing in 387 directly acting antiviral treatment-naive G1-infected individuals (54 with genotype 1a (G1a) and 333 with genotype 1b (G1b)). Liver fibrosis was assessed based on histopathology or ultrasound elastography. Phylogenetic clusters were identified using maximum likelihood models. For statistics, chi-squared or two-sided Fisher's exact tests and multivariate logistic regression models were used, as appropriate. RESULTS: NS3 RAVs were found in 33.33% (18/54) for G1a and 2.62% (8/297) for G1b whereas NS5A variants were present in 5.55% (3/54) G1a and 9.31% (31/333) G1b sequences. Variations in NS5A 31 and 93 codon positions were found only in G1b (4.2% (14/333) for L31I/F/M and 5.39% (17/333) for Y93H). NS5A RAVs were more frequent among patients with advanced liver fibrosis (17.17% (17/99) for F3-F4 versus 6.94% (17/245) for F0-F2; p 0.004) or liver cirrhosis (20.34% (12/59) for F4 versus 7.72% (22/285) for F0-F3; p 0.003). Liver cirrhosis (F4) was associated with higher odds ratio of the NS5A RAVs among HCV-infected patients (odds ratio 2.34, 95% CI 1.004-5.291; p 0.049). NS5A RAVs were less frequent among sequences forming clusters and pairs (5.16% (8/155) versus 11.21% (26/232); p 0.039). CONCLUSIONS: Presence of NS5A RAVs correlated with progression of liver fibrosis and represents de novo selection of variants rather than transmission of drug resistance. Hence, the presence of NS5A RAVs may be a predictor for a long-lasting HCV infection.

Synthesis and characterization of nanocrystalline forsterite coated poly(L-lactide-co-ß-malic acid) scaffolds for bone tissue engineering applications.

In this research, after synthesizing poly(L-lactide-co-ß-malic acid) (PLMA) copolymer, hybrid particles of ice and nanocrystalline forsterite (NF) as coating carriers were used to prepare NF-coated PLMA scaffolds. The porous NF-coated scaffolds were directly fabricated by a combined technique using porogen leaching and freeze-drying methods. The obtained results indicate that the scaffolds were structurally porous with NF particles on their surfaces. When compared to the uncoated scaffolds, the NF coating improved both mechanical properties as well as enhanced bioactivity of the scaffolds. In addition, in vitro biological response of the rat bone marrow stromal cells indicated that NF significantly increased the biocompatibility of NF-coated scaffolds compared with PLMA.

Association of chemokine receptor gene variants with HIV-1 genotype predicted tropism.

OBJECTIVES: As a switch from chemokine (C-C motif) receptor 5 [CCR5 (R5)] to chemokine (C-X-C motif) receptor 4 [CXCR4 (X4)] HIV-1 tropism is associated with symptomatic and AIDS stages of infection, while chemokine receptor gene variants modify the tempo of HIV disease progression, we aimed to analyse the association between pretreatment HIV-1 tropism and chemokine polymorphisms known to restrict disease progression. METHODS: V3 genotype tropism prediction was performed in a group of 221 treatment-naïve patients, with subsequent CCR5 Δ32 (rs333), CCR2 V64I (rs1799864), CCR5 promoter (-627 C/T; rs1799988) and CX3CR1 V249I (rs3732378) genotyping performed in 206 patients. Alleles with a protective effect were assigned positive values while risk alleles were assigned negative values to calculate genetic scores. χ(2) tests, Mann-Whitney U-tests and logistic and linear regression models were used for statistical analyses. RESULTS: R5 tropism was found in 85.5% of patients (n = 189) using a false positive rate (FPR) of 5.75% and in 72.8% of patients (n = 161) using an FPR of 10%. A higher frequency of the 5.75% FPR predicted R5 tropism was associated with the CX3CR1 A allele (P = 0.027). Lower additive genetic scores were associated with an increased frequency of 5.75% FPR predicted R5 tropism (P = 0.0059), with the trend confirmed by logistic regression [odds ratio (OR) 0.5819; 95% confidence interval (CI) 0.3457-0.9795; P = 0.0416]. Viral load tended to increase with decreasing genetic score in the logistic regression analysis (slope = -0.127 ± 0.076; P = 0.095; r(2) = 0.161). CONCLUSIONS: The CX3CR1 A allele and lower genetic scores may restrict the switch of HIV-1 tropism from R5 to X4. This effect may be associated with the amount of co-receptor on the cell surface. Chemokine receptor gene polymorphisms influence both disease progression and tropism variability.

Introduction of pharmacogenetic screening for the human leucocyte antigen (HLA) B*5701 variant in Polish HIV-infected patients.

OBJECTIVE: Prospective pharmacogenetic screening for the human leucocyte antigen (HLA) B*5701 allele can significantly reduce the number of cases of abacavir-related hypersensitivity among HIV-infected patients treated with this drug. The aim of this study was to establish the frequency of the HLA B*5701 variant in HIV-infected Poles. METHODS: The sequence-specific primer (SSP) test was used to assess the feasibility of the introduction of such testing in clinical practice. For this purpose, 234 randomly selected HIV-positive patients were screened using a low-resolution SSP assay, with HLA B*5701-positive results confirmed using a high-resolution test. RESULTS AND CONCLUSIONS: The HLA B*5701 variant was found in 11 of 234 subjects (4.7%). Testing with the selected method proved quick and reliable.

Investigation of a new microcapsule membrane combining alginate, chitosan, polyethylene glycol and poly-L-lysine for cell transplantation applications.

Microencapsulation of living cells may serve as an alternative therapy for patients requiring organ transplants. One of the limiting factors in the progress of such therapy is attaining a biocompatible and mechanically stable polymer. The current study investigates the potential of a novel membrane combining alginate, chitosan, polyethylene glycol (PEG) and poly-L-lysine (PLL) with the objective of proposing a membrane suitable for cell entrapment that may overcome some of the shortcomings of the widely studied alginate-poly-L-lysine-alginate (APA) capsules. The novel microcapsule was formulated using a 1.5% alginate solution coated with 0.05% chitosan, 0.1% PEG and 0.05% poly-L-lysine with a final layer of 0.1% alginate. Microcapsules having a diameter of 450 +/- 30 microm were prepared. Upon citrate treatment, the membrane remained intact and retained its spherical structure. The membrane was able to support liver cell proliferation and the encapsulated cells were capable of secreting proteins. The study demonstrated that the new membrane can be used for cell entrapment. However, further investigations are needed to assess its potential for long term transplantation and usage in the development of bioartificial organs.

[The I/D polymorphism of the ACE gene in children with Henoch-Schoenlein purpura]. / Polimorfizm I/D genu ACE u dzieci z choroba Schönleina-Henocha.

The study group consisted of 24 children with Henoch-Schoenlein purpura (HSP) (13 girls and 11 boys, aged 66-233 months) who were treated in the Ist Pediatric Department between 1980 and 1998. The I/D polymorphism of ACE gene was determined by PCR amplification of genomic DNA with primers flanking the polymorphic region. Our preliminary results suggest lack of association between ACE genotype and kidney function. However, further follow-up studies based on sufficient number of participants are necessary to elucidate the role of ACE polymorphism in appearance and progression of renal symptoms in HSP.

[Survival of patients with the most frequent malignant neoplasms in the population of Krakow in the years 1985-1989]. / Przezycia chorych na najczestsze nowotwory zlosliwe w populacji Krakowa w latach 1985-1989.

In 1985-1989 there was not significant improvement of survival rates for the most common cancer sites in Cracow population. Survival of Cracow patients with colorectal, breast and larynx cancer and chronic lymphatic leukaemia were significantly below European mean. This situation is probably typical for whole Polish population.

[Comparison of 5-year survival rates of patients with the most common malignant neoplasms living in urban and rural areas in the years 1982-1983]. / Porównanie przezyc 5-letnich chorych na najczestsze nowotwory zlosliwe zamieszkalych w miescie i na wsi w latach 1982-1983.

Almost every fifth man and every third woman with malignant neoplasm living in urban and rural areas of the Cracov region, had in the years 1982-1983 the chance to survive 5 years. 5 year survivals of patients from urban and rural areas did not differ significantly. Also in other periods of time between 1976-1977 and 1982-1983 the differences were insignificant. As compared with data taken from other populations in the years 1982-1983, particularly low level of 5 year survivals has been noted in Cracov of male patients with cancer of the colon and in women with cancer of the uterine body. The survivals of patients with malignant neoplasms of the colon in both sexes, of the prostate and kidney in men and of breast in women are strikingly low in Polish patients. Also the survivals of Polish women with carcinoma of the uterine cervix are significantly lower as compared with other countries.

Blood-membrane interaction in hemodialysis leads to increased cytokine production.

Recently much interest has been focused on the role of immunoregulatory cytokines such as interleukin 1 (IL 1) and interleukin 2 (IL 2) during the pathogenesis of immunological as well as inflammatory diseases. Therefore peripheral blood mononuclear cells (PBMC) of eight patients undergoing hemodialysis (HD) were tested for IL 1 and IL 2 production. Before starting HD, cytokine production by PBMC in culture was not altered in comparison to normal healthy controls, however, a significant increase of IL 1 and IL 2 production was observed within the first HD hour which lasted throughout the end of HD. Moreover direct effects of cellulose membranes on PBMC cytokine production as well as serum IL 1 levels have been investigated. Serum IL 1 levels were already elevated before onset of HD and increased further during HD. The discrepancy between PBMC IL 1 production and serum IL 1 levels may be due to the diminished excretion in patients with end-stage renal disease. Since addition of dialysis membrane particles enhanced monocytes to produce more IL 1 as well as lymphocytes to release more IL 2, a direct stimulatory membrane effect is postulated. The increased release of immunoregulatory cytokines may account for some of the pathologic findings observed during hemodialysis.

UV-irradiated epidermal cells produce a specific inhibitor of interleukin 1 activity.

UV irradiation of epidermal cells (EC) in vitro and in vivo leads to an enhanced synthesis of the immunostimulating cytokine interleukin 1 (IL 1). However, UV exposure in vivo also results in local as well as systemic immunosuppression. Therefore, it was tested whether UV-exposed murine EC in culture in addition to IL 1 release an inhibitor of IL 1 activity. Supernatants of UV-irradiated BALB/c EC and of a transformed keratinocyte cell line (Pam 212) were evaluated for their ability to suppress IL 1-mediated thymocyte proliferation. Crude supernatants derived from either UV-exposed or unirradiated EC did not interfere with IL 1 activity. When supernatants were subjected to HPLC gel filtration, fractions eluting at approximately 40 kD significantly blocked the activity of EC-derived IL 1 and murine recombinant IL 1. The release of this inhibitory cytokine (EC-derived contra-IL 1 [EC-contra-IL 1]) was confined to UV-exposed BALB/c or Pam 212 keratinocytes, since no inhibitory activity was detected in supernatants of unirradiated cells. EC-contra-IL 1 also blocked IL 1-induced fibroblast proliferation but did not suppress IL 2 or IL 3 activity. Moreover, EC-contra-IL 1 did not inhibit spontaneous proliferation of a variety of cell lines (Pam 212, P388D1, L 929, EL 4). With the use of chromatofocusing EC-contra-IL 1 exhibited a pI of 8.8, and upon reversed-phase chromatography it eluted within three distinct peaks. Therefore, murine UV-exposed EC, in addition to the production of immunoenhancing cytokines, also may release immunosuppressing mediators and thereby participate in UV-induced immunosuppression. These findings further support the notion that the epidermis may not only be considered as a simple barrier against harmful agents but represents an active element of the immune system.

Inhibition of the induction of contact hypersensitivity by a UV-mediated epidermal cytokine.

UVB exposure (290-320 nm) of mice has been shown to cause systemic suppression of contact hypersensitivity (CHS). Because UVB radiation hardly penetrates the epidermis, epidermal cells have been anticipated to be the site of the initiation of immunosuppression. Supernatants derived from UV-irradiated BALB/c epidermal cell cultures and a keratinocyte cell line (Pam 212) were evaluated for the ability to induce suppression of CHS after i.v. injection to BALB/c mice. Injection of supernatants derived from UV-treated epidermal cells and Pam 212 cells significantly blocked induction but not elicitation of CHS. In contrast, i.v. application of supernatants derived from unirradiated cells did not inhibit CHS. Using high-performance liquid chromatography gel filtration this mediator was shown to be a low-molecular-weight protein (15-50 kD). Moreover UV-mediated inhibitor production seems to be confined to epidermal cells since neither P 388 macrophages nor L 929 fibroblasts released this inhibitory cytokine. Therefore UV radiation may induce epidermal cells to produce an inhibitor of CHS which is distinct from prostaglandins and leukotrienes and may participate in the regulation of UV-mediated local as well as systemic immunosuppression.

Intracranial pressure, cerebral blood flow, and cerebrospinal fluid formation during hyperammonemia in cat.

Intracranial pressure (ICP), cerebral blood flow (CBF), and the cerebrospinal fluid (CSF) formation rate were examined in anesthetized cats during ammonia intoxication. Hyperammonemia, evoked by intravenous infusion of ammonium acetate, caused a significant increase in ICP when the arterial blood ammonia level exceeded 400 mumol X liter-1. A progressive elevation of blood ammonia concentration was followed by a gradual rise in CBF, measured by the xenon-133 clearance technique. At an arterial blood ammonia level exceeding 500 mumol X liter-1, the CBF reached a plateau at 30% above the mean control value. Increase in ICP correlated weakly, but significantly, with the increase in CBF (R = 0.489, p less than 0.005). Elevation of the arterial blood ammonia level to 780.4 +/- 25.5 mumol X liter-1 for 2 hours elicited a significant gradual increase in CSF formation rate, measured by the ventriculocisternal perfusion method with iodine-125-albumin as an indicator substance. A maximum increase in CSF flow of 81% was noted at the end of the ammonium acetate infusion. It is suggested that hyperammonemia increases ICP both by cerebral vasodilatation and by enhancement of the CSF formation rate.